POST-TRANSLATIONAL MODIFICATION and the 20S PROTEASOME SYSTEM of the HALOARCHAEON Haloferax Volcanii

POST-TRANSLATIONAL MODIFICATION AND THE 20S PROTEASOME SYSTEM OF THE HALOARCHAEON Haloferax volcanii

MATTHEW ADAM HUMBARD

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2009

To my wife for her love, understanding, and patience

ACKNOWLEDGMENTS

I would like to acknowledge and thank Dr. Julie Maupin-Furlow for the opportunity to

pursue this project in her lab. Without the independence she granted me to pursue side-projects

and satisfy my intellectual curiosity, I would have never made it. Her constant guidance and

support was the one thing I knew I could count on throughout my graduate education. I would

also like to thank the members of my committee Drs. Valerie dé Crecy-Lagard, K. T.

Shanmugam, James Preston, and Nancy Denslow for their advice, attention, and time. I would also like to thank Dr. Rasche for her helpful input on my project and insights into archaeal physiology. I would like to thank my coworkers in Dr. Maupin-Furlow’s lab especially Drs.

Zhou, Reuter, and Kirkland. Dr. Zhou was instrumental in the creation of acetyltranferase knockout strains and the proteasomal knockout strains, Dr. Reuter assisted me with protein chromatography and activity assays, and Dr. Kirkland helped me with proteomics and held insightful discussions concerning my project and related side-projects. I would also like to thank some undergraduate researchers, Chris Tzikas, Kristin Toscano, Steve Garret, Jessica Coleman,

Dave Krause, Jonathan Pritz, and Cortlin Phillips. Chris and Kristin cloned and expressed different membrane-bound proteases in Haloferax volcanii. Steve Garret and Jessica Coleman worked with the β_strepII construct that was instrumental to the development of this work.

I am especially indebted to Drs. Stan Stevens, Sixue Chen, and Alfred Chung as well as

Scott McClung from the ICBR protein chemistry core for their support with my project. Dr.

Stevens’ experience and knowledge of mass spectrometry protocols and data interpretation were integral in the genesis of my graduate project. Dr. Alfred Chung synthesized artificial peptides for confirmation of phosphopeptides and AQUA studies. Scott McClung trained me to use the

LCQ Deca for personal projects and taught me how to set-up and use the specific equipment, one of the most valuable parts of my graduate education. I also want to thank Dr. Jennifer Busby at

the Scripps Research Institute in Jupiter, Florida for allowing me to come and visit her laboratory to learn about mass spectrometry and run samples on her equipment. This was a valuable trip and set the tone for my graduate project. I would also like to thank Dr. Savita Shanker at the ICBR

DNA sequencing core for her quality work sequencing DNA of constructs and clones.

My graduate career would have never been possible without the advice of Dr. Rajeev

Misra at Arizona State University who gave me my first scientific job and convinced me not to

drop out of college. I can not express how much that has changed the arc of my life and how that

decision has led me to this point.

Finally, I need to thank my family for all their support. I would like to thank my

grandfather Dr. Ralph Kelting who exposed me to biology and the university environment at a

young age. I have aspired to emulate him in as many ways as possible. I want to thank my wife

for her support and patience. I would not have completed this task without her love and

understanding.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 10

LIST OF FIGURES ...... 11

LIST OF ABBREVIATIONS ...... 13

ABSTRACT ...... 17

CHAPTER

1 LITERATURE REVIEW ...... 19

Introduction ...... 19 Proteasome Structure and Function ...... 19 20S Core Particle Structure and Subunit Arrangement ...... 20 Assembly of 20S Core Particle ...... 22 β-subunit Processing ...... 26 Structure and Folding of α-subunits ...... 29 Proteasome Activation ...... 31 19S Regulatory Particle ...... 31 11S Regulator / PA28 ...... 32 PA200 (Blm10) ...... 34 Proteasome Activating Nucleotidases (PAN) ...... 35 Targeting of Proteins for Degradation ...... 36 SsrA-tagging ...... 37 N-end Rule ...... 38 Ubiquitin Conjugation System and Polyubiquitin Chains ...... 40 Pupylation ...... 42 Co- and Post-translational Modification ...... 43 N-terminal Processing and Maturation ...... 44 Deformylation of methionine ...... 44 Methionine removal ...... 45 N-terminal acetylation ...... 46 Acylamino acid hydrolysis ...... 49 Phosphorylation ...... 50 Glycosylation ...... 53 O-linked glycosylation ...... 53 N-linked glycosylation ...... 54 Other Modifications ...... 55 Methylation ...... 55 Acylation ...... 57 Lipidation ...... 57

Post-translational Modification of Proteasomal Proteins ...... 58 Mass Spectrometry ...... 60 Phosphorylation Site Identification by Tandem Mass Spectrometry ...... 62 Quantitative Mass Spectrometry ...... 63 Label-free methodology ...... 63 Label-based methodology ...... 66 Project Rational and Design ...... 69

2 MATERIALS AND METHODS ...... 75

Chemicals, Strains and Media ...... 75 Materials ...... 75 Strains, Media, and Plasmids ...... 75 DNA Techniques ...... 76 Cloning ...... 76 DNA Electrophoresis ...... 77 Plasmid Isolation and Transformation ...... 77 Site-directed Mutagenesis ...... 78 Generation of N-terminal Acetyltransferase Knockouts ...... 78 Genome Analysis ...... 79 RNA Techniques ...... 79 RNA Isolation and RT-PCR ...... 79 qRT-PRC ...... 80 Protein Techniques ...... 80 Protein Expression in Escherichia coli ...... 80 Protein Expression in Haloferax volcanii ...... 80 Protein Quantification ...... 81 Protein Separation and Chromatography ...... 81 Nickel affinity chromatography ...... 81 Strep-tactin chromatography ...... 82 Gel filtration chromatography ...... 82 Peptide Synthesis ...... 82 Two-Dimensional Gel Electrophoresis ...... 83 Immunoblotting ...... 83 Labeling Hfx. volcanii Cells with Orthophosphate in Minimal Media ...... 84 Pulse-Chase ...... 84 Immunoprecipitation (IP) ...... 85 Molecular Modeling ...... 86 Enzyme Assays ...... 86 Peptide Hydrolyzing Activity ...... 86 Kinase Assays ...... 87 Mass Spectrometry ...... 87 Sample Preparation ...... 87 Three Dimensional Ion Trap (LCQ Deca) ...... 88 Hybrid ESI-Q-ToF (ABI QSTAR) ...... 88 Triple Quadrupole (ABI QTRAP) ...... 88 Database Searching and Software ...... 89

3 N-TERMINAL ACETYLATION OF ALPHA-TYPE SUBUNITS OF 20S PROTEASOMES IN Haloferax volcanii ...... 90

Introduction ...... 90 Results ...... 93 Isoforms of 20S Proteasome Subunits ...... 93 Analysis of Proteasome Isoforms by Mass Spectrometry ...... 95 α1 is N-terminally Acetylated ...... 96 Subpopulation of α1 Is Not Acetylated ...... 97 Acetylated Form of α1 Proteins Is Dominant ...... 97 Amino Acid Substitutions in the Penultimate Position of α1 Affect Acetylation and Cleavage of the Initiator Methionine Residue ...... 98 Co-purifying Non-proteasomal Proteins ...... 99 20S Proteasomes Rapidly Isolated by Two-step Affinity Purification from Hfx. volcanii ...... 100 20S Proteasomes Have Different Ratios of Modified α1 Proteins than Whole-cell Lysates ...... 101 CP Peptidase Activity was Enhanced by the α1 Q2A and ∆2-12 Modifications ...... 101 α1 Variants (Q2D, Q2P and Q2T) Accumulated as Heptameric Rings in Haloferax volcanii ...... 102 Complementation of the Hypoosmotic Stress Phenotype of psmA Mutant ...... 103 Cells Expressing α1 Q2T Display a More Thermotolerant Phenotype ...... 104 α2 is N-terminally Acetylated ...... 106 Discussion and Conclusions ...... 107

4 N-TERMINAL PROTEOME AND ACETYLTRANSFERASES IN Haloferax volcanii ..131

Introduction ...... 131 Results and Discussion ...... 133 Phylogenetic Distribution of GNAT Family in Haloferax volcanii ...... 133 Other Nα-acetylated Proteins in Haloferax volcanii ...... 136 Occurrence of Different Amino Acids in the Penultimate and Antepenultimate Positions ...... 139 Conclusions ...... 140

5 PHOSPHORYLATION AND METHYLATION OF SUBUNITS FROM 20S PROTEASOMES AND PROTEASOME ACTIVATING NUCLEOTIDASE A IN Haloferax volcanii ...... 160

Introduction ...... 160 Results and Discussion ...... 161 Isoforms of α1 Modulate During Growth-phase Transitions ...... 161 β Subunit is Phosphorylated ...... 163 PanA is Phosphorylated ...... 165 α2 Subunit is Phosphorylated ...... 166 α1 Subunit is Phosphorylated ...... 166 Variant α1 Proteins Affect Carotenoid Pigmentation and Cell Viability ...... 167

Haloferax volcanii Encodes a Number of Putative Protein Kinases ...... 168 Rio type-I Kinase from Hfx. volcanii Catalyzes Autophosphorylation in vitro ...... 169 Rio type-I Kinase Phosphorylates α1 in vitro ...... 170 α1 Subunit is Methylesterified ...... 170 Conclusions ...... 171

6 SUMMARY AND CONCLUSIONS ...... 198

Summary of Findings ...... 198 Future Directions ...... 201

APPENDIX

A PRIMERS USED IN THIS STUDY ...... 202

LIST OF REFERENCES ...... 206

BIOGRAPHICAL SKETCH ...... 239

LIST OF TABLES

Table page

1-1 List of known post-translational modifications of proteasome subunits...... 73

3-1 Strains and plasmids used in this study...... 113

3-2 Fragments of α1, α2 and β identified by QSTAR MS/MS from a trypsin digest ...... 115

3-3 Fragments of α1, α2 and β identified by QSTAR MS/MS from a sequential trypsin and chymotrypsin digest ...... 116

3-4 Summary of MS/MS-spectral counting and integration with a calculated ratio of Ac1 to N2 forms of α1...... 117

3-5 N-terminal modifications of α1 alter its Nα-acetylation and MAP cleavage pattern in 20S proteasomes and whole cells and influence peptidase activity...... 118

3-6 Three additional proteins identified by ESI-QTOF MS scans of proteasome-enriched samples...... 119

3-7 MS/MS analysis of 20S proteasomes purified by two-step affinity purification...... 120

3-8 MS/MS-identification of the 30-kDa isoform chain predominant in Hfx. volcanii cells expressing the α1 Q2T variant as α1 protein...... 121

4-1 Strains and plasmids used in this study...... 141

4-2 Methionine aminopeptidase and examples of acetyltransferase homologs proposed to play a role in N-terminal cleavage and acetylation...... 146

4-3 Summary of N-terminal peptides and associated proteins of Hfx. volcanii detected by MS/MS...... 152

5-1 Strains and plasmids used in this study...... 175

5-2 Site-directed mutants and phenotypes...... 178

5-3 MS/MS results of Rio1p-Strep fractions...... 179

A-1 Oligonucleotide primers used in this study...... 202

A-2 Oligonucleotide primer pairs for site-directed mutagenesis...... 203

A-3 Oligonucleotide primer pairs for cloning of kinase genes from Hfx. volcanii...... 204

A-4 Oligonucleotide primer pairs for N-terminal acetyltransferase knockouts...... 205

LIST OF FIGURES

Figure page

1-1 Crystal structures of 20S proteasomes from Thermoplasma acidophilium...... 70

1-2 Ubiquitination and pupylation pathways...... 71

1-3 Modified amino acid structures ...... 72

3-1 Isoforms of 20S proteasomal proteins...... 122

3-2 MS/MS Fragmentation of Acet-MoxQGQAQQQAYDR, an N-terminal fragment of the α1 protein of 20S proteasomes...... 123

3-3 MS/MS Fragmentation of Acet-MNRN(D)DKQAYDRGTSLFSPDGRIYQVE, an N- terminal fragment of the α2 protein of 20S proteasomes ...... 124

3-4 20S proteasome CPs are rapidly isolated by two-step affinity purification from Hfx. volcanii ...... 125

3-5 Quantitative mass spectrometry revealed α1 proteins with an acetylated N-terminal initiator methionine (Ac1) were dominant in ‘wild-type’ cells...... 126

3-6 Variant α1 Q2D, Q2P, and Q2T proteins accumulate in cells as heptameric rings...... 127

3-7 Variant α1 proteins either rescue the hypoosmotic stress phenotype of a chromosomal psmA deletion or exacerbate it ...... 128

3-8 Hfx. volcanii cells synthesizing the α1 Q2T variant displayed a thermotolerant phenotype ...... 129

3-9 Two-dimensional gel electrophoresis of cell lysate of Hfx. volcanii GZ130 expressing α1 wild-type (upper) compared to α1 Q2T (lower)...... 130

4-1 GNAT protein phylogenetic tree. Haloferax volcanii proteins compared to bacterial and eukaryotic homologues ...... 144

4-2 Frequency of amino acids in the penultimate position of the N-termini of proteins from the deduced proteome of Hfx. volcanii...... 150

4-3 MS/MS Fragmentation of Acet-SSIELTSSQK, an N-terminal fragment of HVO_1577...... 151

5-1 Phosphatase-sensitive isoforms of α-type 20S proteasomal proteins as a function of Hfx. volcanii growth ...... 181

5-2 MS/MS Fragmentation of RGEDMSMQALpSTL, an internal phosphopeptide of the β subunit of 20S proteasomes ...... 182

5-3 MS/MS fragmentation of the de novo synthesized peptide RGEDMSMQALpSTL. Region corresponding to residues 119 – 131 of the deduced β protein ...... 183

5-4 A PanA-specific phosphopeptide was reproducibly detected by ESI-QTOF analysis of tryptic digestions of PanA purified from Hfx. volcanii DS70 cells ...... 184

5-5 An α2-specific phosphopeptide detected in purified 20S proteasomes by MS/MS ...... 185

5-6 An α1-specific phosphopeptide was detected in purified 20S proteasomes by ESI-Q- ToF with precursor ion scanning ...... 186

5-7 Hypo-osmotic stress response of H26 ΔpanA cells expressing pJAM202c, panA, panA t1018g (PanA S340A) and panB at different NaCl concentrations...... 187

5-8 Phenotype of GZ130 (H26 ΔpsmA) cells expressing various α-type subuntis from Hfx. volcanii...... 188

5-9 Rio-kinases in Hfx. volcanii ...... 189

5-10 Strep_II tagged Rio1p purified from Hfx. volcanii phosphorylation of α1His6 subunits purified from E. coli ...... 190

5-11 MS/MS spectra of a methylesterified peptide fragment of α1 corresponding to residues 105 – 116, YGEPIGIEmethylTLTK ...... 191

5-12 MS/MS spectra of a methylesterified peptide fragment of α1 corresponding to residues 58 – 68, SPLMEmethylPTSVEK...... 192

5-13 MS/MS spectra of a methylesterified peptide fragment of α1 corresponding to residues 23 – 30, LYQVEmethylYAR...... 193

5-14 MS/MS spectra of a methylesterified peptide fragment of α1 corresponding to residues 13 – 22, GITIFSPDmethylGR...... 194

5-15 MS/MS spectra of a methylesterified peptide fragment of α1 corresponding to residues 150 – 163, LYETDPSGTPYEmethylWK...... 195

5-16 Multiple sequence alignment of select α-type 20S proteasome proteins ...... 196

5-17 Multiple sequence alignment of the central region of select β-type 20S proteasome proteins ...... 197

LIST OF ABBREVIATIONS

2DGE Two-dimensional gel electrophoresis

5-FOA 5-Fluoroorotic acid

AARE Acylamino acid releasing enzymes

AcP Acetyl phosphate

ADP Adenosine 5'-diphosphate

AMC 7-amido-4-methyl-coumarin

AMP Adenosine 5'-monophosphate

AP Alkaline phosphatase

Apr Ampicillin resistance

AQUA Absolute quantification

ATCC American Type Culture Collection

ATP Adenosine 5'-triphosphate

BCIP 5-bromo-4-chloro-3-indolyl phosphate p-toluidine

BLAST Basic Local Alignment Search Tool

˚C Degrees Celsius

C- Carboxyl

CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate

CID Collision induced dissociation

CP Core particle or 20S core particle

Da Dalton (atomic mass unit)

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

DTT Dithiothreitol

DUB Deubiquitinating enzyme

Ec Escherichia coli

EDTA Ethylenediaminetetraacetic acid emPAI Exponentially modified protein abundance indices

ESI Electron spray ionization

HABA hydroxyl-azophenyl-benzoic acid

HPLC High pressure liquid chromatography

HRP Horseradish peroxidase

Hv Haloferax volcanii

ICAT Isotope coded affinity tag

IEF Isoelectric focusing

IMAC Immobilized metal-affinity chromatography

IPTG Isopropyl-β-D-thiogalactopyranoside

iTRAQ Isobaric tag for relative and absolute quantification

kDa Kilodalton

Kmr Kanamycin resistance

LB Luria-Bertani broth

LC Liquid chromatography

Succ-LLVY-AMC N-succinyl-leucine-leucine-valine-tyrosine-7-amino-4-methylcoumarin

MALDI Matrix-assisted laser desorption/ionization

MAP Methionine aminopeptidase

MIDAS MRM-initiated detection and sequencing

MRM Multiple reaction monitoring

MS Mass spectrometry

MS/MS (MS2) Tandem mass spectrometry

N- Amino

NBT Nitro blue tetrazolium

NAT N-terminal acetyltransferase

NHS N-hydroxysuccinimide

Ni-NTA Nickel-nitrilotriacetic acid

nm Nanometer

Nvr Novobiocin resistance

O-GlcNAc O-linked β-N-acetylglucosamine

OD Optical density

PAGE Polyacrylamide gel electrophoresis

PAI Protein abundance indices

PAN Proteasome activating nucleotidase

PEP Phosphoenol pyruvate

PCR Polymerase chain reaction

PGPH Peptidylglutamyl-peptide hydrolyzing

Pup Prokaryotic ubiquitin-like protein

PVDF Polyvinyldiflouride

Q Quadrapole

qRT-PCR Quantitative real-time polymerase chain reaction

RNA Ribonucleic Acid

RP-HPLC Reverse phase high pressure liquid chromatography

RPM Rotations per minute

RT Reverse transcriptase

SAM S-adenosyl methionine

SCX Strong cation exchange

SDM Site directed mutagenesis

SDS Sodium dodecyl sulfate

SILAC Stable Isotope Labelling with Amino acids in Cell culture

SIM Single ion monitoring

Spr Spectinomycin resistance

SRM Selective reaction monitoring

Ta Thermoplasma acidophilum

TBS Tris-buffered saline

TBST Tris-buffered saline with Tween-20

TEA Triethylamine

TOF Time of flight

Tris N-tris(hydroxymethyl) aminomethane

Ub Ubiquitin

UV Ultraviolet

XIC Extracted ion chromatogram

V Volt

Vh Volt hour

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

POST-TRANSLATIONAL MODIFICATION AND THE 20S PROTEASOME SYSTEM OF THE HALOARCHAEON Haloferax volcanii By

Matthew Adam Humbard

August 2009

Chair: Julie A. Maupin-Furlow Major: Microbiology and Cell Science

While much of the study of proteasome systems in all organisms, eukaryotes and prokaryotes, focuses on substrate recognition, ubiquitin-like modifiers, or protein targeting, the

20S core particle and regulatory ATPases are subject to a wide variety of modifications that include, but are not limited to, phosphorylation and acetylation. Despite the large number of modifications known, the roles these modifications play within various proteasomes remain unclear, as they remain relatively unstudied. This study focused on identifying and studying specifically the sites of modification at the residue level, including mapping and altering these sites in order to better understand how proteasomes themselves are assembled and controlled.

Through the use of proteomic approaches, we have identified post-translational modification on all three subunits of 20S core particles in Haloferax volcanii as well as on one of the proteasome activating nucleotidases, PanA. In addition to mapping phosphorylation sites to the alpha1, alpha2, beta subunits of the 20S core particle, and PanA, both alpha-type subunits are also N- terminally acetylated. A novel post-translational modification, methylesterification, was also identified on aspartic and glutamic acid residues of the alpha1 protein. This is the first report of methylation of a proteasomal subunit. Phenotypic characterization of variant proteins unable to undergo N-terminal acetylation revealed that the N-terminus of alpha1 plays a role in the stability of the protein, and that this can alter the hypo-osmotic response, growth rate and yield.

Further investigation into N-terminal acetylation in Hfx. volcanii revealed this is a generalized pathway and nearly 30 % of soluble proteins are modified in this manner. Additionally, variant alpha1 proteins that were unable to undergo phosphorylation severely inhibited growth of Hfx. volcanii cells and altered their pigmentation. A candidate serine/threonine kinase from the atypical Rio-kinase family, was shown to catalyze autophosphorylation in vitro and phosphorylate recombinantly purified alpha1 proteins in vitro. Altogether, this study demonstrates that the proteins that make up the proteasomal system in Hfx. volcanii are heavily modified post-translationally, there is a generalized protein N-terminal acetylation pathway at work in the haloarchaeon, and a Rio type-I kinase autophosphorylates as well as phosphorylates alpha1 in vitro.

CHAPTER 1 LITERATURE REVIEW

Introduction

This literature review is designed to present the most current primary scientific literature concerning proteasomes and post-translational modification in the three domains of life. This

review will highlight on the assembly, function, and the role post-translational modification

plays on the regulation of proteasome systems. It will also look at the mechanisms employed by

different organisms with respect to N-terminal maturation, phosphorylation, methylation and

ubiquitin-like modification.

Proteasome Structure and Function

Proteasomes are large, energy dependent, proteases that act as the central house-keeping

protease responsible for protein turnover in the removal of misfolded or damaged proteins within

the cell (Chen et al., 2004). Proteasomes are conserved in archaea, eukaryotes, and in the

Actinobacteria class of bacteria (De Mot et al., 1999; Volker and Lupas, 2002; Wolf and Hilt,

2004; De Mot, 2007). This function, post-translationally controlling the levels of proteins,

places the proteasome in a unique regulatory role. Several biological processes are controlled

through protein degradation by proteasomes. Some of these processes include DNA replication,

cell division, metabolism, and antigen presentation by major histocompatibility complex I (MHC

I) presenting cells of the immune system (Josefsberg et al., 2000; Clarke, 2002; Goldberg et al.,

2002; Kang et al., 2002; Kirkland et al., 2007; Kirkland et al., 2008a). Disregulation of the

proteasome system has been implicated in the progression of several diseases in humans

including Alzheimer’s, Amyotrophic Lateral Sclerosis (ALS), Parkinsons’, and several different

kinds of cancer (Kabashi and Durham, 2006; Hung et al., 2006; van Leeuwen et al., 2006;

Olanow and McNaught, 2006; Zavrski et al., 2007; Hayslip et al., 2007; Stapnes et al., 2007).

Proteasomes are also essential for cellular growth in both eukaryotic organisms (Fujiwara et al.,

1990; Georgatsou et al., 1992; Lee et al., 1992; Nishimura et al., 1993b; Saville and Belote,

1993; Gerards et al., 1994) as well as the haloarchaeon Haloferax volcanii (Zhou et al., 2008).

Proteasomes in the Gram-positive actinobacter Mycobacterium tuberculosis have been shown to be necessary for virulence, as a part of nitric oxide stress response, but are not essential for growth (Darwin et al., 2003).

20S Core Particle Structure and Subunit Arrangement

The 20S core particle of proteasomes has a conserved quaternary structure from species to species. The 20S proteasome consists of a stack of four different seven membered rings made up of subunits either from the α-type or β-type superfamily. The rings are stacked with two

heptameric rings of β-subunits on the inside of the barrel with the outer two rings made up of α-

type subunits (α7β7β7α7) (Figure 1-1. Left panel.) (Lowe et al., 1995; Tamura et al., 1995; Groll

et al., 1997; Zuhl et al., 1997b; Nagy et al., 1998). Eukaryotic organisms, such as

Saccharomyces cerevisiae, encode at least seven different α-type subunits (α1 – α7) and seven

different β-type subunits (β1 – β7). The Eukaryotic proteasomes are made up of

heteroheptameric rings, each ring is made up of seven different subunits (α1 – α7). Each half-

proteasome contains 14 unique subunits, all with a defined space in the structure (Groll et al.,

1997). In higher eukaryotes, a mixed population of proteasomes may exist through the use of γ-

interferon inducible subunits (β1i, β2i, and β5i). Proteasomes that contain these specialized

subunits are called immunoproteasomes (Akiyama et al., 1994; Hisamatsu et al., 1996; Foss et

al., 1998; Rivett et al., 2001) and have reduced activity for cleavage after acidic amino acids

(Gaczynska et al., 1996). Prokaryotic organisms encode far fewer members of the α and β

families. The actinobacter Rhodococcus sp. encodes two α-type (α1 and α2) and two β-type (β1

and β2) subunits. These subunits form heteroheptameric rings much like the yeast (Tamura et

al., 1995; Zuhl et al., 1997a; De Mot et al., 1998). Archaeal organisms usually encode one α- type and one or two β-type subunits, as is the case with Thermoplasma acidophilum (Lowe et al.,

1995; Seemuller et al., 1995). The haloarchaeon Haloferax volcanii is somewhat unique in this respect, encoding two α-type subunits (α1 and α2) and only one β-type subunit (β). The α-type subunits form homoheptameric rings, unlike the yeast and Rhodococcus sp. This allows for several different kinds of proteasomes in a Hfx. volcanii cell. Subpopulations of α1ββα1,

α1ββα2, and α2ββα2 proteasomes have been purified from Hfx. volcanii cells (Wilson et al.,

1999; Kaczowka and Maupin-Furlow, 2003; Reuter et al., 2004; Zhou et al., 2008) (Figure 1-1).

Analysis of the crystal structures of both archaeal and eukaryotic 20S core particles shows that the interior consists of three chambers, the proteolytic chamber found between the two inner

β-rings and two ante-chambers at the interface of the α-ring and the β-rings (Figure 1-1. Right

panel.) (Lowe et al., 1995; Groll et al., 1997; Dorn et al., 1999; Groll et al., 2003). The two antechambers are formed by an aperture in the α-ring, where all substrates must pass. The structure of the antechamber, specifically the α-ring side, excludes folded proteins from entering the catalytic chamber and requires the assistance of ATPases for the recruitment and translocation of substrates. The central chamber of proteasomes houses the catalytic activity.

The N-terminal threonine residues of the active β-subunits are exposed after a processing event in this chamber (Arendt and Hochstrasser, 1997). While prokaryotic proteasomes typically lack the subunit complexity seen in eukaryotic proteasomes, usually encoding one or two β subunits, this is not the case in the heteroheptameric rings of β-subunits in eukaryotic 20S proteasomes

(Zwickl et al., 1992; Heinemeyer et al., 1994). The enzymatic activity of eukaryotic proteasomes is restricted to β1, β2, and β5 or β1i, β2i, and β5i as is the case in the immunoproteasome (Tanaka and Kasahara, 1998). The remaining, inactive, β-subunits are

required for assembly and proper maturation of the active β-subunits (Arendt and Hochstrasser,

1999). Proteasomes can display a variety of proteolytic activities including tryptic, chymotryptic, and postacidic cleavage (Lee et al., 1992; Nishimura et al., 1993b; Gerards et al.,

1994). The result of proteolysis of proteins are short peptides, 3 to 24 amino acids in length.

The fate of these peptides remains unclear. Conflicting hypotheses exist concerning whether the peptides passively diffuse away from proteasomes or are actively removed (Kohler et al., 2001;

Hutschenreiter et al., 2004). The resulting peptides can be used by the cell to regenerate amino acid pools with downstream peptidases or as signaling molecules (Uebel and Tampe, 1999).

Assembly of 20S Core Particle

Maturation of 20S proteasomes is a series of sequential folding, assembly, and processing events. Some assembly steps or intermediates are common to all three domains and others are not. Differences in subunit complexity are reflected in the complexity of assembly pathways for

20S core particles in the three domains of life. Each domain, Archaea, Eukarya, and Bacteria, has different requirements and unique challenges to properly assemble 20S proteasomes.

In the case of some archaeal proteasomes, functional complexes can be assembled entirely in vitro or in recombinant systems. In the case of the archaeon Methanosarcina thermophila, when equimolar amounts of purified α subunits (in the α7 ring form) and β prosubunits (β

subunits with the propeptide intact) are mixed, approximately 50 % of the β subunits are

processed. Electron microscopy revealed 20S proteasomes assembled properly (Maupin-Furlow

et al., 1998). These data show that propeptide processing is dependent on the presence of the α7

ring. The archaeal proteasomes from Thermoplasma acidophilum are also made up of only one

α and one β subunit. Co-expression of both the α and β subunits of T. acidophilum proteasomes

in E. coli yields fully assembled and active 20S proteasomes (Zwickl et al., 1994) as is also the

case with Methanosarcina thermophila (Maupin-Furlow et al., 1998). Additionally, expressing

the α subunit from the crenarchaeote Aeropyrun pernix and the β-precursor subunit from

Archaeoglobus fulgidus results in a proteolytic active chimeric 20S proteasome (Groll et al.,

2003). This spontaneous formation of functional 20S proteasomes indicates that there is no need

for proteasome specific chaperones for proper assembly and the ability to form chimeric

proteasomes comprised of recombinantly expressed subunits from different organisms suggests

that even phylogenetically distant archaea share an assembly pathway for 20S proteasomes.

Not all archaeal proteasomes can be reconstituted in vitro. In the case of haloarchaea,

functional proteasomes have never been reconstituted in vitro for haloarchaea, however assembly

intermediates can still be detected. When α1 subunits are recombinantly expressed in E. coli and

purified in high salt (2 M), heptameric rings (α1(7)) can properly fold (Wilson et al., 1999). A heptameric α7 intermediate can be detected in the case of α1 subunits from Haloferax volcanii

proteasomes. When α subunits from T. acidophilium are expressed in E. coli, they assemble into

heptameric rings (α7) and di-heptameric structures in the cell (α7α7). N-terminal substitution and

deletion analysis of the α subunit of T. acidophilium revealed that a conserved α-helix from residues 22 – 33 is important in forming the stable intermediate heptameric ring. A deletion of

amino acids 2 – 34 (α Δ2-34) is unable to form rings or complete 20S proteasomes. Likewise

with the single amino acid substitution α Glu25Pro is unable to form rings or proteasomes

(Zwickl et al., 1994). A crystal structure of the α-ring made up of α subunits from

Archaeoglobus fulgidus has the same rigid structure whether it is incorporated into the 20S

proteasome or not. The α-ring is believed to be the earliest assembly intermediate in the archaeal

proteasome assembly pathway and is thought to serve as scaffolding to recruit pre-processed β

subunits to form a half-proteasome. The presence of half-proteasomes can be seen when A.

fulgidus β threonine 12 to glycine (Thr12Gly) is used in place of the wild-type β-subunit. 20S

proteasomes did not form properly with this mutation. The crystal structure of the A. fulgidus β

Thr12Gly proteasome revealed two half-proteasomes (with β subunit propeptides uncleaved) had a 4.5 Å gap between them. The gap had regions of disorder that did not resolve in the crystal structure (Groll et al., 2003). This observation suggests that the half-proteasome is an assembly intermediate and dimerization of the half-proteasomes is followed by the propeptide autocleavage of the β-subunits.

Bacterial proteasomes can be found in the Actinobacter class of α-proteobacteria (Tamura et al., 1995; Zuhl et al., 1997a; Zuhl et al., 1997b; De Mot et al., 1998; Nagy et al., 1998; De

Mot, 2007). The first of these bacterial proteasomes to be studied in detail was from

Rhodococcus sp. strain N186/21 (Tamura et al., 1995). Rhodococcus expresses two different α

(α1 and α2) and two different β subunits (β1 and β2). Both β subunits are functional.

Expression of either of the α subunits with either of the β subunits yields a functional 20S proteasome (α1β1, α2β1, α1β2, or α2β2) (Zuhl et al., 1997b). Similar to the archaeal example discussed above, chimeric proteasomes with subunits from different Actinobacter, such as

Frankia, could spontaneously assemble into functional 20S proteasomes (Pouch et al., 2000).

However, when the α-type subunits were expressed alone, no ring structure was observed. This is a departure with the assembly pathway of archaeal proteasomes. When the β subunit variant

β1 Lys33Ala from Frankia sp. was co-expressed with the α subunit, proteasomes were able to form properly, although half proteasomes were also observed (Mayr et al., 1998). The β1

Lys33Ala subunit is unable to cleave off the β1 propeptide, so the propeptide is still present in assembled 20S proteasomes. The half-proteasome is a short lived intermediate in the bacterial proteasome assembly pathway but not the heptameric α rings. There is speculation that the

Rhodococcus proteasome most likely assembles first into heterodimers of α and β subunits, and

the heterodimers oligomerize to form a half-proteasome, consisting of 7 α and seven unprocessed

β subunits. The assembly intermediates of Rhodococcus erythropolis were measured by real- time mass spectrometry confirming that the bacterial proteasome does not pass through the heptameric α-ring as an intermediate (Sharon et al., 2007). Dimers and trimers of α subunits can be detected but the majority of subunits are either assembled into whole proteasomes or are in the short-lived intermediate half-proteasomes. The half proteasomes come together spontaneously and stimulate the cleavage of the propeptides after dimerization.

Eukaryotic proteasomes have maximized the diversity of subunits and as a consequence have the most complicated and specific assembly pathway. Mammalian proteasomes pass

through several intermediates, originally identified as 13S, 15S, and 16S, before their

biochemical composition could be determined (Schmidtke et al., 1997). The first step in

eukaryotic proteasome assembly is similar to the first step in archaeal proteasome assembly, the

formation of the α-ring. In eukaryotic organisms, as opposed to archaeal organisms, this means

the formation of a heteroheptameric ring containing seven different α-type subunits (α1 – α7).

Each of the seven different subunits has a defined place in the structure. Observations of the individual subunits’ in vitro folding and assembly has shown that α7 and α5 of S. cerevisiae can dimerize and form double-ring like structures (Yao et al., 1999b). Similarly, α6, α7, and α1 of S. cerevisiae mixed together results in the formation of a random ring like structure with variable subunit positioning in the rings (Gerards et al., 1997). Since these structures can form randomly, eukaryotes have a devoted set of proteasome specific chaperones that aid in the formation of different assembly intermediates and prevent the malformation of α subunit aggregates or incorrect ring arrangements. Proteasome biogenesis factors (Pba or PAC 1 – 4) are a set of highly conserved eukaryotic chaperones that are responsible for proper α-ring formation

(Rosenzweig and Glickman, 2008b). Pba1 and Pba2 bind to “open” rings that contain up to 5

different α subunits (α1, α2, α5, α6, and α7). The remaining two subunits, α3 and α4, are

inserted last. Yeast cells devoid of α3 can still produce a functional 20S proteasome. In the α-

ring structure, α4 can substitute for α3 in order to close the ring (Velichutina et al., 2004). Once

the α-ring is formed, the sequential addition of different β subunits can occur using the α-ring as a template.

Once the α-ring is correctly assembled, the β-type subunits are incorporated in a specific and sequential order. The α-ring is bound with Pba1 and Pba2; Pba3 and Pba4 bind to the ring to recruit the β2 subunit. Once the β2 subunit is bound, the C-terminal tail of the β2 subunit, Pba1, and Pba2 recruit the β3 (Ramos et al., 2004). β3 brings along β4 and the chaperone Ump1 while displacing Pba3 and Pba4 from the growing structure. The next two subunits are β5 and β6.

They are recruited to the structure by Ump1 followed closely by β1. At this point, the “half proteasome” is complete with the exception of the β7 subunit. Dimerization of the “half proteasomes” is carried out simultaneously to the incorporation of the β7 subunits. This is achieved through the long C-terminal tail of the β7 subunit (Ramos et al., 2004) and a protein complex called Blm10 (PA200) responsible for proteasome activation (Ortega et al., 2005).

Eukaryotic proteasomes are completed when the two “half proteasomes” come together, the propeptides on β-type subunits are cleaved and Pba1, Pba2, and Ump1 are degraded by the proteasome. The order of β subunit insertion into the half-proteasome structure is conserved between yeast and mammals (Hirano et al., 2008). The chaperones responsible for this assembly pathway are also widely distributed throughout eukaryotes (Hirano et al., 2008).

β-subunit Processing

Active β-type subunits of 20S proteasomes belong to the Ntn-hydrolase family. Ntn- hydrolases are typically synthesized as inactive precursors and undergo an autocatalytic internal

cleavage exposing an N-terminal threonine, serine, or cysteine. Threonine (Thr1) serves

universally as the single residue active site for all known active β-type subunits in 20S

proteasomes. A substitution of serine for the active site threonine (Thr1Ser) in Methanosarcina

thermophila 20S proteasomes reduced both the chymotrypsin and post-glutamyl peptidase

activity by 30 and 70 %, respectively (Maupin-Furlow et al., 1998). Likewise, Thermolplasma acidophilium 20S proteasomes containing β subunits with the same substitution (Thr1Ser) has

reduced proteolytic activity against proteins (Kisselev et al., 2000). The propeptides of active β

subunits, and some inactive β subunits as is the case with β7 (N3) (Thomson and Rivett, 1996),

are removed in an autocatalytic event as the final step of proteasome assembly and maturation.

The propeptide of the active β subunits may play multiple roles in cells including supporting initial folding of β subunits, protecting the cells from hydrolase activity (Khan and

James, 1998), preventing Nα-acetylation of the active site (Arendt and Hochstrasser, 1999; Jager

et al., 1999), and aiding in proteasome assembly (Zuhl et al., 1997a). The propeptides of active

β-subunits in 20S proteasomes can vary in length from 4 amino acids to over 70, as is the case

with the γ-interferon inducible β5i (LMP7E2) subunit (Lee et al., 1990).

Some of the propeptides of β-type subunits play a role in proteasome assembly. The propeptide regions of the β subunits in Rhodococcus aid in proteasome assembly. The β1Δpro

(β1 subunit with the propeptide deleted, β1 Δ2-64) still is able to form functional proteasomes in the Rhodococcus sp. However, the assembly efficiency is greatly reduced. Providing the propeptide in trans improved the in vitro assembly of the 20S proteasome (Zuhl et al., 1997b).

Not all propeptides are required for proper assembly as is the case with Thermoplasma acidophilum. The βΔpro (β Δ2-8) subunit from T. acidophilum can be expressed with the α

subunit in E. coli and still assemble into active 20S proteasomes, indicating that the propeptide does not have a role in assembly of the Thermoplasma proteasome (Zwickl et al., 1994).

Propeptides are essential for growth and prevent N-terminal acetylation of active site residues in yeast. The propeptide of β5 (Doa3) is essential for chymotrypsin-like activity and viability of Saccharomyces cerevisiae cells (Arendt and Hochstrasser, 1999). The chymotrypsin-

like activity housed in the β5 subunit is essential to protein degradation and growth (Chen and

Hochstrasser, 1996; Arendt and Hochstrasser, 1997). The β2 and β1 propeptides are dispensable

for cell growth, indicating that they are not necessary for mature 20S proteasome formation.

Even though the modified β2 and β1 subunits are incorporated into 20S core particles, the

mutants expressing the β1ΔLS (β1 Δ2-19 or pre3ΔLS) and β2ΔLS (β2 Δ2-29 or pup1ΔLS)

subunits have a lower growth rate, but viable. Cells expressing both defective subunits have an

increased growth defect. 20S proteasomes containing these leader peptide lacking subunits have

drastically reduced peptidase activity. Proteasomes with β1ΔLS had no peptidylglutamyl-

peptide hydrolyzing (PGPH) peptidase activity and proteasomes with β2ΔLS had a 60 %

reduction in trypsin-like activity. The activity is restored in a nat1 mutant (nat1 encodes Ard1),

an acetyltransferase gene. The acetylation of the N-termini of the different β-subunits results in

the inactivation of the activity by blocking the nucleophile required to catalyze the reaction

(Arendt and Hochstrasser, 1999).

Some prokaryotic organisms encode a single β-type subunit. Others encode multiple β-

type subunits. In the cases where there are more than one type of β subunit, not all the subunits

may be active. In Rhodococcus sp. there are two active β subunits while in the crenarchaeota

Aeropyrum pernix one is active and the other is not (Koonin et al., 2001). In eukaryotic

organisms, four out of the seven β subunits are inactive, β6 (C5), β4 (C7), β3 (C10), and β7 (N3).

Inactive subunits may or may not undergo the propeptide cleavage. The active subunits, β2 (Z),

β1 (Y), and β5 (X), and their immunoproteasome counter-parts β2i (MECL-1), β1i (LMP2), and

β5i (LMP7E2), all undergo the required propeptide autocleavage as part of the final steps of proteasome assembly (Seemuller et al., 1996). The cleavage sites of active eukaryotic β subunits are conserved in yeast (Chen and Hochstrasser, 1995), slime-mold (Schauer et al., 1993), rat

(Lilley et al., 1990), and human (Lee et al., 1990).

Structure and Folding of α-subunits

20S proteasome assembly and maturation continuously excludes the proteolytic active site

from the cytoplasm first by blocking the N-terminal threonine active site with a propeptide and

second by sequestering the active site within the structure after maturation. Conserved amino-

acid residues in the N-termini of α-type subunits of proteasomes create structural elements that

keep proteasomes in an auto-inhibited state by obstructing entry of substrates into the 20S core

particle. The Tyrosine-aspartic acid-arginine motif (YDR) is conserved in all known α-type

subunits in all three domains. The YDR motif makes up the end of a N-terminal α-helix that

extends into the aperture at the center of the α-ring and is an essential component of N-terminal

gating by the α subunits. Deletions or substitutions in this motif results in increases of peptidase

activities of 20S core particles independent of ATPase action (Groll et al., 2000; Benaroudj et

al., 2003; Bajorek and Glickman, 2004). Since this amino acid sequence is present in all α

subunits, gated channel may be a general mechanism for regulating the activity of the

proteasome (Groll et al., 2000).

The rate limiting step in protein degradation by proteasomes is translocation of the

substrate through the α-ring to the active site contained within the central chamber and formed

by the β-rings. Eukaryotic 20S core particles have no protease and relatively low peptidase

activity in the absence of the 19S cap or chemical pretreatment with SDS or heat treatment

(Coux et al., 1996). Peptidase activity of the yeast 20S proteasome is blocked by the N-terminal regions of the α3 subunit. A deletion of the N-terminal α-helix in the α3 (α3 ΔN or α3 Δ2–9) subunit abolishes the N-terminal gating of the 20S core particle. A single substitution of

Asp9Ala in the α3 subunit also increases peptidase activity (Groll et al., 2000). One difference between the crystal structure of eukaryotic and prokaryotic 20S core particles is in the α-rings.

The structures of the 20S core particle from Thermoplasma acidophilum (Lowe et al., 1995) and

Archaeglobus fulgidus (Groll et al., 2003) reveal a common axial channel of 13 Å that connects the interior of the core particle with the surroundings. The X-ray structure of the yeast 20S particle did not contain this channel (Groll et al., 1997). These differences in structures might reflect differences between crystal forms and not actual differences within the cell. The N- terminal 11 amino acids of the A. fulgidus α subunit are not defined by electron density and are less ordered in the 20S proteasome than the 16S “half” proteasome precursor (Groll et al., 2003).

Although α Asp9Asn, Asp9 is present in the YDR motif of A. fulgidus α subunit, in A. fulgidus does not have an obvious change in peptidase activity (Groll et al., 2003), α Δ2 – 12 in T. acidophilum (a deletion of the N-terminal helix including the YDR motif) abolished the need for

ATPase activity of PAN in the degradation of acid-denatured GFPssrA or casein (Benaroudj et

al., 2003). The conserved YDR motif thought to be important in the α-ring gating sterics, is

present in all known archaeal α-type subunits to date. Although the mechanism may not be

exactly the same, archaeal proteasomes may gate their α-ring aperture. Furthermore, cryo-

electron microscopy of the Mycobacterium tuberculosis 20S core particle reveals closed ends

that are dependent on the first 8 residues of the α-subunit. These 8 N-terminal residues diminish peptidase activity of 20S core particles as well (Hu et al., 2008).

Proteasome Activation

Proteasomes, like all energy-dependent proteases with known structures, exist in cells in an auto-inhibited state, sometimes referred to as the latent state. As discussed previously, the outer

α-rings of the 20S core particles act to prevent diffusion of random proteins into the proteolytic core. External forces or proteins have to act on core particles and substrates to stimulate the degradation by active β subunits in the interior of core particles. This can be achieved in vitro with the addition of heat or denaturing agents such as SDS or urea, that denature the substrate proteins or disrupt the axial gating of 20S core particles, or in vivo with the assistance of different protein complexes. This section will discuss the various structures and proteins that have been shown to influence 20S proteasome activity in different organisms.

19S Regulatory Particle

The 19S cap, or PA700, interacts with the α-rings of eukaryotic 20S proteasomes to form

26S or 30S proteasomes in a reversible and ATP-dependent process. The 26S proteasome is a

20S core particle with one 19S cap bound, the 30S proteasome is when both ends are capped with 19S caps. 20S core particles associated with 19S caps are commonly referred to as 26S proteasomes, despite the number of 19S caps in complex. The 19S regulatory particle is responsible for the recognition, binding, unfolding, and translocation of substrates into the proteolytic chamber of the proteasome. Unfolding and translocation of substrates are ATP- dependent processes (Kohler et al., 2001; Navon and Goldberg, 2001; Takeuchi and Tamura,

2004). In yeast, the 19S complex consists of at least 17 unique gene products that make up two different substructures, the lid and the base. The base structure is made up of 6 regulatory

particle triple-A (Rpt) ATPases, Rpt1 to Rpt6, and three regulatory particle non-ATPase (Rpn)

subunits, Rpn1, Rpn2, and Rpn10. The base structure is the ATP-hydrolyzing portion of the

structure and is responsible for the mechanical unfolding and translocation of substrates into the

20S core particle. The lid is attached to the base structure through the Rpn10 subunit of the base.

The functions of the lid portion of the structure include recognition and binding of substrate

proteins (such as polyubiquitination proteins). The lid structure is made up of Rpn3, Rpn 5-9,

Rpn11 and Rpn12.

Although a complete crystal structure of 19S regulatory particles, as well as 26S

proteasomes, has not been resolved for any eukaryotic organism, extensive cryo-electron microscopy and protein-protein interaction studies in yeast, Caenorhabditis elegans, Drosophila melanogaster, and humans have been conducted. The 19S regulatory particles activate the 20S core particles by changing the conformation of N-terminal extensions on the α-type subunits making up the axial gate to the proteasome (Kohler et al., 2001). The base subunits bind to the

α-rings in the 20S core particle. This causes structural changes in the 20S core particle.

Subunits α5 and α6 and the interface between α1 and α7 are joined to the base structure of the

19S RP by the C-terminal tails of Rpt2, Rpt5 and Rpt1 (Smith et al., 2007; da Fonseca and

Morris, 2008). Specifically, the Rpt2 subunit has been shown to physically change the structure of the axial channel of the 20S proteasome by opening it and allowing substrates to enter (Kohler et al., 2001). Rpt1 and Rpt2 of yeast can form hetero-oligomers when recombinantly expressed in E. coli and Rpt4 can form homo-oligomers (Takeuchi and Tamura, 2004). These complexes are enough to hydrolyze ATP and unfold substrates to be exposed to the protease activity of the archaeal 20S core particle in vitro.

11S Regulator / PA28

The 11S regulator, PA28 or PA26 in Trypanosoma brucei (Yao et al., 1999a), stimulates peptidase activity of 20S proteasomes in an ATP-independent manner but does not stimulate proteolysis of folded proteins (Dubiel et al., 1992; Ma et al., 1992). It is a multi-protein complex made up of two different subunits, PA28α and PA28β (Mott et al., 1994; Ahn et al., 1995;

Kohda et al., 1998) and possibly PA28γ (Realini et al., 1994). When recombinantly produced,

PA28α forms a homoheptamer and PA28β remains a monomer (Song et al., 1997). An amino acid substitution in PA28α (N50Y) prevents homoheptameric formation. When the PA28α

N50Y is mixed with PA28β, it forms a heteroheptameric ring composed of three PA28α subunits and four PA28β subunits (Song et al., 1996; Zhang et al., 1999). This heteroheptameric structure is the most likely physiological structure since all three subunits, PA28α, PA28β, and

PA28γ, can activate 20S proteasomes by themselves but the level of activation is greatly increased when PA28α and PA28β are combined (Kuehn and Dahlmann, 1997; Song et al.,

1997). A crystal structure of the Trypanosome PA26 activator and the yeast 20S proteasome in complex was determined (Whitby et al., 2000) as well as the PA26 bound to an archaeal 20S proteasome (Forster et al., 2005). The structure of the axial gate of the α-ring was altered in the combined structures. In the yeast:Trypanosome structure, three of the α subunits, α1, α6, and α7, retained the confirmation previously seen in the 20S core particle structure. The remaining four

α subunits, α2, α3, α4, and α5, showed drastic differences in their N-termini from the 20S structure alone. The four α subunits that normally establish the gating mechanism of 20S proteasomes adopted a new conformation binding to the interior of PA26, thereby opening the aperture to the proteolytic chamber. The archaeal 20S proteasome and Trypanosome PA26 structure revealed that only one side-chain interaction with the α subunits was required for PA26 to stimulate peptidase activity, α Lys66. Amino acid substitutions in position Lys66 to alanine

(α Lys66Ala) and serine (α Lys66Ser) abolished PA26 interaction and stimulation. The idea that this would only interact with one amino acid side chain is supported by the observation that

PA28 activity can be abolished with a single substitution in its C-terminal domain (Ma et al.,

1993; Song et al., 1997; Li et al., 2000).

Although the exact biological role of the PA28 activator is debated, the fact that the expression of PA28αβ is induced by γ-interferon could indicate that it plays a role in modulating the activity of the immunoproteasome. The three specific subunits of the immunoproteasome,

β1i, β2i and β5i, are also induced by γ-interferon (Ahn et al., 1995; Groettrup et al., 1995).

Therefore, PA28αβ may play an important structural or functional role in the immunoproteasome

(Rechsteiner et al., 2000). It has also been suggested, since 11S regulators can bind to 26S proteasomes, they may facilitate the diffusion of peptides out of the proteolytic chamber. This function could potentially be the physiological role of the activator in the immunoproteasome.

The length of the product peptide generated by 20S proteasomes is determined by the amount of time the product spends in the proteolytic chamber (Tanaka and Kasahara, 1998). If the 11S regulator opens the opposite axial gate, larger products (peptides 8 – 10 amino acids in length), compatible with MHC I presentation could diffuse out of the core particle and be used for antigen presentation (Tanaka and Kasahara, 1998).

PA200 (Blm10)

PA200 (Blm10) is a 200 kDa protein that binds to the outer ring of the 20S core particle similar to the 19S regulatory particle (Ortega et al., 2005). It is wide spread in mammalian tissues but also found in lower eukaryotes such as yeast. PA200 is a large protein made up of

HEAT repeats and forms a α-solenoid structure (Schmidt et al., 2005). PA200 in yeast was originally thought to only aid in the assembly pathway or mature 20S proteasomes by bringing together two half-proteasomes (Fehlker et al., 2003). Negative-stain electron microscopy showed that PA200 interacts with the mature core particle forming a cap over the 20S proteasome channel (Schmidt et al., 2005; Iwanczyk et al., 2006). Additional work showed that

PA200 binds to mature and enzymatically active 20S core particles and opens the axial channel formed by the α-subunits, enhancing peptidase activity but not protease activity (Iwanczyk et al.,

2006). Current purposed roles and physiological function of PA200 is to regulate substrate translocation or product release of 20S proteasomes. These hypotheses are supported by the ability of PA200 to distinguish between open and close gate conformations of the mature 20S core particle. Since PA200 preferentially binds open state core particles, PA200 binding to one may facilitate gate opening at the trans end of the core particle. 20S core particles with two

PA200s bound have reduced peptidase-activity and hybrid RP-CP-PA200 complexes have a lower rate of protein degradation (Lehmann et al., 2008). These two observations support the model where PA200 binding affects the trans axial gate.

So far, this section has discussed three examples of protein complexes, 19S RP, PA28, and

PA200, that are able to simulate protease activity of 20S core particles. All three protein complexes bind directly to the α-ring of core particles alter the structure of the axial gate formed by the α-ring. Although this may not be the only way to affect proteolytic activity of 20S core particles, this strategy of affecting α subunit conformation is a conserved mechanism for activation in these three examples. Other strategies may include unfolding of proteins so they can pass into the proteolytic chamber of the proteasome without affecting the α-ring structure.

Proteasome Activating Nucleotidases (PAN)

Similar to the Rpt subunits within the base of the 19S regulatory particle of eukaryotic 26S proteasomes, archaeal species encode for ATPase subunits thought to regulate 20S proteasome function (Zwickl et al., 1999; Wilson et al., 2000). Some of these archaeal regulatory particles are referred to as PAN (Proteasome Activating Nucleotidases) and share sequence homology to the ATPase subunits of the 19S RP in eukaryotes (Zwickl et al., 1999). PAN complexes can even interact with eukaryotic proteasomes (Smith et al., 2005). The conserved C-terminal motif hydrophobic-X-tyrosine (HbXY), where X is any amino acid, has been shown to be essential for

PAN proteasome function (Smith et al., 2007; Zhou et al., 2008). It is believed that this C- terminal tail is required for functional association of PAN complexes with the 20S core particle.

Some archaea encode more than one PAN protein. Two PAN proteins, PanA and PanB,

have been identified in the haloarchaeon Hfx. volcanii (Reuter et al., 2004) and two in

Methanosarcina mazei (Medalia et al., 2006). Another homologue from Methanococcus

jannaschii has been shown to increase the activity of proteasomes from Methanosarcina

thermophila and Thermoplasma acidophilum (Zwickl et al., 1999; Wilson et al., 2000). The

PAN proteins, PanA and PanB, from Hfx. volcanii have been shown to form heterohexamers as

well as homododecamers (Reuter et al., unpublished) and the M. jannaschii PAN assembles into

dodecamers that can associate with 20S proteasomes (Wilson et al., 2000). PAN proteins can

bind and unfold proteins in vitro without additional components. This unfolding event is ATP

dependent (Benaroudj and Goldberg, 2000; Navon and Goldberg, 2001). Following the

unfolding event, proteins bound by PAN are translocated into the catalytic chamber of the 20S

proteasome to be degraded (Smith et al., 2005). PAN is hypothesized to be involved in substrate

selection and delivery to 20S core particles. Binding to substrates by PAN stimulates ATP

hydrolysis, unfoldase activity, and translocation of the denatured peptide into the proteolytic

chamber of the proteasome (Benaroudj et al., 2003). Substrates of the PAN complexes are fed

into the proteolytic chamber C- to N-terminus after being mechanically unfolded on the surface

of the ring (Navon and Goldberg, 2001). Not all archaea encode PAN proteins. Archaea that are

apparently lacking PAN, may encode different AAA ATPases thought to have similar function,

such as VAT or Cdc48 as is the case for Thermoplasma acidophilum (Gerega et al., 2005).

Targeting of Proteins for Degradation

The ability to quickly turnover specific proteins as well as damaged or misfolded proteins

is an essential function in all cells. Bacteria, archaea, and eukarya have evolved specific

pathways that target certain proteins for destruction. Generalized pathways of protein

degradation exist for damaged or misfolded proteins, but directed and targeted approaches that

include a myriad of post-translational tagging or modifications are also employed. This section

will review four different targeting mechanisms employed in different organisms that use a co-

or post-translational tag to signal degradation.

SsrA-tagging

A targeting mechanism used by bacterial cells involves tagging proteins that have stalled at

the ribosome due to lack of charged tRNA, a transcription error, or as a strategy to regulate

cellular metabolism through proteolysis (Abo et al., 2000). This involves addition of a short 11 amino acid tag to the C-terminus of protein that targets it for degradation (Komine et al., 1994;

Gottesman et al., 1998; Gillet and Felden, 2001). This tag is called SsrA for “small stable RNA

A” encoded protein. The ssrA molecule is a tmRNA or a transfer-messenger RNA and acts as

both a transfer RNA and a messenger RNA. The ssrA is charged with an alanine (A) and enters

a stalled ribosome at the A site. The original, stalled, piece of mRNA is displaced by the

tmRNA that codes for an additional 10 amino acids. The result is an eleven amino acid C-

terminal tag (AANDENYALAA) (Karzai et al., 2000). SsrA-tagged proteins are recognized by

either adapter molecules, such as SspB (Wah et al., 2002), or proteases, such as ClpAP, ClpXP,

FtsH, and Lon (Gottesman et al., 1998; Smith et al., 1999; Farrell et al., 2005; Herman et al.,

1998). The adapter molecule SspB is not required for degradation of SsrA-tagged substrates in

E. coli but it enhances the rate of degradation by the ClpXP complex (Flynn et al., 2001; Wah et al., 2002). Mutations in the ssrA sequence resulting in amino acid substitutions alanine 10 to aspartic acid (Ala10Asp) and Ala11Asp greatly reduce the efficacy of the targeting (Gottesman et al., 1998). These substitutions interfere with the SsrA / ClpXP interaction. The ClpXP complex binds residues 1, 2, and 8 – 10 in the SsrA tag. The adapter molecule SspB binds

residues 1 – 4 and residue 7 (Flynn et al., 2001). Even though the SsrA tagging system seems

unique to the bacteria, SsrA-tagged proteins can be degraded by 20S proteasomes and PAN complexes from archaeal species (Benaroudj and Goldberg, 2000; Navon and Goldberg, 2001;

Benaroudj et al., 2003; Medalia et al., 2006). GFP-SsrA is a common substrate used for proteolysis assays for archaeal proteasomes (Benaroudj and Goldberg, 2000; Benaroudj et al.,

2003; Reuter and Maupin-Furlow, 2004; Medalia et al., 2006; Benaroudj and Goldberg, 2000).

Since PAN proteins can thread substrates in a C- to N-terminus direction into the proteasome

(Navon and Goldberg, 2001), perhaps there is a conserved C-terminal degradation signal in the

archaea.

N-end Rule

The N-end rule is a protein targeting pathway that connects the N-terminal amino acid of a protein to the in vivo stability, or half-life, of that protein with respect to proteolysis (Gonda et

al., 1989; Varshavsky, 1996). Although currently the N-end rule has not been demonstrated in any archaeal organism, the pathway is conserved between bacteria and eukaryotes (Mogk et al.,

2007). The machinery involved in the N-end rule pathway may vary from domain to domain but the recognition of the N-terminal motifs is highly conserved. The N-end rule pathway involves a series of post-translational modifications at the N-terminal amino acid of a protein, eventually leading to the degradation of the protein by either the proteasome / ubiquitin pathway in eukaryotes or the ClpAP / ClpS system in bacteria (Mogk et al., 2007).

With respect to the N-end rule, N-terminal amino acids can be described as primary, secondary, or tertiary destabilizing residues. These classifications are based on the number of post-translational modifications the residues undergo before being targeted to proteolytic machinery. The primary destabilizing residues in mammals are phenylalanine, tryptophan, tyrosine, leucine, arginine, lysine, histidine, and isoleucine (Varshavsky, 1996). They do not

require any additional post-translational modification before entering the proteasome degradation

pathway. In the case of eukaryotic organisms, the primary destabilizing residue tagged

substrates enter the proteasome-ubiquitin pathway by interacting with E3 ubiquitin ligases called

N-recognins (Tasaki et al., 2005; Meinnel et al., 2006; Xia et al., 2008). In bacteria, there are

fewer primary destabilizing residues. Even though there are fewer, the primary destabilizing

residues in bacteria are conserved (Phe, Trp, Tyr, and Leu) (Varshavsky, 1996). Proteins with a

primary destabilizing residue at the N-terminus are targeted to the ATP-dependent proteolytic system of ClpAP and may use the adapter ClpS (Erbse et al., 2006; Hou et al., 2008).

Secondary stabilizing residues, lysine and arginine in E. coli and aspartic and glutamic acid in Vibrio vulnificus and eukaryotic cells, require a primary destabilizing residue be attached to the primary amine at the N-terminus of the protein. In the E. coli example, this is carried out by the L/FK,R-transferase (Aat) and results in a leucine (primary destabilizing residue) as the N-

terminal residue with the secondary, lysine or arginine in this example, in the N-terminal penultimate position of the protein (Abramochkin and Shrader, 1995). The L/FK,R-transferase can also transfer phenylalanine, methionine, and tryptophan to the N-terminus of a protein less efficiently, but the dominant physiological role is to transfer a leucine (Abramochkin and

Shrader, 1995). In V. vulnificus, there is an additional transferase, LD,E-transferase (Bpt) that

catalyzes the formation of the peptide bond between the N-terminal acidic residues, aspartic or

glutamic acid, with leucine (Graciet et al., 2006). Aspartic acid and glutamic acid are the only secondary destabilizing residues in eukaryotic systems and in these systems arginine is

conjugated to the N-terminus of aspartic acids or glutamic acids by R-transferases (Ate1) (Tasaki and Kwon, 2007).

Tertiary destabilizing amino acids are only found in eukaryotes to date and require two post-translational modifications before entering the proteasome degradation pathway. The tertiary destabilizing residues are asparagine, glutamine, and cysteine (cysteine is only a tertiary destabilizing residue in mammals) (Varshavsky, 1996). Both asparagine and glutamine undergo enzymatic deamidation to aspartic and glutamic acid, respectively, by the enzyme Ntan1 (Baker and Varshavsky, 1995). Aspartic acid and glutamic acid are secondary destabilizing residues and are substrates for R-transferases (Ate1). Cysteine is modified through a reaction with nitric oxide to form oxidized cysteine (CysS-O3H). This oxidized cysteine is a substrate for the Ate1

R-transferase (Varshavsky, 2008).

Ubiquitin Conjugation System and Polyubiquitin Chains

The ubiquitin conjugation system is the primary targeting mechanism for protein degradation in eukaryotic cells. Ubiquitin is a conserved 76 amino acid protein and is covalently attached through a series of enzymatic reactions to proteins at lysine residues or the N-terminus and targets them for degradation by 26S proteasomes in eukaryotic organisms (Hershko and

Ciechanover, 1998). The addition of ubiquitin to proteins is a multi-step process that is carried

out by a series of enzymes. The chemistry that activates and transfers the ubiquitin protein to a

target protein appears to have evolved from the thiamine biosynthesis and molybdopterin

synthesis pathways (Hochstrasser, 2000; Hochstrasser, 2009). Step one is carried out by the Ub-

activating enzyme, E1. The C-terminal glycine residue of ubiquitin is activated by forming an

intermediate ubiquitin adenylate through the hydrolysis of ATP and the release of

pyrophosphate. A cysteine residue on E1 substitutes for the adenylate resulting in a thioester

linkage and the release of adenosine monophosphate (AMP). The activated ubiquitin protein is

then transferred to another cysteine residue in an E2 enzyme (ubiquitin carrier proteins or

ubiquitin-conjugating enzymes) resulting in a new thioester bond. The ubiquitin-ligated E2

enzyme physically interacts with an E3 enzyme that is bound to a substrate. One of two different events can occur. The E3 enzyme can present a substrate directly to the E2 and the substrate is ubiquitinated by the E2 or ubiquitin can be transferred to the E3 and onto a free primary amine in the bound protein. In either case, the ubiquitin is linked to either the ε-amine of a lysine residue or a free α-amine of the N-terminus resulting in an atypical isopeptide bond (distinct amide).

This process may be repeated, ligating ubiquitin to ubiquitin until a polyubiquitin chain consisting of four or more ubiquitin molecules is formed (Figure 1-2). In some cases, another enzyme designated E4 (accessory factor) is needed to elongate the ubiquitin chain (Koegl et al.,

1999). The second ubiquitin in the chain is connected to the first at a lysine residue in the primary sequence of ubiquitin, usually Lys48. There are examples of polyubiquitin chains being assembled on Lys6, Lys 11, Lys 27, Lys29, Lys33, Lys 63, but these events seem to be more unique and not always associated with protein degradation (Li and Ye, 2008). The classic signal for degradation by 26S proteasomes is a polyubiquitin chain consisting of at least four ubiquitin molecules connected through Lys48. The four ubiquitin chain signals 19S regulatory particles to bind and unfold the substrate. The complexity of this process is reflected in the enzyme complexity. While a typical organism may have only one or two different E1 enzymes, there can be dozens of different E2 and hundreds of E3 ligases encoded in a single genome (Hershko and

Ciechanover, 1998; Huang et al., 2004; Li and Ye, 2008). The E3 enzymes seem to provide the selectivity of substrates with each E3 enzyme having a narrow range of possible substrates.

Several 19S regulatory particle subunits can bind ubiquitin, increasing the concentration of ubiquitinated substrates at 26S proteasomes. Rpt5 subunits of base structures in 19 RP are involved in ubiquitin recognition (Lam et al., 2002). There is some debate how important the

Rpt5 interaction with ubiquitin is, since there are several other candidates that may be more

available for initial interactions with substrates. The non-ATPase subunits Rpn10 and Rpn1 can bind and interact with ubiquitinated proteins (Young et al., 1998; Elsasser et al., 2002). More recently, Rpn13 is thought to be the subunit in the lid portion of the regulatory particle that is involved in initial interaction with ubiquitin-tagged substrates (Husnjak et al., 2008; Schreiner et al., 2008).

Ubiquitin itself is not degraded in this process. Eukaryotic organisms synthesize deubiquitinating enzymes (DUBs), or ubiquitin-specific proteases, that remove the ubiquitin from substrates while they are at the 19S cap. Deubiquitinating enzymes serve multiple roles in the cell including processing ubiquitin to an active form, proof reading of polyubiquitin chains, and regenerating ubiquitin by removing polyubiquitin chains from proteins as they are translocated into 20S core particles (Amerik and Hochstrasser, 2004). Deubiquitinating enzymes play an important regulatory role with ubiquitin as they initiate ubiquitin immediately after it is translated by removing the N-terminal leader sequence and regenerates it so it is not degraded by the proteasome.

Pupylation

The existence of an ubiquitin like molecule in prokaryotes has been speculated in the past

(Durner and Boger, 1995; Maupin-Furlow and Ferry, 1995; Bienkowska et al., 2003; Spreter et al., 2005). The discovery of Pup (prokaryotic ubiquitin-like protein) demonstrates that

Mycobacterium uses a similar protein tag to modify proteins, possibly target them for degradation by the 20S proteasome. The pup gene is in an operon with the proteasome 20S core particle genes prcB (β) and prcA (α) and is conserved in the Actinobacter class of bacteria. The

C-terminal sequence, Lys-Gly-Gly-Gln, is 100 % conserved throughout the Actinobacter and is similar to the C-terminus of ubiquitin, Arg-Gly-Gly. The 6.9 kDa connector protein called Pup has been shown to be covalently linked to proteins and target them for degradation by the 20S

proteasome (Iyer et al., 2008; Pearce et al., 2008). Pup is covalently attached to the ε-amine of a

lysine side-chain of a substrate after the C-terminal glutamine is deaminated to a glutamic acid.

This deamination is carried out by Dop, a deaminase encoded in M. tuberculosis. Dop binds

ATP as a cofactor but does not hydrolyze it in the process of deamination (Striebel et al., 2009).

The free carboxylic acid is phosphorylated in an ATP-dependent manner and the phosphate group is displaced by the target protein (Iyer et al., 2008). This process is catalyzed by a carboxylate-amine ligase called PafA. PafA is required for the conjugation of Pup to proteasome substrates in Mycobacterium (Pearce et al., 2008) (Figure 1-2). There are dozens of speculated targets for puplyation (Mukherjee and Orth, 2008) and subsequent degradation by the 20S proteasome in Mycobacterium, but there are also other questions surrounding this mechanism.

For example, it is unclear if being modified by one Pup protein is sufficient for degradation or if poly-Pup chains, as is the case with ubiquitin, must form for efficient degradation. Also, it is unclear whether there are Pup specific proteases, depupylating enzymes, that remove Pup proteins or chains from substrates before they are degraded. Pupylation is an exciting new post- translational modification found in the Actinobacter that could be associated with protein turnover.

Co- and Post-translational Modification

During or after translation, polypeptides undergo a series of maturation events that may include proteolytic processing, folding, localization, and chemical modification before they are mature, functional proteins. There are over 200 known protein modifications that can occur across the three domains of life (some of the modifications discussed in this section can be seen in Figure 1-3). These modifications can be post-translational or co-translational, stable or reversible. Protein modification is responsible for increased protein diversity across all proteomes. Considering post-translational modifications, proteomes may be two or three orders

of magnitude more complex than the corresponding genome would predict (Walsh et al., 2005).

The majority of proteins that undergo a modification often times are subjected to multiple

modifications (Yang, 2005). Some of these modifications include disulfide bond formation, glycosylation, lipidation, biotinylation, phosphorylation, methylation, acetylation, and nitrosylation. Nearly every amino acid can undergo a side chain modification with the exceptions of leucine, isoleucine, valine, alanine, and phenylalanine. Some stable modifications are required for proper folding or stability of a protein. Other transient modifications are used to modulate the function of proteins. This section will discuss common post-translational modifications that occur in the different domains of life emphasizing the mechanisms of modification and the enzymes that catalyze the reactions.

N-terminal Processing and Maturation

All RNA-encoded protein synthesis begins at the N-terminus. The N-terminal residues of growing polypeptide chains are the first portions of proteins exposed to the enzymatic activities of the cytoplasms of organisms. Residue-specific alterations to the N-terminus of proteins can occur co-translationally and post-translationally. Modifications such as removal of initiator methionine, deformylation of formyl-methionine in bacteria, modification of α-amino group by acetylation, or propeptide cleavage are examples. The removal of the initiator methionine and the addition of an acetyl group to the α-amino of the polypeptide are two modifications that affect the majority of proteins in eukaryotic cells (Brown and Roberts, 1976).

Deformylation of methionine

Formylated methionine is the initiating residue in bacterial translation. Neither eukaryotes

(with the exception of protein synthesis that takes place in the mitochondria or chloroplast) or archaea use formyl-methionine as an initiating residue (Ramesh and RajBhandary, 2001).

Deformylation is the co-translational removal of the formyl-group on methionine and takes place

on nearly all polypeptides in bacterial cells (Fry and Lamborg, 1967; Adams, 1968; Giglione and

Meinnel, 2001; Giglione et al., 2004). Peptide deformylases catalyze the cleavage of the N-

formyl group from nascent N-formyl-methionine polypeptides in bacteria and organelles.

Peptide deformylases are related to amino-peptidases and belong to the superfamily of HEXXH-

containing metalloproteases. All the metalloproteases of this large family use two histidine

residues of the HEXXH motif as metal ligands. Peptide deformylases constitute a new subfamily in which cysteines are the third metal ligand (Meinnel et al., 1996).

Methionine removal

Initiating methionine removal by methionine aminopeptidases is an essential co- translational process that occurs in all organisms and compartments within the cell where protein translation takes place across all three domains of life (Bradshaw et al., 1998; Giglione et al.,

2004). This process is carried out by a devoted set of peptidases called methionine aminopeptidases (MAP). MAP activity is the major source of amino acid diversity at the N-

terminus of proteins, and 80 % of all proteins undergo this modification in the cell (Waller, 1963;

Brown, 1970; Matheson et al., 1975). There are two families of methionine aminopeptidases,

MAP1 (type 1) and MAP2 (type 2). The different MAP families have different phylogenetic

distributions. Archaea, such as Pyrococcus furiosus and Haloferax volcanii, have one MAP2

while bacteria, such as Escherichia coli, have one MAP1 (Lowther and Matthews, 2000).

Higher eukaryotic organisms can have both MAP1 and MAP2 in the cytoplasm and MAP1 in

organelles. Although these two families of MAP enzymes have only low levels of sequence

identity, they have similar three-dimensional crystal structures (Lowther and Matthews, 2000).

Whether or not a protein is a MAP substrate is dictated by the nature of the N-terminal

(second) penultimate amino acid. The radius of gyration of the second amino acid defines

whether or not the initiating methionine can be removed (Hirel et al., 1989). When small amino

acids, such as glycine, alanine, proline, serine, threonine, valine and cysteine, are found in the

penultimate position of a protein, the methionine is efficiently removed from the N-terminus.

Other amino acids, asparagine, aspartic acid, leucine and isoleucine, can also have their initiator methionine removed but at a lower efficiency (Hirel et al., 1989).

N-terminal acetylation

Nα-acetylation is a common co-translational modification in eukaryotic organisms with 50

– 90 % of soluble proteins acetylated on N-terminal serine, alanine, glycine or threonine residues after the removal of the initiating methionine (Brown and Roberts, 1976; Driessen et al., 1985;

Polevoda and Sherman, 2003b). In contrast, bacterial N-terminal acetylation is rare and thought to be post-translational (Waller, 1963; Tanaka et al., 1989). Recent reports in the haloarchaea have demonstrated that N-terminal acetylation may be more common in archaea than in bacteria with an estimated 20 – 30 % of all soluble proteins being putative substrates (Falb et al., 2006;

Humbard et al., 2006; Aivaliotis et al., 2007; Mackay et al., 2007; Kirkland et al., 2008b).

Both bacteria and eukaryotic organisms synthesize multiple N-terminal acetyltransferases

(NAT). In Escherichia coli, there are three N-terminal acetyltransferases; RimI, RimJ, and

RimL (Yoshikawa et al., 1987; Tanaka et al., 1989). There are three major N-terminal acetyltransferases found in Saccharomyces cerevisiae (Polevoda and Sherman, 2003a). They are designated NatA, NatB, and NatC and act on specific substrates.

NatA. NatA is the primary NAT in yeast cells with multiple substrates identified in vivo and over 2500 predicted protein targets. NatA substrates appear to be degenerative, encompassing a wide range of sequences, especially those with N-terminal residues of serine or alanine. Approximately 90 % of proteins with a serine, threonine, glycine, or alanine in the N- terminal position are acetylated in yeast (Driessen et al., 1985; Polevoda and Sherman, 2003b) and a third, auxiliary subunit Nat5p (San) (Gautschi et al., 2003). NatA activity requires two

subunits, Ard1p and Nat1p (Mullen et al., 1989; Park and Szostak, 1992). The probable role of

Nat1p is substrate binding and positioning of NatA at the ribosome and Ard1p is the catalytic

subunit. The nat1 and ard1 mutants were unable to acetylate proteins in vivo including those

with serine or alanine as the N-terminal amino acid. The mutants were tested against 24 proteins

known to be acetylated in vivo by two-dimensional gel analysis (Polevoda et al., 1999; Polevoda

and Sherman, 2003b). The mutants shared similar growth phenotypes including temperature-

sensitivity, slower growth on non-fermentable sugars, failure to enter G0, defects in sporulation,

and sensitivity to salt and detergents (SDS) (Mullen et al., 1989). Even though mutants that

affect NatA activity have a pleiotropic phenotype in yeast, the activity is not essential.

Interestingly, the NatA activity is essential in Trypanosoma brucei during the mammalian and insect-stage of development (Ingram et al., 2000). The exact mechanism that makes this activity essential in T. brucei is not currently known. It is speculated that it is required for proper chromatin remodeling or telomeric silencing. Nat5p is not essential or required for the acetylation of the 24 model NatA substrates . Knock-outs of the nat5 gene in yeast does not share the phenotypes of the ard1 or nat1 mutants (Polevoda and Sherman, 2003a). It is possible that Nat5p acetylates a subset of not yet identified targets. Recent studies have identified possible substrates for Nat5p containing NatA complexes in humans (Arnesen et al., 2006).

NatB. NatB acetyltransferase contains the catalytic subunit Nat3p and the auxiliary subunit Mdm20p. Both are required for NatB activity (Polevoda et al., 2003). NatB substrates have common N-terminal sequences of Met-Met-, Met-Asn-, Met-Asp-, or Met-Glu-. All Met-

Asp- and Met-Glu- eukaryotic proteins that have been analyzed have been shown to be N- terminally acetylated. NatB acetylates two essential proteins, actin and tropomyosin in yeast. In addition, it has been shown to acetylate the small subunit of ribonucleotide reductase Rnr4p,

ribosomal proteins S21 and S28 (Arnold et al., 1999), 26S proteasomal subunits Pre1p, Rpt3p, and Rpn11p (Kimura et al., 2003). Out of all three of the N-terminal acetyltransferase activities in yeast, mutations in either nat3p or mdm20 have the most severe phenotypes. It was originally believed to be essential for cell viability (Kulkarni and Sherman, 1994). Further examination revealed that cells were defective in acetylation of two essential proteins, actin and tropomyosin.

This defect led to the temperature and osmotic sensitivity, inability to utilize non-fermentable carbon sources, and reduced mating, and increased sensitivity to DNA damaging agents

(Polevoda et al., 2003). Acetylation of tropomyosin is required for association with actin filaments and lack of actin acetylation decreases sliding velocity and disrupts cytoskeleton function (Jeong et al., 2002). The disruption of the cytoskeletal network leads to the pleiotropic phenotype of nat3p and mdm20 mutations.

NatC. The catalytic subunit of NatC is Mak3p and encoded by the mak3 gene. NatC is made up of three subunits Mak3p (catalytic), Mak10p and Mak31p (Polevoda and Sherman,

2001). All three subunits are required for the activity of NatC. Knock-out strains for the genes responsible for the NatC activity have slower growth rates at higher temperatures when grown on non-fermentable sugars. The substrates for NatC in the cell are peptides beginning with an N- terminal methionine and followed by an isoleucine, leucine, tryptophan, or phenylalanine (Met-

Ile, Met-Leu, Met-Trp, and Met-Phe). The activity of NatC is required for the acetylation of the viral major coat protein, gag, with an Ac-Met-Leu-Arg-Phe- N-terminus as well as two 26S proteasomal subunits Pup2p and Pre5p (Kimura et al., 2003). NatC activity is clearly not essential, yet the activity is conserved through eukaryotes and similar substrates are acetylated by the enzyme complex in different species (Polevoda and Sherman, 2003a).

RimI, RimJ, and RimL. Only four proteins from E. coli are known to be Nα-acetylated.

They are the ribosomal subunit L7 (L12 is the unacetylated form) (Isono and Isono, 1981), S5

(Cumberlidge and Isono, 1979), S18 (Isono and Isono, 1980), and the elongation factor Tu (Arai et al., 1980). Three N-terminal acetyltransferases have been identified in E. coli, RimI, RimJ, and RimL. These three acetyltransferases are responsible for the Nα-acetylation of L12, S5 and

S18 respectively. The bacterial acetyltransferases apparently do not form a complex with other proteins as the NAT genes of eukaryotic organisms, but act alone. Purified RimL can catalyze the acetylation of the primary N-terminal amino of L12 without an accessory proteins (Miao et al., 2007). Since RimL can act in vitro on folded protein substrates, it is believed that the modification of bacterial proteins by N-terminal acetylation is post-translational rather than co- translational. Additionally, the presence of both L7 and L12 (the acetylated and unacetylated forms of L7 protein) on mature ribosomes also indicates that the acetylation is post-translational.

Acylamino acid hydrolysis

Acylamino acid-releasing enzymes (AAREs) are able to remove acetylated amino acids from the N-terminal end of peptides and proteins (Radhakrishna and Wold, 1989; Krishna and

Wold, 1992; Sokolik et al., 1994). The AAREs have been found in both eukaryotes and archaea but not in bacteria (Ishikawa et al., 1998; Mori and Ishikawa, 2005; Perrier et al., 2005). The phylogenetic distribution follows the trend of generalized protein acetylation pathways. In vitro studies reveal these enzymes can cleave Acetyl-Ala, Acetyl-Thr, Acetyl-Met, Acetyl-Gly, and

Acetyl-Ser from the N-terminus of proteins and peptides (Sokolik et al., 1994). There are two additional peptide hydrolases that can remove acetylated amino acids from the N-terminus of peptides, acetyl-Met and acetyl-Cys hydrolases, that are involved in actin processing (Sheff and

Rubenstein, 1992a; Sheff and Rubenstein, 1992b). It is unclear whether these enzymes are

related to AARE in structure or function. The exact roles of AARE are currently not known in

the cell but they could be involved in recycling amino acids for future synthesis.

Phosphorylation

Protein phosphorylation has been referred to as the flagship of reversible post-translational modification and is common in all three domains of life (Johnson and Barford, 1993). The vast majority, if not all, cellular processes are affected by phosphorylation of proteins.

Phosphorylation of proteins regulates DNA replication, transcription, translation, metabolism, transport of proteins and metabolites, cell division, protein degradation, and serves as a relay chemical in signal transduction pathways in both prokaryotes and eukaryotes (Kennelly, 2002;

Kennelly, 2003; Eichler and Adams, 2005). Phosphorylation can occur on the side chains of

amino acids serine, threonine, tyrosine, aspartic acid, arginine, lysine, cysteine, glutamic acid,

and histidine. In the case of ser, thr, tyr, and asp, phosphorylation events create a phosphoester

bond with the hydroxyl group. Histidine is different. Histidine residues are phosphorylated on a nitrogen atom in the imidazole ring resulting in phosphoramidite bonds (P–N). There are two nitrogen atoms that can serve as the acceptor of the phosphate group, atom 1 and 3, resulting in the formation of 1-phosphohistidine or 3-phosphohistidine. A phosphate group can be donated from the γ-phosphate of adenosine triphosphate (ATP), the β-phosphate of adenosine diphosphate (ADP), phosphoenolpyruvate (PEP), acetyl-phosphate (AcP), polyphosphate, or another phosphorylated protein.

Kinases catalyze the addition of a phosphate group to a substrate. Kinases are one of the largest group of known post-translational enzymes with 518 putative protein kinases identified in the human genome alone (Venter et al., 2001; Manning et al., 2002; Johnson and Hunter, 2005).

The complexity and large number of protein kinases implies a large number of substrates, ranging in the thousands. It is estimated that one third of all intracellular proteins in eukaryotic

organisms exist in different phosphorylation states resulting in approximately 20,000 distinct

phosphoproteins within the cell. In eukaryotes, kinases are classified by structural motifs in the

primary amino acid sequence or by substrate. There is a kinase superfamily of enzymes and 13

atypical protein kinase (aPK) subfamilies that do not fit into a typical eukaryotic protein kinase

(ePK). Typical eukaryotic protein kinases contain a catalytic domain of 250 – 300 amino acids

that bind nucleotides, bind the peptide substrate, and carry out the phosphoryl transfer reaction.

Kinases that carry out diverse functions in the cell still have this conserved catalytic domain.

Recent genomic data has revealed the atypical protein kinase families that share little to no

homology to ePK (LaRonde-LeBlanc and Wlodawer, 2005b).

Histidine / Aspartic Acid Kinases. Histidine and aspartic acid phosphorylation are part of the signal-transduction cascade known as two-component systems. Two-component signal transduction systems are widespread in bacteria and archaea but appear to be restricted to plants, yeast, fungi and protozoa in eukaryotes (Wolanin et al., 2002). The histidine kinase in a two- component system can be embedded in the membrane of the cell. In response to external stimuli, the kinase uses ATP to autophosphorylate on a histidine residue, creating a phosphoramidate bond (P–N), before transferring the phosphate to an aspartic acid residue on a second protein, known as a response regulator (Klumpp and Krieglstein, 2002). The phosphorylated response regulator often times is a transcription factor and directly affects transcription within the bacterial or archaeal cell.

Although two-component systems have not been found in animals, there are examples of histidine phosphorylation. Phosphorylation on histidine residues has been demonstrated for histone H4 in rats (Chen et al., 1977), p36 (Motojima and Goto, 1993), and p38 (Hegde and Das,

1987). There is also an atypical protein kinase family that shares sequence similarity to histidine

kinases, the branched-chain α-ketoacid dehydrogenase kinase (Popov et al., 1992). The role of histidine phosphorylation in mammalian cells is currently unknown, although it is speculated to be an important signaling mechanism in liver regeneration, cell signaling, differentiation, and cell motility (Klumpp and Krieglstein, 2009).

Serine / Threonine Kinases. Serine / threonine kinases transfer phosphate groups to create phosphoserine and phosphothreonine through the formation of phosphoester bonds.

Phosphorylation of serine and threonine are the most common forms of phosphorylation with about 90 % of the phosphorylation events occurring on the hydroxyl side-chain of serine and 9 % on threonine. Every organism has examples of serine/threonine kinases in their genome with nearly 500 in the human genome alone (Venter et al., 2001). In addition to the typical eukaryotic protein kinase superfamily, there are 10 additional serine/threonine kinase families that contain kinases that control transcription (Le Douarin et al., 1996), ribosome biogenesis (LaRonde-

LeBlanc and Wlodawer, 2005b), stress response (Abraham, 2004), metabolism (Popov et al.,

1997), heat shock (Shemetov et al., 2008), apoptosis (Izquierdo and Valcarcel, 2007), translation

(Roberts et al., 2006), and signal transduction (Radziwill et al., 2003).

A typical serine/threonine kinase will have two major domains, the N-terminal domain containing a β-sheet and one α-helix and a C-terminal domain that is primarily α-helices. There is a hinge region between the two domains. This allows for movement of the two domains in relation to one-another in response to binding of substrates. There are several sub domains associated with kinase structure, including a nucleotide binding domain (typically GXGXXG), a catalytic loop (with Asp / Asn residues for phosphate transfer), a metal-binding loop (DFG loop), and in some cases an activation loop (APE) (Hanks and Hunter, 1995). Phosphorylation of the

activation loop is a common mechanism to negatively regulate kinase activity (Johnson et al.,

1996).

Tyrosine Kinases. Tyrosine phosphorylation is the least common type of phosphorylation that takes place on proteins. Less than 1 % of all protein phosphorylation events result in phosphotyrosine (~0.05 – 0.5 %) (Sefton et al., 1980; Hunter, 1998; Machida et al., 2003). The archetype of an eukaryotic tyrosine kinase is the membrane receptor that autophosphorylates or phosphorylates a binding partner at the beginning of a signal transduction cascade. Examples of bacterial and archaeal tyrosine phosphorylation have also been detected, although it is unclear if the role of tyrosine phosphorylation is conserved across all domains. Tyrosine phosphorylation of a cellular receptor in eukaryotic cells is closely associated with dimerization and the subsequent transfer of the phosphate to another kinase closely physically associated with the receptor kinase (Hunter, 1998).

Glycosylation

Protein glycosylation describes a large number of post-translational modifications that regulate protein targeting and enzymatic function. It is estimated that nearly 50 % of all eukaryotic proteins are glycosylated (Apweiler et al., 1999; Morelle et al., 2009). Eukaryotic glycosylation begins in the lumen of the endoplasmic reticulum and finishes in the Golgi apparatus. Glycosylation was originally believed to be restricted to the Eukarya. It is now known that bacteria and archaea can undergo both N-linked and O-linked glycosylation.

O-linked glycosylation

O-gylcosyl is a modification that takes place on serine and threonine residues in eukaryotic proteins. Often, the modification consists of the addition of monosaccharides, usually N- acetylglucosamine (GlcNAc). The addition of the monosaccharide is typically catalyzed by a devoted O-GlcNAc transferase located in the Golgi apparatus and subsequently removed by a

devoted hydrolase (Moloney et al., 2000a). There are few examples of polysaccharide additions

in O-linked glycosylation, such as with the Notch protein (Moloney et al., 2000b). The Notch

protein is important in eukaryotic cells as an extra-cellular signaling protein and can be modified

by chains of two or three different monosaccharides. These different glycosylation events play

important roles in the maturation and function of the Notch signaling pathway.

Both archaeal and bacterial S-layer proteins can undergo O-linked modification as well.

An O-linked glycosylation pathway has been detected in the bacteria Campylobacter jejuni and

C. coli (Szymanski et al., 2003). Even though only a few targets have been identifed, the pathway appears to be similar to the eukaryotic pathway (Szymanski et al., 2003). Both

Halobacterium salinarum and Haloferax volcanii have S-layer proteins that are modified on threonine residues with a disaccharide galactose-glucose (Mescher and Strominger, 1976;

Sumper et al., 1990), and Sulfolobus acidocaldarius cytochrome B558/566 contains an O-linked

mannose subunit (Hettmann et al., 1998). Little is known about the mechanism and enzymology

of O-linked glycosylation in archaea or its relation to eukaryotic or bacterial pathways.

N-linked glycosylation

In eukaryotic cells, N-linked glycosylation is both more common and more complex than

O-linked glycosylation (Mechref and Novotny, 2002). The glycan chain is assembled in the

endoplasmic reticulum and transferred to an asparagine residue within the sequence motif

Ser/Thr-X-Asn by a oligosaccharyltransferase (Trombetta and Parodi, 2001). The glycan chain

is made up of seven different monosaccharides. This heptasaccharide structure is found on all

eukaryotic N-linked glycosylation substrates. The transfer of the saccharide chain to the

asparagine residue is through a polyisoprenol-based lipid carrier dolichol pyrophosphate (Parodi

and Martin-Barrientos, 1977). Additional modifications can take place on the glycan chain after

it has been attached to the target protein including the addition and subtraction of monosaccharides (Walsh et al., 2005).

It is believed that the process of N-linked glycosylation that takes place in eukaryotes evolved from the archaeal system (Burda and Aebi, 1999). This is supported by the observations that antibiotics that interfere with N-linked glycosylation in eukaryotes also interfere with N- linked glycosylation in archaea. Antibiotics such as bacitracin and tunimycin both inhibit the dolichol carriers in eukaryotes and inhibit N-linked glycosylation in the archaeon Halobacterium salinarum (Wieland et al., 1980). Interestingly, the same antibiotics do not interfere with N- linked glycosylation of the S-layer protein of another haloarchaeon Haloferax volcanii (Eichler,

2001). This difference is likely due to variations in the attachment pathways between the two organisms.

Other Modifications

There are literally hundreds of covalent modifications that can take place on different residues within a protein. The incredible number and diversity of post-translational modifications makes it impossible to exhaustively cover in this section. The following examples are of common post-translational modifications that either occur in archaeal organisms, are related to protein stability, or are involved in protein targeting that have not been discussed previously.

Methylation

Even though several of proteins are post-translationally methylated, protein methylation remains a rather understudied modification. A protein can be methylated on the amine groups of alanine, arginine, glutamic acid, histidine, lysine (α and ε), and proline, the hydroxyl groups of glutamic acid and aspartic acid, or the thiol group of cysteine (Nochumson et al., 1978; Paik and

Kim, 1986). In all of the examples listed previously, the methyl group is donated from S-

adenosyl methionine (SAM). O-linked methylation is reversible (Paik et al., 1975). N-linked

methylation is irreversible as is the case with the trimethylation of the N-terminus of the E. coli

protein L11 (Dognin and Wittmann-Liebold, 1980).

Both bacteria and archaea methylate proteins involved in chemotaxis as a result of external

stimuli. Halobacterium salinarum uses methylation to regulate separate taxis responses for

phototaxis and chemotaxis (Hildebrand and Schimz, 1990; Hou et al., 1998). Some N-ε- methylation events (irreversible methylation of side chain amino group of lysine) are thought to increase the thermal stability of a protein and lessen the tendency to aggregate (Febbraio et al.,

2004). DNA-binding and ribosomal proteins are also methylated. Histone proteins are also heavily methylated (Cheung et al., 2000). This pattern of methylation of histone proteins is conserved in archaeal organisms. The Sul7 family of histone proteins found in Sulfolobus

species are methylated on lysine residues (Baumann et al., 1994; Knapp et al., 1996;

Wardleworth et al., 2002).

Another mechanism of methylation is the isoaspartate methylation pathway. This pathway

is involved in protein repair and over-expression increases heat-shock response in Escherichia

coli (Kindrachuk et al., 2003). Isoaspartyl residues form spontaneously through deamidation of

asparagine or isomerization of aspartic acid. The carbonyl-group of the amino acid attacks the

amine of the amino acid backbone forming a five-membered L-succinimidyl ring. This ring

spontaneously opens to either aspartic acid or the atypical L-isoasparyl residue. The rate of

formation of the isoasparyl residue is three times the rate for the formation of aspartic acid

(Johnson et al., 1987). Methylation of isoaspartate by peroxisome proliferator-activated receptor-interacting protein with methyltransferase domain (PIMT) increases the rate at which the isoasparyl residue returns to the succinimide ring form. The ring rehydrolyzes to either

aspartate or isoaspartate. If the ring resolves to isoaspartate, the process repeats (Vigneswara et

al., 2006). Although this process may seem inefficient, after several rounds of methylation, the

damaged protein can regain function (Johnson et al., 1987).

Acylation

There are several different kinds of acylation that can take place on a protein. The most common forms of acylation are acetylation of ε-amino group of lysines and α-amino groups of

N-termini, myristoylation of N-terminal glycines, and palmitoylation of cysteine residues.

Having already discussed N-terminal acetylation, the following section will focus on the acyl-

modification lipidation.

Lipidation

Lipid modification of proteins includes protein isoprenylation, palmitoylation, glycosylphophatidylinositol lipid anchoring, N-myristoylation, and several other modifications.

Isoprenylation has been observed in the haloarchaea Halobacterium salinarum and Haloferax volcanii (Sagami et al., 1994; Konrad and Eichler, 2002). N-myristoylation is an irreversible cotranslational modification where a myristate (14 carbon) is covalently linked to an N-terminal glycine residue of a protein (Johnson et al., 1994). The myristate molecule is attached to the N- terminal glycine through an amide bond after the initiating methionine is removed by a methionine aminopeptidase (Utsumi et al., 2001; Maurer-Stroh et al., 2002). The enzyme that catalyzes the addition of the myristate is a N-myristoyl transferase (NMT). NMT transfers the myristate group from the intermediate compound myristoyl-CoA, with release of CoA in the process. Proteins modified by N-myristoylation are typically found in the membrane. The hydrophobicity of the myristate group helps to localize the proteins to the membrane and can aid in hyrdrophobic protein-protein interactions (Farazi et al., 2001). Palmitoylation of proteins is the addition of a C16 chain from a fatty acyl-CoA donor to a cysteine residue, rather than an N-

terminal glycine. This modification is referred to as S-palmitoylation (Bijlmakers and Marsh,

2003). While it is not widespread in eukaryotic organisms, the modification plays an important role for the proper localization and function of Ras GTPase signaling (Resh, 1999).

Post-translational Modification of Proteasomal Proteins

Subunits of 20S core particles and associated proteins are subject to several forms of post- translational modification. In addition to the propeptide processing that some β subunits undergo, subunits from 20S and 26S proteasomes undergo Nα-acetylation, phosphorylation, S-

glutathionation (Demasi et al., 2003), O-linked glycosylation (Zachara and Hart, 2004; Zhang et

al., 2003a), and N-myristoylation (Lee and Shaw, 2007). Even though the functions of some of

the previously mentioned modifications are unknown, the large number of modifications

indicates the proteasome system is heavily regulated (summarized in Table 1-1).

All seven α-type subunits from yeast 20S proteasomes (α1, α2, α3, α4, α5, α6, and α7) and

two β-type subunits (β3 and β4) are N-terminally acetylated (Kimura et al., 2000). Additionally,

20S core particle α-type subunits from haloarchaea Halobacterium salinarum and Natronomonas

pharaonis are also N-terminally acetylated (Aivaliotis et al., 2007; Falb et al., 2006), including

the two α-type subunits for the haloarchaeon Haloferax volcanii (α1 and α2) (Humbard et al.,

2006). The crystal structures of 20S core particles and α-rings show that the N-terminal α-helix of the α-type subunits points into the aperture, creating a steric gate. 20S proteasomes purified from a nat1 yeast strain had a two-fold increase in chymotrypsin-like peptidase activity (Kimura

et al., 2000). Analysis of the N-terminal acetylation of the α subunits purified from this nat1

strain revealed that 5 out of the seven were no longer modified. These data suggest acetylation

of α subunits in 20S core particles is important in establishing or maintaining the regulatory gate

formed by the α-ring. In addition to modifying the α-type and β-type subunits of 20S core

particles in yeast, ten out of the seventeen subunits of 19S regulatory caps in yeast are also Nα-

acetylated: Rpt3, Rpt4, Rpt5, Rpt6, Rpn2, Rpn3, Rpn5, Rpn6, Rpn8, and Rpn11 (Kimura et al.,

2003).

Phosphorylation of subunits from 20S core particles and 19S regulatory particles is well

established. Four out of the six Rpt subunits are phosphorylated in the 19S regulatory particle,

Rpt2, Rpt3, Rpt4, and Rpt6, as well as one Rpn subunit, Rpn8. The exact roles of many of the

phosphorylation events is unclear at this time. It has been speculated that the phosphorylation of

subunits in response to γ-interferon may influence the shift to the immunoproteasome (Bose et

al., 2001; Rivett et al., 2001; Bose et al., 2004). Phosphorylation of the 19S regulatory particle

Rpt6 has been shown to stimulate 19S association with the 20S to form the 26S proteasome

through the interaction with α3. Dephosphorylation stimulates the disassembly (Satoh et al.,

2001).

Rpt2 subunits from 19S regulatory particles are N-myristoylated in Saccharomyces

cerevisiae after the initiating methionine is removed (Kimura et al., 2003). Rpt4 subunits from

several organisms are predicted, by primary amino acid sequence, to be N-myristoylated. The organisms predicted to have myristoylated N-termini are Caenorhabditis elegans, Gallus gallus,

Drosophila melanogaster, Homo sapiens, Mus musculus, Schizosaccharomyces pombe, and

Saccharomyces cerevisiae (Maurer-Stroh et al., 2002). N-myristoylation has been shown to aid

in the translocation of proteasomes between nuclear envelope and the endoplasmic reticulum.

This translocation event may even be coupled to a phosphorylation of a serine residue on Rpt4

found near the N-terminus. The exact role of this modification in the 26S proteasome is

unknown, but N-myristoylated is known to affect protein-protein and protein-membrane

interactions (Johnson et al., 1994; Farazi et al., 2001).

Other modifications of proteasomal subunits have been seen such as O-linked β-N- acetylglucosamine (O-GlcNAc) and S-glutathionylation. Recent reports demonstrate a number of subunits from both Drosophila (Sumegi et al., 2003) and mammalian proteasomes (Zhang et al., 2003a) are glycosylated with O-GlcNAc moieties. This modification is quite widespread in

Drosophila with 5 out of 19 subunits of the 19S cap and 9 out of 14 of the subunits of the core particle being reversibly modified in this way (Sumegi et al., 2003). The modification of mammalian proteasomes by O-GlcNAc negatively regulates proteasome activity and blocks the degradation of the transcription factor Sp1 (Zhang et al., 2003a). The changes in different levels of glycosylation of the proteasome correspond with periods of low nutrition. The lower the nutrition, the less glycosylation, the more Sp1 can be degraded by the proteasome (Zhang et al.,

2003a). An additional post-translational mechanism of negatively regulating the proteasome is the S-glutathionylation of yeast 20S proteasomes in response to oxidative stress (Demasi et al.,

2001; Demasi and Davies, 2003; Ishii et al., 2005). While the demonstration of this modification is solely in vitro, it makes sense to temporarily and reversibly inhibit the activity of the 20S proteasome in yeast immediately following an oxidative stress. A temporary decrease in proteasome activity would prevent the degradation of redox signaling factors such as AP-1.

Also, preemptively modifying the proteasome subunit cysteines with S-glutathion would prevent the formation of irreversible oxidation of thiol groups to Cys-SO2H or Cys-SO3H, allowing the

proteasome to eventually be reactivated in the absence of oxidative stress (Demasi et al., 2003).

Mass Spectrometry

Tandem mass spectrometry has emerged in the past decade as a reliable and sensitive

method for identification and analysis of small molecules such as peptides and post-translational

modifications. Tandem mass spectrometry typically utilizes site-specific proteases, such as

Trypsin or AspN, to fragment proteins into peptides of predictable size (Olsen et al., 2004). The

resulting peptides may or may not be separated by high-pressure liquid chromatography before

analysis by a mass spectrometer. The work flow for tandem mass spectrometry is a series of steps where the sample interacts with an ion source, a mass analyzer, a collision chamber, a

second mass analyzer, and a detector. Different mass spectrometers have different types of ion

sources, mass analyzers, collision chambers, and detectors. Peptides are ionized and enter a detector that measures the masses of the most abundant peptides. After the masses are measured, the peptides are selectively moved into a collision chamber where they are fragmented again

through a process called collision induced dissociation (CID). The masses of the resulting

fragments are measured in another mass analyzer. The output of the second mass analyzer is

called an MS/MS or MS2. Different strategies for ionization may include matrix assisted laser

desorption ionization (MALDI) or electron spray ionization (ESI) (Bakhtiar and Nelson, 2000).

Different mass analyzers can be used in combination with the different ionization strategies such

as quadrupoles (Q) or time-of-flight tubes (TOF). In tandem mass spectrometry, least two mass

analyzers are needed. A mass spectrometer can have the same mass analyzers as is the case for a

MALDI-TOF/TOF or an ESI-QqQ or have different mass analyzers such as ESI-Q-TOF. In combination with other proteomic techniques, such as two-dimensional gel electrophoresis or strong cation exchange chromatography (SCX), a large number of proteins can be separated and identified by mass spectrometry. Recent advances in proteomics and specifically mass spectrometry have allowed scientists to obtain accurate quantifiable readings of protein levels in different samples. This section will review and highlight the role mass spectrometry has played in the identification of phosphorylation sites as well as review the current state of the art of quantitative mass spectrometry techniques.

Phosphorylation Site Identification by Tandem Mass Spectrometry

Advances in tandem mass spectrometry (MS/MS or MS2) have made the high-throughput identification of multiple phosphorylation sites on hundreds of proteins simultaneously possible

(Resing and Ahn, 1997; Sickmann and Meyer, 2001). Prior to tandem mass spectrometry, scientists relied on the use of radioactive phosphates, two-dimensional gel electrophoresis, or N- terminal sequencing to determine sites of phosphorylation. While not only being technically difficult, they often required the use of radioactive phosphate and therefore limited the types of analysis that were available. Recent advances in immobilized metal affinity chromatography

(IMAC) (Posewitz and Tempst, 1999; Zhou et al., 2000; Ficarro et al., 2002; Raska et al., 2002;

Nuhse et al., 2004), dyes and stains (Jacob and Turck, 2008), and use of phosphospecific antibodies (Zhang et al., 2002) have allowed for the high-throughput identification of hundreds of phosphorylation sites. The incorporation of phosphopeptide selective techniques with tandem

MS/MS has created an exponential increase in the total number of phosphorylation sites identified. In 2003, nearly 500 novel phosphorylation sites were identified on proteins. That is greater than the sum of all the phosphosites identified in the primary literature from 1990 to

2001. Of those nearly 500 phosphosites, almost 70 % of them were identified by MS/MS experiments (Loyet et al., 2005).

Label-based methods for the detection of phosphopeptides have been used to map the positions of phosphate groups by taking advantage of the β-elimination reaction phosphate groups undergo in alkaline conditions. The β-elimination of phosphoserine and phosphothreonine results in the formation of the amino acids dehydroalanine and β- methyldehydroalanine (Knight et al., 2003). These resulting alkene groups can be used as nucleophiles for the attachment of various chemicals that are easily detected such as fluorophores (McLachlin and Chait, 2003). Specifically, the fluorescence affinity tag (or FAT)

that consists of cysteamine linked to a fluorescent rhodamine tag, has been used to selectively

modify and tag phosphoproteins for offline purification prior to MS/MS analysis (Stevens, Jr. et

al., 2005). All of these phospho-specific tools have given rise to increased understanding of

global levels of phosphorylation in vivo and further fuels understanding of post-translational

modification.

Quantitative Mass Spectrometry

Proteomic researchers not only want to identify what proteins or what modifications are present in an organism, but also to rank proteins in order of abundance or relative quantity to one another. The ability to quantitatively measure the difference between biological states is fundamental to a detailed understanding of cellular biology. Early attempts at quantifying proteomic data involved the use of protein score to rank abundance (Allet et al., 2004). A

protein is scored by the number of times a peptide from that protein is identified in a mass

spectrometry run, the higher the score the more abundant. This initial strategy proved ineffective

in providing repeatable quantitative data (Ishihama et al., 2005). New strategies have been

developed in the past decade that provide comparative and absolute quantification of proteins in

complex mixtures. There are two basic strategies of quantitative mass spectrometry, using

unmodified proteins and comparing two samples under the same conditions (label-free) or using

tags, chemical or isotopes, to take direct measurements of protein amounts (labeling based

methods).

Label-free methodology

There are several practical situations where direct comparison of two different samples is

required in the absence of isotopic labeling or chemical tagging. To address this need, there are

two different quantitative techniques that do not involve the use of any kind of label. The first

involves counting and comparing the number of spectra of a protein, and the second compares

the intensity of the mass spectrometric signal of precursor ions belonging to a specific protein.

The first strategy is called spectral counting and is a quantitative method that involves comparing the number of MS/MS spectra assigned to each protein. Examples of this technique using idealized proteins spiked into yeast extract demonstrates that spectral counting is correlative to protein quantity over two orders of magnitude (Liu et al., 2004). The correlation is strong enough to compare proteomic measurements with estimated protein copy numbers in yeast cells (Ghaemmaghami et al., 2003). There are some obvious disadvantages and limitations to spectral counting. While spectral counting can provide a general idea of the relative abundance of a protein between two samples, it cannot be used to compare two different proteins in the same preparation. Also, spectral counting is correlated less with protein abundance as the mixture becomes more complex. Since many proteomics researchers use the bench-mark of two peptides per protein, spectral counting usually under-estimates the differences in protein levels.

Additional statistical tools and techniques have been added to this practice to make it more accurate (Gilchrist et al., 2006; Washburn et al., 2001). Since there is a direct correlation between the numbers of peptides identified in a single run to the abundance of that protein, a common way to estimate the dominant proteins in a complex mixture is called protein abundance indices (PAI). PAI can use any observed parameter in a mass spectrometry run, but combines multiple readings from all observed peptides. The relationship between protein quantity and peptide identifications was observed to be logarithmic. Correlating protein quantity to peptide numbers is referred to as exponentially modified PAI (emPAI) (Ishihama et al., 2005).

The second strategy involves the use of peak intensity of the first MS run to estimate relative protein quantity. This strategy is often referred to as extracted ion chromatogram (XIC)

counting (Bondarenko et al., 2002). Extracted ion chromatogram based methods are more

reliable and sensitive than spectral counting alone. The principle is similar in that the intensity

of a signal is still measured, but rather than measuring the number of times the mass

spectrometer records a peptide hit, the nature of the chromatography peak is analyzed in order to

make a comparison. The area under the curve for a specific peptide can be linearly related to the

relative quantity of that protein. Two peptides from different chromatographic preparations can

be directly and accurately compared. There is no way to determine the MS detector response to

a random peptide due to difference in ionization properties but a strategy that utilizes the peak

areas of three of the most abundant peptides of a protein has proven to be quantitative. This

process combines XIC with PAI and is termed xPAI (Rappsilber et al., 2002). By averaging the

signal of multiple peptides from different proteins in the same mass spectrometry run, a rough

idea of the relative quantity of those two proteins can be estimated. This has proven to be more

accurate than just spectral counting alone when comparing two dissimilar proteins in the same

sample.

Thorough comparisons of these different approaches have been performed (Old et al.,

2005; Anderson and Hunter, 2006; Ono et al., 2006; Bantscheff et al., 2007). The overall conclusions of these studies reveal that the two techniques, spectral counting and XIC, can be used to make different kinds of comparisons. Spectral counting, with or without emPAI, is more accurate at comparing proteins from two different runs than XIC based methods, and peak area intensities (XIC based methods) are more accurate at estimating the differences between two proteins in the same sample than spectral counting alone (Old et al., 2005). Together, spectral counting and XIC based methods give researchers valuable insight into relative protein quantities between samples and within the same sample.

Label-based methodology

Additional methodologies for the quantitative analysis of protein samples have been

developed that use tags consisting of either a chemical that modifies peptides or stable isotopes

to alter the molecular weight of peptides. Stable isotope labeling refers to a set of strategies that

involves the incorporation or labeling of proteins of peptides with “heavy” isotopes of carbon

(13C), nitrogen (15N), oxygen (18O) or hydrogen (deuterium, 2H). There are four basic strategies

for the incorporation of “heavy” stable isotopes into mass spectrometry samples, (i) adding an

isotopically labeled peptides that mirrors a physiological one, (ii) incorporation of isotopes

during enzymatic digestion of protein, (iii) reacting proteins or peptides with isotopically labeled

markers, or (iv) having the isotopes incorporated by living cells metabolically. Examples of each

of these strategies will be discussed further.

The addition of a synthetic peptide to a sample that was synthesized with an isotopically

labeled amino acid (15N-alanine for example) is a way to measure the absolute quantity of

peptide in the sample. This strategy is called Absolute quantification (AQUA) (Desiderio and

Kai, 1983; Gerber et al., 2003; Kirkpatrick et al., 2005). AQUA is widely accepted as the most analytical mass spectrometry technique for the measurement of peptide quantity in a sample. A synthetic peptide labeled with stable isotopes of known sequence is spiked into a sample. The quantity of the AQUA peptide is known. Since the AQUA peptide has the exact same sequence as a peptide previously identified in the sample, the ionization and detection should be the same for both. This removes the variability normally associated with comparing two unique peptides.

This technique not only allows for the relative comparison of peptide quantities in the sample, but since the exact amount of AQUA peptide added to the sample is known, the exact amount of peptide in the sample can be measured (Desiderio and Kai, 1983). AQUA has several

disadvantages, most notably the synthesis of the synthetic peptide can be tedious and expensive.

Furthermore, each AQUA experiment requires new peptides to be synthesized, and different

experiments and peptides cannot be directly compared. Even with these drawbacks, AQUA

remains the most standard method for quantification of mass spectrometry data.

The second, albeit unpopular, strategy to incorporate a label is to perform the protease

18 18 digestion in the presence of H2 O. During a trypsin digestion, O is incorporated into the

18 peptide during cleavage if the digestion is performed in the presence of H2 O (Mirgorodskaya et

al., 2000; Yao et al., 2001). A problem with this technique is the variability in the amount of 18O

that is incorporated into the peptide. One or both of the oxygen atoms may be exchanged for the

solvent 18O at the C-terminal end of the peptide. In addition, some proteases will only

incorporate one 18O into the peptide such as Lys-N. This will result in only a 2 Da shift in

molecular weight and such a small shift in weight is not adequate for quantitative measurement.

Chemical tagging approaches are popular ways to quantify different proteomic samples.

Different reagents can be reacted with the sulfhydryl, amine, and carboxyl groups of amino

acids. Isotope coded affinity tag (ICAT) was one of the first commercially available tags (Gygi

et al., 1999). The ICAT reagent consists of a cysteine-reactive group and a polyether linker

region with 8 deuteriums (heavy) or 8 hydrogens (light) and a biotin molecule for the recovery of

the modified peptides. Two different samples will be reacted with either the light or the heavy

ICAT reagent. The modified proteins are mixed and digested with a protease such as trypsin.

The labeled peptides are isolated in an avidin column and analyzed by MS. The fact that ICAT

reagents only react with sulfhydryl groups is both an advantage and a disadvantage. Cysteine is

a relatively infrequent amino acid so a complex mixture is significantly simplified after the

avidin chromatography. A simplified mixture aids in producing accurate quantifiable data.

However, some proteins contain only one or no cysteines. For these proteins, quantifiable data

will not be generated in an ICAT experiment (Wu et al., 2006).

Another tagging strategy derivitizes α-amine groups of N-termini and ε-amine groups of lysine residues. This tagging method is called isobaric tag for relative and absolute quantification (iTRAQ) (Ross et al., 2004). The iTRAQ labeling system is a set of reagents that use N-hydroxysuccinimide (NHS) chemistry to react to the amines but each of the different (up to 8) iTRAQ reagents give a unique reporter ion in the MS/MS scan (114 Da – 121 Da). In an

ICAT experiment, protein pools were labeled, iTRAQ reagents are typically added to pools of peptides since modification of lysine residues prevents cleavage by trypsin. Since each of the tags are a different mass and give a similar fragmentation spectrum, the samples can be mixed to reduce variability from run to run (Wu et al., 2006).

The last strategy that will be discussed with be in vivo incorporation of stable isotopes.

There are two basic approaches. The first involves growing cells in media that contains “light” nitrogen (14N) or “heavy” nitrogen (15N). Two different cultures are grown, one in the heavy and

one in the light nitrogen. After both are grown, the cultures are mixed and processed for

downstream MS analysis. Both the heavy and the light peptides are present in each run so the

ratio of the two peptides is directly compared (Conrads et al., 2001). Mass shifts due to this

technique can be difficult to predict since the labeling can be in both the amino-backbone of the

protein as well as the amino acid side chains. The second approach for in vivo labeling of

proteins is called stable isotope labeling with amino acids in cell culture (SILAC) (Ong et al.,

2002). SILAC uses 13C-lysine, 2H-leucine, 13C-tyrosine, or 13C / 15N-arginine as the heavy

isotopes. SILAC is more often used than the 14N / 15N method previously discussed because the

mass defects are easier to predict making data analysis less difficult. The workflow is very

similar. The heavy amino acids are added to cultures that are actively growing. After the growth

(and labeling) is completed, the samples are mixed and processed as one sample for mass

spectrometry. The different masses can be directly compared in the mass spectrometry run. The

most common strategy for SILAC is the use of a “heavy” arginine (either 13C or 15N) since every

tryptic fragment would contain a predictable mass defect due to the presence of the C-terminal arginine.

Project Rational and Design

The objective of this study is to identify post-translational modifications of the subunits of

20S proteasomes and PAN proteins from Haloferax volcanii. Due to the ease of genetic manipulation as well as the relative simplicity of the proteasome system, Hfx. volcanii is an ideal

model organism to study archaea and proteasomes. Since little was known concerning post-

translational modifications of proteins in archaea or proteasome subunits, the first goal was to

establish how the different subunits were modified. Once the nature of the modification was

known, the location on each of the proteins was identified. Once the modification and site of the modification was known, mutations were introduced into the genes in order to perturb the primary amino acid sequence surrounding the modification, and blocking or disrupting the modification. Once successful, cell expressing variant proteins were assayed for a variety of phenotypes including temperature tolerance and osmotic stress. Proteins were purified and assayed for activity and presence of the modifications. Finally, bioinformatic techniques

identified candidate genes responsible for the modifications. Candidate enzymes were tested for

their ability to modify the wild-type or variant proteins in vitro.

Figure 1-1. Crystal structures of 20S proteasomes from Thermoplasma acidophilium (PBD:1PMA) (Lowe et al., 1995). The 20S proteasome is in an α7β7β7α7 configuration with a central channel joining three inner cavities. The central cavity is lined by the catalytic sites, which mediate the hydrolysis of peptide bonds. The axial pores on each end of the cylinder restrict access of substrate. Graphic rendered with Pymol.

Figure 1-2. Ubiquitination and pupylation pathways. Ubiquitin is activated by E1 and transferred to an E2 enzyme. Depending on the E2 enzyme, the ubiquitin will either be transferred to an E3 and then a substrate, or directly transferred to a substrate bound by a RING protein. A polyubiquitin chain will form on the substrate, targeting it for degradation by the proteasome. The Pup protein is deaminated by Dop and phosphorylated, possibly by PafA. The phosphorylated Pup is covalently attached to a substrate protein that may be degraded by the proteasome.

N-terminal acetylation

O Gln O Gln H H H 2NH C CH N 3CH N N CH N C CH N H H Met O Gly O Met O Gly

Phosphorylation of Amino Acids

NH OH OH CH3 O OH N

–O O– P O NH N O O O CH3 O P O– O N N – P O O P – – –O PO O– OO– O O 3-phosphohistidine –O Phosphoserine O Phosphoaspartate 1-phosphohistidine P Phosphothreonine O O– O– Phosphotyrosine

N--acetylation -N-alkylation O

CH3 NH3+ NH CH3 NH3+ NH

O-linked Glycosylation

O OH OH OH HN HN HN HN OH O UDP-Glucose O O O O CH 3 CH3 Lysine N-acetyl-Lys Lysine N-alkyl-Lys Threonine Glc-O-Thr

N-myristoylation

O O H 3CH N 2NH 11 N-terminus O N-myristoly-Gly-protein

Figure 1-3. Modified amino acid structures. Modifications, N-terminal acetylation, phosphorylation, N-ε-acetylation, ε-N-alkylation (methylation), O-linked glycosylation, and N-mystristoylation, are in red.

Table 1-1. List of known post-translational modifications of proteasome subunits.

Subunit Organism Modification Enzyme Reference α7 Candida albicans ~P CK2 (Pardo et al., 1998) α6 Candida albicans ~P CK2, rhCK2-α (Fernandez et al., 2002) α3 Candida albicans S248~P CK2 (Fernandez et al., 2002) α5 Candida albicans ~P CK2 (Fernandez et al., 2002) (Horiguchi et al., 2005; α7 Carassius auratus ~P Tokumoto et al., 2000) Y120~P, A1 N- (Horiguchi et al., 2005; α2 Carassius auratus acetylalanine Tokumoto et al., 2000) (Frentzel et al., 1992; Haass et α1 Drosophila melanogaster Y103~P al., 1989) (DeMartino et al., 1991; Kristensen et al., 1995; α1 Homo sapiens N-acetylmethionine Kristensen et al., 1994; Tamura et al., 1991) (Claverol et al., 2002; Kristensen et al., 1995; ~P S249, N- α3 Homo sapiens CKII Kristensen et al., 1994; Tamura acetylserine et al., 1991; Beausoleil et al., 2004) (Kristensen et al., 1995; α4 Homo sapiens ~P CKII Kristensen et al., 1994; Tamura et al., 1991) (Kristensen et al., 1995; Y120~P N- α2 Homo sapiens Kristensen et al., 1994; Tamura acetylalanine et al., 1991) (DeMartino et al., 1991; α5 Homo sapiens S56~P Beausoleil et al., 2004) S250~P, N- α7 Homo sapiens acetylated α1 Mus musculus N-acetylmethionine (Elenich et al., 1999) N-acetylserine α3 Mus musculus (Elenich et al., 1999) S249~P N-acetylalanine, α2 Mus musculus (Seelig et al., 1993) Y120~P α5 Mus musculus S56~P (Elenich et al., 1999) (Umeda et al., 1997a; Umeda et α1 Oryza sativa S~P CKII al., 1997b) α1 Rattus norvegicus N-acetylmethionine (Tokunaga et al., 1990) N-acetylserine (Castano et al., 1996; Tokunaga α3 Rattus norvegicus S249~P, S250~P, et al., 1990) S243~P α4 Rattus norvegicus ~P (Castano et al., 1996) α5 Rattus norvegicus S56~P α6 Rattus norvegicus alkylation N-acetylalanine, α2 Rattus norvegicus (Tokunaga et al., 1990) Y120~P α7 Rattus norvegicus ~P β3 Rattus norvegicus alkylation (Nishimura et al., 1993a) (Emori et al., 1991; Groll et al., (S,T)~P, Nα- α2 Saccharomyces cerevisiae NAT1 1997; Iwafune et al., 2002; acetylated Kimura et al., 2000)

Subunit Organism Modification Enzyme Reference (Emori et al., 1991; Groll et al., Y~P, Nα-acetylated, 1997; Iwafune et al., 2004; α7 Saccharomyces cerevisiae S258~P, S263~P, CK II, NAT1 Iwafune et al., 2002; Pardo et S264~P, ~P al., 1998; Kimura et al., 2000) N-Myristoylation, (Emori et al., 1991; Groll et al., α3 Saccharomyces cerevisiae NAT1 Nα-acetylated 1997; Kimura et al., 2000) (Heinemeyer et al., 1994; α6 Saccharomyces cerevisiae Nα-acetylated MAK3 Kimura et al., 2000) (Chen and Hochstrasser, 1995; α5 Saccharomyces cerevisiae Nα-acetylated MAK3 Groll et al., 1997; Kimura et al., 2000) α1 Saccharomyces cerevisiae Nα-acetylated NAT1 (Kimura et al., 2000) (Chen and Hochstrasser, 1995; (S,T)~P, Nα- α4 Saccharomyces cerevisiae NAT1 Groll et al., 1997; Iwafune et al., acetylated 2002; Kimura et al., 2000) (Groll et al., 1997; Kimura et al., β4 Saccharomyces cerevisiae Nα-acetylated NAT3 2000) (Groll et al., 1997; Kimura et al., β3 Saccharomyces cerevisiae Nα-acetylated NAT1 2000) (Tokumoto et al., 1999; Wakata α7 Xenopus laevis ~P et al., 2004) N-acetylalanine, (Tokumoto et al., 1999; Wakata α2 Xenopus laevis Y120~P et al., 2004)

CHAPTER 2 MATERIALS AND METHODS

Chemicals, Strains and Media

Materials

Organic and inorganic analytical grade chemicals were from Fisher Scientific (Atlanta,

GA) and Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated. Restriction

endonucleases and DNA-modifying enzymes were from New England BioLabs (Beverly, MA).

Desalted oligonucleotides were from Integrated DNA Technologies (Coralville, IA). Hi-Lo

DNA molecular weight standards were from Minnesota Molecular, Inc. (Minneapolis, MN).

Agarose, real-time polymerase chain reaction (RT-PCR) materials and Precision Plus™ protein

molecular mass were purchased from Bio-Rad (Hercules, CA). Radioactive chemicals were purchased from Perkin Elmer (Waltham, MA), and Strep-Tactin Superflow resin was purchased from Qiagen (Valencia, CA). Site-directed mutagenesis materials were from Invitrogen

(Carlsbad, CA). Polyvinyl difluoride membranes for Western blots were from Amersham

Biosciences (Piscataway, NJ).

Strains, Media, and Plasmids

Strains and plasmids used in each of the studies are listed in Tables contained within each chapter. Escherichia coli strain DH5α (Life Technologies) was used for routine cloning, and

GM2163 (New England Biolabs) was used for purification of plasmid DNA for transformation of Hfx. volcanii as previously described (Cline and Doolittle, 1992). Hfx. volcanii strains were grown in ATCC 974 medium at 42 ˚C and 200 rpm or in minimal medium (lactate and glycerol) adapted from Allers et. al. (Allers et al., 2004) for radiolabeling studies consisting of 2.5 M

NaCl, 85 mM MgCl2·6H2O, 85 mM MgSO4·7H2O, 55 mM KCl, 5 mM CaCl2·2H2O, 5 mM

NH4Cl, 4.5 mM NaBr, 1.5 mM NaHCO3, 10 mM KPO4 pH 7.5, 30 mM Tris pH 7.5, 0.25 %

(v/v) dl-lactic acid, 15 mM succinic acid, 0.025 % (v/v) glycerol. Media were supplemented

with 2 nM MnCl2·4H2O, 1.5 nM ZnSO4·7H2O, 8 nM FeSO4·7H2O, 2 nM CuSO4·5H2O, 65 μg/ml

histidine, 50 μg∙ml−1 leucine, 50 μg∙ml−1 methionine, 50 μg∙ml−1 tryptophan, 50 μg∙ml−1

−1 −1 −1 glycine, d50 μg∙ml L-pantothenic acid, 50 μg∙ml uracil, 40 μg∙ml thymidine and

hypoxantine, 800 ng∙ml−1 thiamine 100 ng∙ml−1 d-biotin. E. coli strains were grown in Luria-

Bertani broth at 37 ˚C. Media were supplemented with 100 μg∙ml−1 of ampicillin or 0.1 μg∙ml−1

of novobiocin as needed. For salt stress tests, the concentration of NaCl was reduced to 1, 1.125,

1.25, 1.375 and 1.5 M from the typical 2.25 M NaCl of the ‘normal’ ATCC 974 medium.

Inoculum for the salt stress test was generated by growing cells in ‘normal salt’ ATCC 974

medium. Cells were streaked from -80 ºC glycerol stocks onto fresh plates, grown twice to log-

phase in 2 ml medium and used as an inoculum for the salt stress test. Each subculture was

inoculated to an initial OD600 of 0.01 to 0.02. Experiments were performed in triplicate and the

mean ± S.D. was calculated. Casamino acid medium used in the construction of acetyltransferase

knockouts consisted of 2.4 M NaCl, 80 mM MgCl2·6H2O, 175 mM MgSO4·7H2O, 50 mM KCl,

120 mM Tris buffer (pH 7.5), 3 mM CaCl2, and 0.5 % (w/v) casamino acids. Media were supplemented with 50 μg∙ml−1uracil and / or 100 μg∙ml−1 5-fluroorotic acid as needed.

DNA Techniques

Cloning

Genes were amplified by PCR using DNA polymerases including Thermalase

(Invitrogen), Accuprime, or Phusion (New England Biolabs). The fidelity of all cloned PCR-

amplified products was confirmed by DNA sequencing using the dideoxy termination method

with Perkin-Elmer/Applied Biosystems and LICOR automated DNA sequencers (DNA

Sequencing Facilities, Interdisciplinary Center for Biotechnology Research and Department of

Microbiology and Cell Science, University of Florida). Products and vectors were cut with

restriction enzymes NdeI, BlpI, KpnI, BbvCI, BamHI, EcoRI or XbaI according to

Manufacturer’s specifications (New England Biolabs) as needed. Digested vector DNAs were

treated with shrimp alkaline phosphatase (Roche Scientific) for one hour at 37 ˚C. For blunt end

cloning, Vent polymerase was used to fill in BlpI overhangs. The resulting products were treated with T4 polynucleotide kinase (PNK) as per Manufacturer’s instructions (New England

Biolands). Ligation reactions were preformed using T4 DNA ligase at 16 ˚C for 16 – 24 hours

(New England Biolabs). For cloning products encoding an in-frame 3` Strep_II tag, PCR

products and pJAM816 (Humbard et al., 2009) were cut with NdeI and KpnI. Digested pJAM816 was treated with shrimp alkaline phosphatase (Roche Scientific) for one hour at 37 ˚C.

Products were ligated using T4 DNA ligase as previously described.

DNA Electrophoresis

Sizes of PCR products and plasmids were analyzed by electrophoresis using 0.8 – 2 %

(w/v) agarose gels in TAE buffer (40 mM Tris acetate, 2 mM EDTA, pH 8.5) with Hi-Lo DNA molecular weight markers as standards (Minnesota Molecular, Minneapolis, MN). Gels were photographed after staining with ethidium bromide at 0.5 μg∙ml−1 with a Mini visionary imaging

system (FOTODYNE, Hartland, WS).

Plasmid Isolation and Transformation

Plasmids were isolated by Qiagen Miniprep kit according to Manufacturer’s protocols

(Qiagen Inc., Valencia, CA). When applicable, plasmids were purified from agarose slices by

QIAquick gel extraction kit (Qiagen). Hfx. volcanii strains DS70, H26, and different mutants

were transformed with plasmid DNA isolated from E. coli GM2163 (dcm- dam-) according to

Cline et. al. (Cline and Doolittle, 1992).

Site-directed Mutagenesis

Site-directed mutagenesis was performed using the QuikChange Site Directed Mutagenesis

kit (Stratagene) as per Manufacturer’s instructions with the following modifications. Pfu DNA

polymerase (Stratagene) was used for generation of all mutations with exception of psmC mutations in which Phusion DNA polymerase (New England Biolabs) was used. An elongation time of 7 minutes and 30 seconds was used for the generation of 11 kb products. Restriction enzyme digests were carried out with DpnI (Stratagene or New England Biolabs).

Oligonucleotides used in site-directed mutagenesis reactions are listed in Table A-2 and were not

HPLC purified.

Generation of N-terminal Acetyltransferase Knockouts

Generation of N-terminal acetyltransferase gene knockouts (HVO_1954 and HVO_2709) followed the previously described protocols of Allers et. al. (Allers et al., 2004). Primers used in the gene deletions are listed in Table A-4. The PCR products of the chromosomal regions of both genes (± 500 base pairs) were treated with T4 polynucleotide kinase (PNK) and blunt-end cloned into the EcoRI site of plasmid pTA131. Inverse PCR was used to remove the coding region of the genes (start to stop codons). The inverse PCR products were self-ligated and transformed into E. coli. Inverse PCR clones were purified and transformed into Hfx. volcanii strain H26 (uracil auxotroph). Transformations were plated on CA-medium without uracil.

Cells that successfully incorporated the plasmid into the chromosome survived the uracil

auxotrophy. Transformants were challenged in uracil containing medium with 5-fluoroorotic

acid (5-FOA). The plasmid recombines out of the chromosome either restoring the wild-type

copy of the gene or deleting the target gene. 5-FOA resistant colonies are checked for the presence or absence of the target gene by PCR of the chromosomal region using primers 700 bp

± target gene.

Genome Analysis

NCBI Local BlastP (Altschul et al., 1997) with BioEdit sequence editor software v7.0.4.1

(Hall, 1999) was used to compare the theoretical Hfx. volcanii DS2 proteome

(http://archaea.ucsc.edu/, April 2007 version) (unpublished). Phylogenetic and molecular

evolutionary analyses of the primary sequences of proteins were conducted using MEGA v3.1

(Kumar et al., 2004). Pairwise and multiple sequence alignment was performed using Clustal W

(Thompson et al., 1994). Evolutionary distances were estimated from the protein sequences

using the p-distance substitution model. Consensus tree inference was by neighbor-joining with bootstrap phylogeny test (1000 replicates; 64238 seed) and pairwise gap deletion. Regions of high variability and low complexity were filtered out of the search using Gblocks server

(Castresana, 2000).

RNA Techniques

RNA Isolation and RT-PCR

Total RNA was isolated using the RNeasy Mini kit for bacteria (Qiagen) as previously reported (Reuter et al., 2004), with the following modifications. One milliliter of cells (OD600 =

0.8) was harvested by centrifugation and lysed in 100 μl of nuclease free dH2O. Lysed cells were treated with Dnase I (Sigma) according to Manufacturer’s protocol. RNA samples applied to the RNeasy Mini RNA isolation column were centrifuged for an additional 2 minutes before elution to further dry out the membrane. Quality of RNA was determined by 0.8 % (w/v) agarose gel electrophoresis in 1 × TAE buffer after heating the RNA samples to 50 ˚C for 15 minutes. Total RNA concentration was determined from A260nm. Total RNA was used to

generate cDNA using the iScript cDNA synthesis kit (Bio-Rad).

qRT-PRC

Quantitative real-time PCR was performed using the iQ SYBR Green Supermix (Bio-Rad)

and iCycler MyiQ real-time PCR detection system (Bio-Rad). qRT-PCR was performed as both

a dilution series of the cDNA library and as triplicate samples of a dilution in the linear range of

detection for both the psmA and ribL transcripts. A standard curve using a PCR product specific

for the psmA gene was performed to measure the linear range of detection of the iCycler.

Primers specific for the ribL, a ribosomal protein gene, coding region were used as a control.

The primers for the psmA gene were previously reported (Reuter et al., 2004). The ratio of ribL

from the two samples was calculated and the resulting scalar was used to correct the transcript

levels of the psmA gene from the different samples.

Protein Techniques

Protein Expression in Escherichia coli

Hfx. volcanii proteins were expressed in E. coli BL21 (DE3) with pSJS1240 (expressing

rare tRNA genes for protein expression). Plasmids and strains used for synthesis of 20S

proteasome subunits are listed in Table 3-1 or Table 5-1. Strains were grown in Luria-Bertani

broth at 37 ˚C and 200 rpm. Media were supplemented with 100 μg∙ml-1 of ampicillin, 50 μg∙ml-

1 of kanamycin, 50 μg∙ml-1 spectinomycin, or 50 μg∙ml-1 of chloramphenicol as needed. Cells

were grown to mid-log phase (OD600 = 0.5 – 0.8) for 1 hour and induced with isopropyl-β-D-

thiogalactopyranoside (IPTG) at a final concentration of 400 μM for 3 – 5 hours.

Protein Expression in Haloferax volcanii

20S proteasome proteins and protein kinases were expressed in Hfx. volcanii strains from plasmids listed in Tables 3-1, 4-1, and 5-1. Cultures were grown in ATCC 974 medium at 42 ˚C

and 200 rpm in Fernbach flasks to late stationary phase (OD600 = 2.5 – 3). Media were

supplemented with or 0.1 μg∙ml-1 of novobiocin or 4 μg∙ml-1 mevinolin as needed.

Protein Quantification

Protein concentrations were determined by Bradford method (Bradford, 1976) with bovine

serum albumin as the standard (Bio-Rad) with the following modifications. Reaction volume for

the colormetric assay was scaled down to 200 μl. Bradford reagent (40 μl) was added to a 1:10

to 1:50 dilution of protein sample and incubated for 1 – 10 minutes. The assay was linear

between 100 and 800 μg∙ml-1 of protein.

Protein Separation and Chromatography

Cells were harvested by centrifugation at 6000 × g for 30 minutes and lysed by passing through a French pressure cell (3500 PSI) in preparation for downstream chromatography. Cell lysates and chromatography fractions were monitored for purity by staining with Coomassie blue

R-250 or SYPRO Ruby (Invitrogen) after separation by reducing 12 % SDS-PAGE according to

Laemmli (Laemmli et al., 1970). Molecular mass standards for SDS-PAGE included phosphorylase b (97.4 kDa), serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa) (Bio-Rad).

Nickel affinity chromatography

Cells were harvested by centrifugation (10,000 × g, 20 minutes, 4 °C) and resuspended in

60 ml of 20 mM Tris buffer (pH 7.2) supplemented with 2 M NaCl (buffer A) and 5 mM imidazole. Cells were passed through a chilled French pressure cell at 20,000 lb∙in-2. Cell debris

was removed by centrifugation (16,000 × g, 20 minutes, 4 °C). The filtrate obtained with a 0.45

μM-pore-size filter (Nalge Nunc International, Rochester, NY) was applied to a 5 ml Ni2+-

Sepharose column (HiTrap chelating; Amersham Biosciences) equilibrated with buffer A and

washed with a step gradient consisting of 5 and 60 mM imidazole in buffer A. Fractions

containing His-tagged proteins were eluted in buffer A with 500 mM imidazole.

Strep-tactin chromatography

Strep_II tagged (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) proteins were purified by application of cell lysate onto a 0.2-ml Strep-Tactin column (Qiagen) equilibrated with 20 mM Tris buffer

(pH 7.5) with 2 M NaCl (buffer L). The column was washed with 10 ml of buffer L, and proteins were eluted in 20 mM Tris buffer (pH 7.5) with 2 M NaCl and 1.5 mM desthiobiotin

(buffer E). Fractions containing protein were pooled and dialyzed against buffer A using D-

Tube Dialyzer (Novagen). Column resin was regenerated by washing 3 times with 5 column volumes of 100 mM Tris buffer pH 8.0 with 150 mM NaCl, 1 mM EDTA, and 1 mM HABA

(hydroxy-azophenyl-benzoic acid) (buffer R).

Gel filtration chromatography

Protein samples (0.5 ml per run) were applied to a Superose-6 HR 10/30 gel filtration

column (Amersham Biosciences) equilibrated in 20 mM Tris buffer (pH 7.2) with 2 M NaCl at a

flow-rate of 0.1 – 0.3 ml∙min.-1. Molecular mass standards for gel filtration included cytochrome

C (12 kDa), carbonic anhydrase (29 kDa), serum albumin (66 kDa), alcohol dehydrogenase (150 kDa), β-amylase (200 kDa), apoferritin (443 kDa), thyroglobulin (669 kDa), and Blue Dextran

(2000 kDa) (Sigma). Elution of protein was monitored by UV absorbance at 280 nm.

Peptide Synthesis

The synthetic phosphopeptide RGEDMSMQALpSTL (where pS represents phosphoserine) used for comparison to a phosphopeptide of the β protein was generated using fluorenyl methyoxycalbomyl-amino acid chemistry on an ABI 432A peptide synthesizer.

Peptide purity and molecular weight were measured after synthesis with MALDI-TOF. The

NH2-MQGQAQQQAYDK-biotin was synthesized in the same way for N-terminal

13 15 acetyltransferase assays. L-alanine-N-FMOC (U- C3 and N) was purchased from Cambridge

Isotope Laboratories Incorporated (Andover, MA) and used for the synthesis of two AQUA

peptides, Acetyl-MQGQAQQQAYDR and QGQAQQQAYDR, for quantitative mass spectrometry standard controls (heavy isotope amino acids are underlined and bold).

Two-Dimensional Gel Electrophoresis

Two dimensional gel electrophoresis was preformed as previously described (Karadzic and

Maupin-Furlow, 2005; Kirkland et al., 2006) with the following modifications, 2 ml of cells at

OD600 = 1, were centrifuged at 10,000 × g at 4 ˚C for 15 minutes and lysed in dH2O. The lysed

cells were treated with DNase I for 30 minutes at room temperature. Proteins were precipitated

from cell lysate by mixing 100 % cold acetone with the cell lysate. The resulting protein pellet

was washed 5 times with 100 % cold acetone and resuspended in with 10 % glycerol in 20 mM

Tris buffer (pH 7.2). Total protein (250 μg) was loaded onto an 11 cm ReadyStrip IPG Strip

(Bio-Rad) with a pI range of 3.9 – 5.1. Isoelectric focusing was performed using a Protean IEF

Cell (Bio-Rad) for 40,000 Vh. Proteins were separated on the second dimension using Criterion

Precast 12.5 % SDS-PAGE gels (11 cm with IPG +1 well) (Bio-Rad) at 16 ˚C for 90 minutes.

Proteins were stained in gel with SYPRO Ruby (Invitrogen) according to manufacturer. Gels were imaged on a Typhoon (Amersham), and spots were excised manually and identified by mass spectrometry.

Immunoblotting

For immunoblotting, purified proteins and cell lysates were separated by denaturing (SDS) and reducing (β-mercaptoethanol) PAGE gels (Laemmli et al., 1970). After separation, proteins were transferred to Hybond-P membranes (Amersham Bioscience, Piscataway, NJ) using 10 mM

2-N-morpholinoethanesulfonic acid (MES) buffer at pH 6.0 with 10 % (v/v) methanol at either

20 V for 16 hours or 200 V for 90 minutes. Transferred membranes were blocked with 10 %

(w/v) milk solution in Tris-buffered saline (TBS) for 1 – 16 hours with rocking. Primary antibodies, polyclonal α-α1, α-α2, and α-PanA generated in rabbit (Kaczowka and Maupin-

Furlow, 2003), were suspended in 5 % (w/v) milk in Tris-buffered saline with 0.05 % Tween-20

(TBST) at the following dilutions: 1:2000 α-PanA, 1:5000 α-α1, and 1:5000 α-α2. Primary antibody solution was incubated with the membrane for 60 – 90 minutes rocking. Primary antibody solution was washed 2 × with TBS for 15 minutes. The secondary antibody was goat anti-rabbit IgG – Alkaline phosphatse (AP). Blots were developed in AP-buffer (100 mM Tris

buffer (pH 9.5), 100 mM NaCl, and 5 mM MgCl2) with 400 μM nitro blue tetrazolium (NBT) and 380 μM 5-bromo-4-chloro-3-indolyl phosphate p-toluidine (BCIP) and imaged using a Versa

Doc 1000 with Quantity One v. 4 Software (Bio-Rad).

Labeling Hfx. volcanii Cells with Orthophosphate in Minimal Media

Cells grown to log phase in 1 – 3 ml (13 × 100 mm2 tubes, 42 ˚C, 200 rpm) were used to

inoculate 100 ml cultures of lactate minimal medium in 500 ml flasks by a 1:100 dilution. These

larger cultures are grown to OD600nm = 0.6 - 0.8 (log phase) (42 ˚C, 200 rpm). The cells were washed with phosphate-free medium and concentrated to 10 % their original volume. This cell suspension was incubated at 42 ºC in a standing water-bath with 10 μCi · ml-1 32P (Perkin Elmer

NEX053H) for up to 20 hours. Cells were washed with phosphate-free lactate minimal medium three times and stored at –20ºC until further analysis.

Pulse-Chase

For pulse-chase, Hfx. volcanii cells were grown in 200 ml of minimal (Hv-Min) medium in

500 ml flasks to an OD600 of 0.4 to 0.6 (200 rpm, 42 ºC). Cells were collected by centrifugation

at 10,000 × g for 15 minutes at 40 ºC and resuspended in 80 ml of prewarmed (42 ºC) Hv-Min

medium. Cells were incubated at 42 ºC for 1 hour with shaking in 500 ml flasks (200 rpm).

Radioactively labeled methionine and cysteine (500 μCi of 35S-Met and 35S-Cys mixture)

(EXPRE35S35S Protein Labeling Mix, Perkin Elmer) were added to the culture and incubated for

an additional 20 minutes (42 ºC, 200 rpm). Samples were divided into 8 - 10 ml fractions in 15

ml conical tubes. Translation was arrested immediately for one tube (0 minutes) by addition of 1

ml of “stop” solution (22 % [w/v] sodium azide, 100 μg/ml puromycin) and incubated on ice.

The remaining 7 samples were incubated with 1 ml of “chase” solution (8 mg/ml “cold”

methionine and cysteine, 100 μg/ml puromycin, 1 mg/ml protease inhibitor cocktail) at 42 ºC and

200 rpm. Each sample was arrested at appropriate time intervals (1, 3, 5, 10, 30 and 60 minutes

and 12 hours) by the addition of 1 ml of “stop” solution and chilling on ice. Each 10 ml sample

was collected by centrifugation at 8,000 × g at room temperature for 10 minutes. Pellets with

approximately 1 ml of medium were resuspended and re-pelleted in 2 ml microcentrifuge tubes at 14,000 × g at room temperature for 5 minutes. Cell pellets were frozen at -80 ºC for a maximum of 48 hour prior to immunoprecipitation (IP).

Immunoprecipitation (IP)

For IP, protein A-Sepharose beads (Pharmacia) [10 mg per ml phosphate buffered saline

(PBS) with 0.01 % (w/v) sodium azide] were aliquoted into 1.8 ml Eppendorf tubes (75 μl per

tube), washed once with cold PBS and resuspended in 1 ml cold PBS. The beads were charged

by addition of 10 - 12 μl of polyclonal antiserum for 4 - 12 hours at 4 ºC with continuous agitation and washed 5 × with cold PBS to remove unbound antibody. Cell pellets from pulse-

chase labeling (described above) were prepared for IP by resuspension in 150 μl of denaturing

lysis buffer (1 % [w/v] SDS, 50 mM Tris-Cl, pH 7.4, 5 mM EDTA, 10 mM DTT, 1 mM PMSF,

300 mM NaCl), boiling for 10 minutes, and addition of 1.35 ml non-denaturing lysis buffer (1 %

[v/v] Triton-X-100, 50 mM Tris-Cl, pH 7.4, 5 mM EDTA, 0.02 % [w/v] sodium azide, 10 mM

iodoacetamide, 1mM PMSF, 300 mM NaCl). Lysate was incubated with ~500 units of

Benzonase (2 μl) (Sigma-Aldrich) at room temperature for 30 minutes with occasional mixing.

Samples were clarified by centrifugation at 10,000 × g for 5 minutes, and clarified lysate was

added to the charged beads. The lysate/bead mixture was incubated at 4 ºC with rocking for 3

hours. Beads were washed 5 × with IP wash buffer (0.1 % [v/v] Triton-X-100, 50 mM Tris-Cl,

pH 7.4, 300 mM NaCl, 5 mM EDTA, 0.02 % [w/v] sodium azide, 0.1 % [w/v] SDS and 0.1 %

[w/v] deoxycholine) and one final time with cold PBS. SDS reducing dye (20 μl of 100 mM

Tris-Cl, pH 6.8, 10 % [v/v] beta mercaptoethanol, 2 % [w/v] SDS, 10 % [v/v] glycerol, 0.6

mg/ml bromophenol blue) was added. Samples were boiled for 10 minutes and centrifuged at

14,000 × g for 5 minutes to remove the beads. Proteins in the supernatant were separated by 12

% SDS-PAGE (200 V for 45 minutes), and their migration was compared to Precision Plus

Protein dual color standards (Bio-Rad). Gels were incubated in fixing solution (10 % methanol,

7 % acetic acid) for 30 minutes and an intensifier solution (1M salicylic acid) for 1 hour. Gels were washed with H2O, dried under vacuum for 1.5 - 2 hours, and exposed to radiographic film

for a minimum of 10 days at -80 ºC. Band intensity was quantified using a Versa Doc 1000 with

Quantity One v. 4 Software (Bio-Rad).

Molecular Modeling

Crystal structure files were downloaded from RCSB and the protein data bank. Three dimensional analysis of structure was done on Rasmol (Sayle and Milner-White, 1995), Pymol, and VMD (Humphrey et al., 1996) structure viewers. Homology based structure determination was done by using SWISS-MODEL (Peitsch, 1996).

Enzyme Assays

Peptide Hydrolyzing Activity

Chymotrypsin-like peptide-hydrolyzing activity of 20S proteasomes was assayed in buffer

E with 20 mM N-Suc-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (LLVY-AMC). Peptide substrate was solubilized in 100 % (v/v) DMSO at 5 mM prior to dilution for assay (20 μM final). Release of 7-amino-4-methylcoumarin was monitored by determining the increase in fluorescence over time (excitation λ of 350 nm; emission λ of 460 nm) with an Aminco

Fluorocolorimeter (Spectronic Instruments) or with Bio-Tek Syntergy HT 96-well plate reader

(Bio-Tek Instruments). Assays were performed at 37 ºC and 65 ºC. Assay conditions of pH 7.5,

2 M NaCl and 65 ºC were previously determined optimal for Hfx. volcanii 20S proteasomes

(Wilson et al., 1999).

Kinase Assays

Kinases were purified to electrophoretic purity by Strep-Tactin chromatography as

described above. Purified kinases were dialyzed using a D-tube dialysis tube (Novagen) into 20 mM Tris buffer (pH 7.2) with 2 M NaCl (or 2 M KCl) at 4 ˚C. Kinase activity was measured by incubating purified enzyme (x μg) with different concentrations of Mn2+ and Mg2+ (0 – 250 mM)

for one hour in the presence of 200 – 400 μCi of [γ-32P] adenosine 5’-triphosphate (Perkin

Elmer) in a reaction volume of 20 μl. Kinase reactions were inactived by addition of 20 μl

reducing SDS-PAGE buffer and separated by 12.5 % SDS-PAGE gels. Gels were stained with

Coomassie brilliant blue G and dried to Whatman paper. X-ray film was exposed to the dried gel for 1 – 10 days.

Mass Spectrometry

Sample Preparation

Protein fractions from Ni2+-Sepharose or Strep-Tactin chromatography were either

separated by reducing 12 % SDS-PAGE or provided as in-solution samples for mass

spectrometry. Proteins were stained in gel with Bio-Safe Coomassie (Bio-Rad). Protein bands

corresponding to the molecular weights of the proteins of interest (such as the subunits of 20S

proteasomes, 20 - 30 kDa) were excised from the gel and destained with 100 mM NH4HCO3 in

50 % (vol/vol) acetonitrile (4 °C, overnight). Protein samples were reduced, alkylated in-gel or in-solution, and digested with sequencing grade trypsin, AspN, chymotrypin, or Glu-C

(Promega).

Three Dimensional Ion Trap (LCQ Deca)

The subset of data used to analyze the N-terminal proteome was generated on a Thermo

LCQ Deca quadrupole ion trap mass spectrometer (LCQ DECA MS) in line with a 5 cm × 75 μm inner diameter PepMaxTM C18 5 μm / 300 Å capillary column (LC Packings). Sequest Analysis

software was used to interface with the LCQ Deca and run protocols. All runs had a 30 or 60

minutes gradient from 5 – 50 % acetylnitrile with a flow-rate of 10 – 20 μl∙minute-1. Parent ion

scans (MS) were followed by three or four data dependent MS/MS scans.

Hybrid ESI-Q-ToF (ABI QSTAR)

Capillary reverse-phase high-performance liquid chromatography (HPLC) separation of

the protein digests was performed using a PepMaxTM C18 column (75 µm inside diameter, 15 cm

length; LC Packings, San Francisco, CA) in combination with an Ultimate capillary HPLC

system (LC Packings, San Francisco, CA) operated at a flow rate of 200 nl∙minute-1. A gradient

(60 minutes) from 5 to 50 % (v/v) acetonitrile in 0.1 % (v/v) acetic acid was used. Tandem mass spectrometry (MS/MS) analysis was performed using a hybrid quadrupole time-of-flight (QTOF) instrument (QSTAR) equipped with a nanoelectrospray source (Applied Biosystems, Foster City,

CA) and operated with Analyst QS 1.1 data acquisition software.

Triple Quadrupole (ABI QTRAP)

Quantitative mass spectrometry techniques were performed using a triple-quadrupole tandem mass spectrometry analyzer (QTRAP) equipped with a nanoelectrospray source (Applied

Biosystems). Capillary reverse-phase high-performance liquid chromatography (HPLC) separation of the protein digests was performed using a PepMapTM C18 column (75 µm inside

diameter, 15 cm length; LC Packings, San Francisco, CA) in combination with an Ultimate

capillary HPLC system (LC Packings, San Francisco, CA) operated at a flow rate of 200

nl∙minute-1. A gradient (60 minute) from 5 to 50 % (v/v) acetonitrile in 0.1 % (v/v) acetic acid

was used. A control peptide (amino acids 13 – 22 of the α1 protein, GITIFSPDGR, which is

unmodified) was included in all samples to minimize variability. Peptides were targeted for

precursor ion scanning using a multiple reaction monitoring program (MRM) developed in the

MIDAS workflow software system. Relative quantities of different peptides were measured by

integration of the respective peaks on the initial MS scan using the integration function on

Analyst QS 1.2.

Database Searching and Software

Peptides were identified using MASCOT algorithms (Perkins et al., 1999) that searched a

custom, non-redundant database based on the hypothetical proteome of translated open-reading

frames from the Hfx. volcanii genome (Eisen, professional communication April 2007, http://archaea.ucsc.edu/). Probability-based MOWSE scores were estimated by comparison of search results against estimated random match population and are reported as ~10 × log10(p), where p is the absolute probability. Individual ion scores greater than 32 indicates identity or extensive homology (p<0.05). Carbamidomethylation was used as a fixed modification due to sample preparation. Variable modifications that were searched included deamidation of asparagines and glutamine, N-terminal acetylation, oxidation (single and double) of methionine, and phosphorylation of serine, threonine, or tyrosine.

CHAPTER 3 N-TERMINAL ACETYLATION OF ALPHA-TYPE SUBUNITS OF 20S PROTEASOMES IN HALOFERAX VOLCANII

Introduction

Proteolysis is important in regulation and protein quality control. Energy-dependent proteases are crucial to early stages of protein degradation. In eukaryotes and archaea, proteasomes are the central energy-dependent proteases of the cytosol and are essential for growth (Rosenzweig and Glickman, 2008a; Zhou et al., 2008). The catalytic component of proteasomes, the 20S core particle, consists of four heptameric rings of α- and β-type subunits stacked as a barrel in an α7β7β7α7 configuration. The active sites responsible for peptide bond hydrolysis are formed by the N-terminal Thr residues of β-type subunits and are sequestered within the central channel of the barrel-like structure. Energy-dependent triple-A ATPases, including regulatory particle triple-A ATPases (Rpt) in eukaryotes and proteasome-activating nucleotidases (PAN) in archaea, mediate the unfolding and translocation of substrate proteins through the α-rings for degradation within the 20S core (Smith et al., 2007; Rosenzweig and

Glickman, 2008a).

One major difference between eukaryotic and prokaryotic 20S core particles is in the crystal structure of the channel opening formed by the α-rings. Due to partial disorder of the α- subunit N-termini, the site of substrate entry appears open at the ends of the 20S cylinders of

Thermoplasma acidophilum (Lowe et al., 1995), Archaeglobus fulgidus (Groll et al., 2003) and

Mycobacterium tuberculosis (Hu et al., 2008). In contrast, X-ray structures of the 20S core particles of yeast (Groll et al., 1997) and bovine (Unno et al., 2002a; Unno et al., 2002b) do not contain this opening. Instead the extreme N-termini of the α2, α3 and α4 subunits and the loop structure of α5 fill the central pore in a gate-like structure.

Evidence suggests that all 20S proteasomes are gated, and the major differences observed in the state of the α-ring gate in crystal structures are not physiological. For example, the N-

terminal 11 amino acids of the A. fulgidus α subunit, which are not defined by electron density in

the 20S proteasome structure, are more ordered in the 16S “half” proteasome precursor (Groll et

al., 2003). Furthermore, cryo-electron microscopy of the M. tuberculosis 20S core particle

reveals closed ends that are dependent on the first 8 residues of the α-subunit and which diminish

peptidolytic activity. Consistent with this, deletion of the N-terminal α-helix (Δ2 - 12) of the T. acidophilum 20S proteasome α-subunit abolishes the need for an ATPase (i.e., PAN) in the degradation of acid-denatured GFP-SsrA or casein (Benaroudj et al., 2003). In addition, the conserved YDR motif thought to be important in the sterics of α-ring gating, is present in all archaeal α-type subunits to date (Groll et al., 2003). Thus, although the mechanism may not be exactly the same, prokaryotic proteasomes are thought to gate their α-ring aperture.

A gated channel formed by the N-termini of α-rings may be a general mechanism for regulating the activity of proteasomes. The rate limiting step in proteasome-mediated protein degradation is translocation of substrates through the α-rings to the active sites contained within the β-rings (Kohler et al., 2001). This mechanism is supported by the finding that eukaryotic

20S core particles have no peptidolytic activity in the absence of Rpt proteins or mild chaotropic agents such as SDS or heat treatment (Coux et al., 1996). Furthermore, peptidase activity of the yeast 20S proteasome is blocked by the N-terminal regions of the α3 subunit. Deletion (Δ2-9) or single substitution (D9A) of N-terminal residues of α3 derepresses this peptidase activity (Groll et al., 2000).

An additional gating mechanism could be employed by post-translational modifications of the N-termini of the α-type subunits. The α-type subunits of 20S proteasomes are modified by

Nα-acetylation in several eukaryotes and haloarchaea including Haloferax volcanii (Tokunaga et

al., 1990; Kimura et al., 2000; Kimura et al., 2003; Falb et al., 2006; Humbard et al., 2006). In

yeast, N-acetyltransferase 1 (NAT1) is responsible for the Na-acetylation of five of the α-type subunits (α1, α2, α3, α4, and α7). Proteasomes purified from a nat1 mutant have two-fold higher chymotrypsin-like peptidase activity in the absence of SDS compared to wild-type suggesting that Nα-acetylation enhances closure of the α-gate (Kimura et al., 2000). In Hfx. volcanii, both

α1 and α2 are Nα-acetylated on their initiator methionine residue with a subset of α1 not

acetylated and instead cleaved by an apparent methionine aminopeptidase. A large scale

proteomic survey reveals Nα-acetylation is common to other proteasomal α-type proteins of the

haloarchaea (Falb et al., 2006). In this previous survey, the ratios of Nα-acetylated and cleaved forms of the α-type proteins were quantified by spectral counting and estimated to be around 3:1 and 4:3 for Halobacterium salinarum and Natronomonas pharaonis, respectively (Falb et al.,

2006). So far, this existence of two unique forms of α subunit N-termini in the cell simultaneously has only been observed in the haloarchaea.

In this chapter, both α1 and α2 are demonstrated to be Nα-acetylated on their initiator

methionine residue with a subset of α1 not acetylated and instead cleaved by an apparent

methionine aminopeptidase. Quantitative MS/MS was used to precisely determine the ratio of

the Nα-acetylated to cleaved forms of the α1 protein of 20S proteasomes in Hfx. volcanii. Site-

directed mutagenesis was also used to examine how the penultimate residue of α1 influences its

Nα-acetylated state. Residues that perturbed this state had profound phenotypic consequences

including a substantial increase in the levels of α1 protein and an enhanced tolerance of cells to

heat and hypoosmotic stress.

Results

Isoforms of 20S Proteasome Subunits

Many subunits of 26S and 20S proteasomes from eukaryotes have isoforms that are generated by post-translational modification and can be separated by 2-DE. In order to

investigate this in archaea, an Hfx. volcanii strain which synthesizes a C-terminal histidine

tagged α1 protein (α1-His) of 20S proteasomes (Kaczowka and Maupin-Furlow, 2003) was used to enrich for proteasomal complexes by nickel affinity chromatography. Based on previous work

(Kaczowka and Maupin-Furlow, 2003), samples enriched for proteasomes using this method and

strain are proteolytically active and are composed primarily of 20S proteasomes and heptamers

of α-type subunits. Less abundant assemblies of α1 and α2 with molecular masses smaller than heptameric rings are also observed. Therefore, this general approach was used to enrich for proteasomes from log phase cells for further analysis. Samples were separated by 2-DE using an immobilized pH gradient from 3.9 to 5.1 prior to reducing 12 % SDS-PAGE (Figure 3-1).

Protein spots which migrated similar in molecular mass to the previously described α1, α2 and β subunits of 20S proteasomes (37.5, 34.5 and 30 kDa, respectively, based on SDS-PAGE)

(Wilson et al., 1999) were digested in-gel by trypsin and the corresponding peptide mixture analyzed by tandem mass spectrometry. All of these spots were identified as either α1, α2 or β

20S proteasomal proteins of Hfx. volcanii as indicated in Figure 3-1.

Based on these results, all three of the Hfx. volcanii 20S proteasomal proteins (α1, α2 and

β) migrated as isoform chains (Figure 3-1). These proteasome-fractions were composed primarily of α1 and β subunits, consistent with the low levels of α2 protein present in log phase

Hfx. volcanii cells (Wilson et al., 1999; Kaczowka and Maupin-Furlow, 2003). Approximately eight isoforms of the α1 protein were identified (Figure 3-1; a to h). Although the α1 isoforms were most likely a mixture of α1 proteins with and without the C-terminal histidine tag, this

alone did not account for the large number of α1-specific isoforms detected in the chain. In

addition to α1, four to five distinct isoforms of the β protein were detected (Figure 3-1; i to m).

Based on SDS-PAGE, four of these isoforms (indicated as i, j, k and m) were similar in

migration to the mature β subunit versus the β precursor. This differentiation is based on a

previous study (Wilson et al., 1999) which used Edman degradation to determine the N-terminal

residue of the 20S proteasomal β subunit to be Thr49 (residue number according to the deduced

protein sequence). Thr49 is highly conserved with the active site Thr of other β-type

proteasomal proteins, which undergo autocatalytic processing to remove an N-terminal

propeptide and expose the active site Thr during 20S proteasome assembly (Seemuller et al.,

1996; Maupin-Furlow et al., 1998). The propeptide of the Hfx. volcanii β-precursor is rather large and accounts for 5,427 Da of the molecular mass of the polypeptide. This difference in molecular mass between the β subunit and β precursor protein enables separation of these two proteins by SDS-PAGE. Thus, the three most basic spots of the β isoform chain (i, j, k) and the highly acidic spot (m) are attributed to β subunits which have undergone processing. The spot with slightly higher molecular mass (30.1 kDa; spot l) may be a partially processed β subunit or a non-β protein which copurifies during the proteasome-enrichment procedure and migrates too close to the mature β subunit to differentiate it via MS analysis of the protein spot. Although the levels of α2 are lower in the cell prior to stationary phase than the α1 and β proteins, two distinct isoforms of α2 were detected (Figure 3-1, n and o). Based on these results, the number of α- and

β-type isoforms detected was significantly greater than the three polypeptides deduced from the complete genome sequence (α1, α2, and β). The identification of these multiple α1, α2 and β isoforms suggested that the proteasomal polypeptides are modified in the cell by mechanisms which alter their pI.

It should be noted that the uppermost band of 66-kDa directly associated with the nickel-

Sepharose column, based on its purification from not only the His-tag expression strain

(DS70/pJAM204) but also parent strain DS70. This protein was recently identified as PitA

(Bab-Dinitz et al., 2006), and contains an N-terminal chlorite dimutase-related domain and C-

terminal antibiotic biosynthesis monooxygenase-related domain linked by a central histidine region which most likely accounts for its divalent metal affinity. In contrast to the PitA protein, purification of the predominant lower bands composed of α1, α2 and β was specific for the α1-

His expression strain.

Analysis of Proteasome Isoforms by Mass Spectrometry

To enhance the number of peptide fragments detected by mass spectrometry and increase

the likelihood of detecting post- or co-translational modifications of the proteasome isoforms, the

proteasome-enriched fractions were separated by reducing 1D (vs. 2D) SDS-PAGE. The α1, α2

and β proteins were excised from the gel as bands, reduced, alkylated, and cleaved with either

trypsin, sequentially with trypsin and chymotrypsin or with Glu-C endoprotease. This approach

enhanced the protein sequence coverage when compared to analysis of protein spots from 2D

gels (data not shown), most likely due to the higher amount of protein isolated from the 1D gels.

The peptide fragments resulting from the protease digests were separated by reversed-phase

HPLC and analyzed by ESI-QTOF MS. Using this approach, the trypsin digest of the α1-His

purified sample resulted in 37.9 % coverage of β, 47.8 % coverage of α2, and 45.2 % coverage of

α1 (Table 3-2 and 3-3) with Mascot total protein scores of 2162, 338, and 161, respectively

(coverage and scores based on complete polypeptide deduced from GenBank nos. AF126262,

AF126261 and AF126260) (Wilson et al., 1999). In combination with a trypsin and

chymotrypsin sequential digest, total coverage of α1 and β was increased to 54.7 % and 56 %,

respectively. No additional peptides were detected for α2 using this sequential approach. The

Mascot total protein scores for the sequential tryptic and chymotryptic digests of β, α2 and α1

were 763, 422 and 328, respectively. Summaries of these Mascot search results are presented in

Table 3-2 and 3-3. The Glu-C endoprotease was used to enhance coverage of the N-terminus of

α2.

α1 is N-terminally Acetylated

The ions that corresponded to the α1 N-terminus included both Nα-acetylated and unmodifed forms. The ion corresponding to the N-terminal fragment of the α1 subunit with the

Nα-acetylation had an individual MOWSE score of 76 and an Expect of 5.5e-7. It contained 12 amino acids, from 1 to 12 (MQGQAQQQAYDR), and had a monoisotopic mass of 1480.67 Da.

It eluted from RP-HPLC between 20.01 minutes and 20.35 minutes as a doubly charged ion

(741.343, 2+). The peptide contained an oxidation on methionine 1 and an N-terminal acetylation site. The MS/MS fragmentation of the ion produced a complete y-ion series as well as several key b-ions including b(1) with a mass of 190.05 Da (Figure 3-2). This mass corresponded to a methionine with an oxidation to methionine sulfoxide (147.0354) plus an acetylation (42 Da). Additional b- and a-ions supported this oxidation / acetylation mass. There were several internal ion fragmentations resulting in major peaks in the spectrum. The major peaks are labeled and listed in inset table in Figure 3-2. It should be noted that the N-terminally acetylated ion was obtained in multiple experiments each time with multiple ions. In addition, an unmodified N-terminal peptide for the α1 subunit was also identified. This suggested either not all of the α1 subunits in the cell were acetylated or the acetyl group was removed during collision-induced dissociation (CID). This second possibility is unlikely since the unmodified fragment had a unique elution time from HPLC. If the acetyl group was lost in CID, the peptide should be chemically indistinguishable from the modified one prior to CID. It is far more likely that the unmodified and modified α1 subunit exists in a cell simultaneously. Since, the Ni2+-

Sepharose chromatography fractions of cells expressing a hexahistidine tagged α1 included both

α1 rings and 20S proteasomes, we were unable to determine whether both the unmodified and

acetylated forms of α1 were present in functional 20S proteasomes.

Subpopulation of α1 Is Not Acetylated

In addition to the acetylated α1 peptide fragment, a tryptic peptide ion of α1 was also

detected which spanned residues 2 to 12 (QGQAQQQAYDR). It was a doubly charged ion (m/z

646.8) with a Mascot individual ion score of 43 and an E value of 1 e-3. The MS/MS

fragmentation spectrum contained a complete y-ion series as well as four unique b-ions

supporting an N-terminal cleavage event. This result suggested that not all of the α1 proteins in the cell are acetylated, and a subset may instead be cleaved by methionine aminopeptidase.

Thus, N-terminal acetylated and cleaved variants of α1 appear to exist in the cell simultaneously.

Acetylated Form of α1 Proteins Is Dominant

In order to better understand the relative ratios of these forms, quantitative mass spectrometry of α1-His6 proteins purified from Hfx. volcanii DS70 (pJAM204) cells was performed. Initially, spectral counting was combined with a comparative integration of peak area of each of the isotopic envelopes of the two N-terminal peptides. The ratio of the Ac1 to N2 form of α1 were 103:1 (Table 3-4), heavily favoring the acetylated (Ac1) to the unacetylated methionine-removed form (N2). To confirm the integration intensity of the MS scan from the Q-

ToF instrument, different instrumentation, a triple quadrupole, was used with a multiple reaction monitoring (MRM)-initiated detection and sequencing workflow (MIDAS). Using this software, the mass spectrometer only detects the ions that are defined by a set of parameters. The ions defined were for the two forms of the N-terminal peptide (733.79, 2+ for Ac1 and 647.17,2+ for

N2) and for additional selectivity, product ions from the second MS scan (MS/MS) were used to identify the correct species. The resulting filtering identified two peptides that matched those

defined mass / charge ratios and product ions. The calculated ratio of these two ions was 100:1

(Figure 3-5).

Amino Acid Substitutions in the Penultimate Position of α1 Affect Acetylation and Cleavage of the Initiator Methionine Residue

As a result of N-terminal processing and protein maturation by methionine aminopeptidase

(MAP) and Nα-acetyltransferase (NAT) enzymes, at least four different forms of a protein are

possible. These include proteins that retain their initiator methionine residue in either an Nα-

acetylated (Ac1) or unmodified form (N1) as well as those which undergo MAP cleavage

resulting in a cleaved methionine (N2) which can also be Nα-acetylated (Ac2). Unlike bacteria,

formyl-methionine has not been identified as an initiating residue in archaea (Ramesh and

RajBhandary, 2001). These final N-terminal states of a protein are thought to be governed by the nature of the first three amino acids, the initiating methionine, the penultimate and the antepenultimate amino acid (Polevoda and Sherman, 2003b). Taking this into consideration, a series of amino acid substitutions were introduced into the α1 protein through site-directed mutagenesis of the psmA gene. The resulting substitutions in α1 were Q2A, Q2D, Q2P, Q2S,

Q2T, Q2V and a deletion of the N-terminal α-helix (ΔQ2-R12) resulting in fusion of the initiating methionine to G13. All of the α1 protein variants were expressed in an Hfx. volcanii

strain with the wild-type psmA gene deleted from the chromosome (GZ130) and all of the

variants had a C-terminal hexahistidine (His6) tag. Variant α1 proteins were purified from whole

cell lysates by nickel affinity chromatography alone. Chromatography fractions enriched for α1

variants were subjected to tryptic digest and mass spectrometry analysis by ESI-Q-ToF (ABI

QSTAR). The acetylation patterns of the different α1 variants were measured by spectral

counting (Table 3-5) as described above for wild type α1. The results revealed variant α1 Q2A

was acetylated on the second amino acid, alanine, after removal of the initiator methionine (Ac2)

100 % of the time. Variant α1 ΔQ2-R12 (G13) was not modified by either acetylation or

methionine removal (N1). Variants α1 Q2S and Q2V were acetylated on the initiating

methionine (Ac1) similar to wild-type. Variants α1 Q2D, Q2P and Q2T were a mixture of

cleaved and uncleaved, acetylated and unacetylated forms of the protein (N1, Ac1, N2, Ac2)

with the ratios of these different forms varying among different chromatography preparations

(Table 3-5).

Co-purifying Non-proteasomal Proteins

In addition to the 60-kDa nickel binding protein PitA already discussed above, three

additional proteins were identified by tandem spectrometric analysis of the proteasome-enriched

samples. These included HVO_1577, HVO_2108, and HVO_2645 (Table 3-6) of calculated

molecular masses 19377.70, 20752.38, and 32702.80 Da. Whether these proteins co-purify with

α1-His or purify by nickel affinity chromatography alone remains to be determined. Proteins of

molecular masses in the range of 15 to 40 kDa were not detected by SYPRO Ruby stained SDS-

PAGE gels of proteins purified by nickel affinity chromatography from parent strain DS70.

However, mass spectrometry can be more sensitive at protein detection than gel stains due to the

diversity of protein chemical properties.

The high content of His residues in HVO_2108 and HVO_2645 as well as the prediction

that these proteins bind metal (Table 3-6) is consistent with the likelihood that these proteins

bind the nickel column and not the α1-His protein. It is less clear regarding HVO_1577, which

is not predicted to bind metal. This latter protein contains a DUF293 domain and is a distant

homolog of the heat shock transcriptional repressor HrcA, recently shown to bind and regulate a

bacterial GroEL (Wilson et al., 2005). Thus, it is tempting to speculate that HVO_1577 may

associate with proteasomes.

20S Proteasomes Rapidly Isolated by Two-step Affinity Purification from Hfx. volcanii

To reduce contaminant background and to further purify 20S proteasomes for kinetic studies, an efficient, two-step affinity purification protocol was developed to rapidly isolate 20S proteasomes from Hfx. volcanii. It involved fusing a StrepII tag to the C-terminus of the β subunit and a hexahistidine (His6) tag to the C-terminus of the α1 subunit. The tagged proteins were produced from a synthetic operon [psmB-strepII (β-StrepII) followed by psmA-his6 (α1-

His6)] using the strong Halobacterium cutirubrum rRNA P2 promoter and T7 phage

transcriptional terminator on a plasmid provided in trans. To examine this approach, cell lysates of Hfx. volcanii DS70 expressing the synthetic operon were first subject to nickel affinity chromatography to isolate α1-His6 proteins and complexes. The second column, StrepTactin (a

streptavidin variant), purified β-StrepII subunits in complex with α1-His6 proteins. The purity of the resulting fractions was determined by tandem mass spectrometry (MS/MS) (Table 3-7) in

addition to SDS-PAGE and Superose 6 gel filtration (Figure 3-4). Based on MS/MS, only α1, α2

and β protein fragments were detected. Likewise, Coomassie blue staining of fractions separated

by reducing SDS-PAGE revealed only 20S proteasome-specific protein bands including: a

doublet of β and β-StrepII at ~ 20 kDa, a doublet of α1 and α1-His at ~ 30 kDa and α2 at 26kDa

(Figure 3-4). The appearance of only one major peak in the gel filtration chromatogram at the

molecular weight of 20S proteasomes (600 kDa) further supports the conclusion that the

fractions were homogeneous for 20S proteasomes containing tagged α1 and β proteins and

untagged α2 proteins. This purification strategy proves sufficient for rapid and effective

purification of 20S proteasomes from Hfx. volcanii cells under native conditions and is similar to

a previously reported technique for purifying 20S proteasomes from yeast cells (Wang et al.,

2007).

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20S Proteasomes Have Different Ratios of Modified α1 Proteins than Whole-cell Lysates

Measuring the relative levels of acetylation in purified 20S proteasomes containing the variant α1 proteins revealed that nearly 100 % of the α1 proteins that successfully assembled into

20S proteasomes were in the Ac1 form (acetylated on the initiator methionine residue) (Table 3-

5). The α1 variants Q2A, Q2S, Q2V, Δ2 – 12 and wild-type did not display any difference in acetylation patterns whether or not they were successfully incorporated into 20S proteasomes. In contrast, α1 variants Q2D, Q2T, and Q2P were a mixture of acetylated (Ac1), unacetylated methionine removed (N2), and acetylated on the second amino acid (Ac2) forms. When 20S proteasomes were isolated containing these variants, only the Ac1 form of the α1 proteins were detected by MS/MS. The other forms (N1, Ac2 and N2) were either as α1 monomers or associated in non-20S proteasome complexes. Thus, although modification of the penultimate residue can perturb the acetylation pattern of the α1 protein; the dominant N-terminal form of α1 in 20S proteasomes is Ac1. These results suggest that either the Ac1 form of α1 is preferentially selected for incorporation into 20S proteasomes or 20S proteasome structural elements are required for proper acetylation of the N-terminal initiator methionine residue of α1.

CP Peptidase Activity was Enhanced by the α1 Q2A and ∆2-12 Modifications

The influence of the N-terminal modifications of α1 on the chymotrypsin-like activity of the various CPs was examined at conditions previously found optimal for proteasomes of α1β composition purified from Hfx. volcanii DS2 (i.e., Tris-HCl buffer at pH 7.5 with 2 M NaCl at

65 ˚C) (Wilson et al., 1999). Assays were also performed at 37 ˚C to assess the effect of temperature. Not surprisingly, CPs containing the α1 Δ2-12 variant showed a 3- to 5-fold increase in activity compared to wild-type at both temperatures examined (Table 3-5). The N- terminal α-helix thought to make up the regulatory gate of the CP was deleted in this α1 variant

(∆2-12), and this change is likely to allow the passive diffusion of cleavable peptides into the

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proteolytic core. Interestingly, the Q2A variant which has only modest changes in its N-terminus

including the removal of the initiator methionine and acetylation of the exposed N-terminal

alanine was also enhanced (nearly 2-fold at 65 ˚C) in its CP peptidase activity, suggesting it too

has a more ‘open’ gate conformation than wild-type. CPs containing the α1 Q2V, Q2S and Q2T

variants displayed activity comparable to wild-type at both temperatures examined, consistent with the Nα-acetylated state of these CPs which is similar to wild-type.

α1 Variants (Q2D, Q2P and Q2T) Accumulated as Heptameric Rings in Haloferax volcanii

We also noted during purification that α1 variant proteins Q2D, Q2T and Q2P, that were

detected in multiple N-terminal forms (N1, Ac1 and/or N2), accumulated to much higher levels

than the α1 wild-type or other α1 variants when expressed in Hfx. volcanii (Figure 3-6A).

Analysis of cell lysate by 2-D gels revealed a 30-kDa isoform chain predominated in the Hfx.

volcanii GZ130 cells (grown at 37 ºC) expressing α1 Q2T compared to wild-type α1 (Figure 3-

9). This isoform chain was identified by MS/MS to be solely composed of α1 protein (Table 3-

8), highlighting the extreme excess of α1 protein in these α1 Q2T-expressing cells. Additional

differences in cell lysate separated by 1D gels were also observed including an ~70 kDa protein

that was more abundant and 37 kDa protein that was less abundant in the α1 variant strains than

wild type (Figure 3-6A). However, these differences appeared modest compared to the α1

protein excess of the 2D-gels (Figure 3-9).

To investigate the quaternary structure of the excess α1, the variant and wild-type α1

proteins enriched by Ni2+-Sepharose chromatography were compared by Superose 6 gel filtration

chromatography (Figure 3-6B). For cells expressing wild-type α1, the majority of this α1 protein was associated in CPs. In contrast, the dominant quaternary form of the α1 variant proteins that appeared in excess (Q2D, Q2T and Q2P) was of molecular mass consistent with heptameric rings (170 kDa), with the levels of the 600-kDa CPs similar to wild-type (Figure 3-6B). Since β

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subunits are required for proteasome assembly, the highly abundant α1 protein variants appear to

be trapped in an assembly or degradation intermediate of heptameric rings. qRT-PCR analysis revealed no dramatic difference in the levels of psmA-specific transcript among the various strains (e.g., cells expressing α1 wild-type vs. α1 Q2D) (Figure 3-6C), suggesting the enhanced levels of α1 protein in the variant strains are a result of a post-transcriptional event such as protein half-life or translation efficiency.

Complementation of the Hypoosmotic Stress Phenotype of psmA Mutant

Hfx. volcanii requires high concentrations of salt to grow (1.7 to 2.5 M NaCl)

(Mullakhanbhai and Larsen, 1975). Recently characterized strains of Hfx. volcanii that are

lacking in proteasomal subunit genes, most notably psmA (α1), display an increased sensitivity to

hypoosmotic environments, low salt containing media (Zhou et al., 2008). For the psmA mutants, this increased sensitivity to low salt media can be rescued by the addition of a psmA- his6 gene in trans (plasmid pJAM204) (Zhou et al., 2008). In order to understand how the individual substitutions in the penultimate residue of the α1 protein were affecting the cell, strains expressing α1 variant proteins were grown in rich medium with varying concentrations of

NaCl (Figure 3-7). As previously reported, while the ∆psmA strain (GZ130) is fully complemented to wild-type by providing psmA-his6 gene in trans, the negative control

(pJAM202c vector alone) failed to rescue the hypo-osmolarity phenotype caused by deletion of the psmA gene. Interestingly, both genes encoding the α1 Q2A and α1 Δ2 – 12 variants had dominant negative phenotypes with even greater growth inhibition when expressed in trans than the psmA knockout (GZ130) alone (Figure 3-7). In contrast, GZ130 strains expressing α1 Q2D and α1 Q2T were less sensitive to osmotic stress than wild-type. Effectively, strains with α1

Q2D or α1 Q2T grew at lower concentrations of NaCl than wild-type cells (Figure 3-7).

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Cells Expressing α1 Q2T Display a More Thermotolerant Phenotype

Curiosity about the possibility of a temperature-sensitive phenotype of cells producing

proteasomal CPs with an N-terminal truncated α1 (i.e., ΔQ2 – R12 and Q2A) led us to measure

the temperature sensitivity of Hfx. volcanii GZ130 (∆psmA) cells expressing the various α1

proteins in trans. Strains expressing the N-terminal truncated α1 variants did not display a

significant temperature sensitive phenotype compared to strains expressing wild-type α1.

However, to our surprise, strains expressing α1 Q2D and α1 Q2T variants grew to a higher cell

density at 50 ºC than those expressing wild-type α1 in both H26 parent and GZ130 ∆psmA mutant strain backgrounds, revealing that this growth phenotype at 50 ºC associated with the α1

variants was dominant over the genomic encoded wild-type α1 protein. To further analyze this

phenomenon, a growth curve was determined for cells which synthesized α1 Q2T vs. wild-type

at 50 ºC (Figure 3-8A). Cells with α1 Q2T grew to 6-times the final density of those with wild-

type α1 (Figure 3-8A). The growth rate of the ∆psmA cells expressing wild-type α1 and α1 Q2T

from plasmids was further analyzed at temperatures ranging from 37 to 50 ºC (Figure 3-8B). At

all temperatures measured, cells expressing the variant α1 Q2T had a significantly higher growth

rate than cells expressing wild-type α1. It should also be noted that growth at 50 ºC of the

∆psmA strain expressing psmA-his6 (α1) in trans (GZ130-pJAM204) was comparable to H26,

the parent strain of this study (data not shown). Likewise, the Arrhenius plot for cells expressing

wild-type α1 in this study is in good agreement with that previously published for growth of Hfx.

volcanii DS70 (Robinson et al., 2005).

It is surprising that such a dramatic increase in overall growth rate, cell yield and

tolerance of stress (low salt and high temperature) was observed for the α1 Q2T variant

compared to wild-type, since the difference between the two strains expressing these variants at

the genetic level is only two nucleotide substitutions in the penultimate codon of the 5’-end of

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the psmA gene resulting in a single amino acid substitution. While the thermostability of

extremophilic proteins is relatively common, including optimal activity of the Hfx. volcanii CP at

65-75 ˚C (Wilson et al., 1999), increasing the growth rate, growth yield and stress tolerance of

the entire organism by making a subtle change to a structural gene is less common. There are a

few examples of accomplishing this by over-expressing heat shock proteins, Hsp70 (Nollen et

al., 1999; Nakamoto et al., 2000), ShsP (Takeuchi, 2006), or by uncoupling the regulation of heat-shock inducible transcription factors (hsf) (Wagstaff et al., 1998) in various organisms.

Oddly, the connection between proteasomal activity and acquired stress tolerance appears inversely proportional with the more active, potentially ungated and unregulated, proteasomal

CPs (i.e., Q2A and ∆2-12 variants) rendering the Hfx. volcanii cells less tolerant of stress than those with gated CPs. Interestingly, this type of inverse relationship correlates with previous studies in eukaryotic cells including the observed increase in thermotolerance of yeast after inhibition of proteasomal CP activity, apparently due to an induced heat-shock response (Bush et

al., 1997; Lee and Goldberg, 1998). In our study, the increased stress tolerance of Hfx. volcanii

α1 Q2T cells does not appear to be a product of enhanced CP peptidase activity. Both wild-type

and Q2T α1 containing CPs had similar peptidase activities at both moderate and high

temperatures (37 and 65 ˚C, respectively) (Table 3-5) and their thermostability was comparable,

based on preincubation at 75 to 80 ˚C for 30 min and activity assay at 65 ˚C (data not shown).

Although 2-D gel electrophoresis did not indicate cells expressing the α1 Q2T variant were locked into a constitutive heat-shock phenotype when grown at 37 ºC, it did reveal unusually

high levels of α1 protein that were also detected throughout α1 purification (Figure 3-6). Based

on these findings, we propose that the high levels of the α1 Q2T rings may interact with and

stimulate the protein folding activity of chaperones such as those of the triple-A ATPase family

105

or more directly act as a chaperone to stabilize proteins needed for cell proliferation and/or stress

response. Chaperone activities have been found to be closely associated with energy-dependent

protease pathways such as the proteasome, Clp and FtsH systems (Gottesman et al., 1997;

Suzuki et al., 1997; Yano et al., 2005). It is also possible that the Q2T strain is in an induced

heat-shock state based on the observed increase in the levels of a 70-kDa protein and decrease in

the levels of a 37-kDa protein compared to wild-type by 1-D PAGE (Figure 3-6A); however,

these differences could not be resolved by 2-D PAGE to facilitate rapid protein identification.

α2 is N-terminally Acetylated

Based on the finding that the α1 protein was Nα-acetylated, it was speculated that α2 may also include this modification. In order to enhance the probability of observing peptides containing the N-terminus of α2, this protein was digested with Glu-C endoprotease. The rationale for using Glu-C (vs. trypsin) was based on the abundance of basic residues in the N- terminal region of α2 (e.g., Arg3 and Lys6). Trypsin was predicted to cleave carboxyl to these basic residues and generate short N-terminal peptide ions that would not be readily detected by

LC-MS/MS. The Glu-C endoprotease digest of α2 protein generated a quadruply charged ion

(m/z 776.6) which contained the first 26 amino acids of the deduced protein sequence (Acetyl-

MNRNDKQAYDRGTSLFSPDGRIYQVE).

This peptide had a Mascot individual ion score of 24 and an E value of 0.1. The MS/MS fragmentation spectrum of this N-terminal peptide (Figure 3-3) contained primarily b-type ions including a 185.1 Da ion which corresponded to an acetylated methionine and several y-type ions. The peptide contained an acetyl group on the N-terminus, and the fourth amino acid

(Asn4) was deamidated. The low Mascot ion score and corresponding high expect value were likely a consequence of the length and amino acid composition of the peptide. However, the ability to manually assign all the peaks in the spectrum and the consistency of the data support

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the validity of this identification as an acetylated N-terminal peptide of α2. In contrast to α1, all of the N-terminal fragments of α2 detected by MS/MS were acetylated.

Discussion and Conclusions

This study demonstrates that both α-type proteasomal subunits (α1 and α2) in Hfx. volcanii are Nα-acetylated and that a subpopulation of α1 proteins are not acetylated but rather missing the initiator methionine. To elaborate on the Nα-acetylation of the α1 proteins, the acetylated

(Ac1) and unacetylated (N1) forms of α1 protein were quantified. The acetylated form was shown to be in 100-fold excess of the unacetylated form in whole cells and purified 20S proteasomes or CPs. Amino acid substitutions in the N-terminal penultimate position and removal of the N-terminal α-helix of the α1 protein were used to probe Nα-acetyltransferase and

methionine aminopeptidase (MAP) activities of Hfx. volcanii. Deletion of the N-terminal α-helix

(∆Q2-R12) and substitution of the penultimate glutamine for an alanine residue (Q2A) had the most profound influence on these activities. The N-terminal deletion (∆Q2-R12) rendered α1

unavailable for modification by Nα-acetyltransferase based on the detection of α1 only in the N1

form. Likewise, although the Q2A modification did not impair Nα-acetylation of α1, it did alter

the site of this modification to the N2 position based on detection of α1 only in the Ac2 form.

This latter result suggests that the M-A bond of the Q2A variant is a much better substrate for

MAP cleavage than the M-Q bond of wild-type α1, consistent with the preferences of purified

MAP enzymes (Wiltschi et al., 2009). In contrast to ∆Q2-R12 and Q2A, the Q2D, Q2P, and

Q2T variants did not alter the prevalence of α1 proteins in the Ac1 form in the CPs; however,

these latter site-directed mutations did result in an increase in the population of unacetylated N1

and/or N2 forms of α1 as rings in whole cells. Thus, although the population of Nα-acetylated α1

proteins was impacted by these latter amino acid substitutions, the acetylated state of the α1

proteins within the CP did not change.

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Cells expressing the different α1 N-terminal variants had a collection of phenotypes and

CP activities that correlated with the ratios of α1 in the Nα-acetylated or unacetylated state. For example, CPs purified from strains producing α1 variants completely devoid (i.e., α1 ΔQ2-R12) or impaired in the position of Nα-acetylation (i.e., Q2A in the Ac2 vs. Ac1 form) had enhanced

levels of peptidase activity suggesting the gate of the CP was in a more open state than wild-type

allowing better access of substrate to the proteolytic active sites harbored within the central

chamber. Interestingly, the Hfx. volcanii strains producing these ‘open’ CPs displayed reduced

growth in low salt medium suggesting that gating of the CP (likely to protect the cell from

uncontrolled proteolysis) is important for cell viability under these stressful conditions. All of

the other site-directed mutations examined did not impact the level or residue position of the Nα-

acetylation of α1 in CPs nor did they enhance (some actually reduced) the CP peptidase activity,

suggesting the CP gate is not artificially opened by these mutations. Although these latter strains

produced apparently ‘gated’ CPs, a number of them (i.e., those producing α1 Q2D, Q2T, Q2P)

accumulated unusually high levels of α1 rings in various unacetylated states (N1 and N2 forms).

Of these, two strains were further examined (i.e., α1 Q2D and Q2T) and found to display higher

growth rates and improved tolerance to heat and hypoosmotic stress compared to wild-type.

These phenotypes were dominant even when the α1 variant was co-produced with wild-type α1,

suggesting the accumulation of α1 rings can significantly alter the proliferation and survival of

cells.

Before systematic surveys of Nα-acetylated proteins in the archaea had occurred, it was

unclear whether the acetyl groups that modified the N-termini of the proteasomal α1 and α2

proteins, as detected in this study, are common or rare events in Hfx. volcanii. Co-translational

Nα-acetylation is common in eukaryotes with 50 to 90 % of all proteins acetylated on N-terminal

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serine, alanine, glycine, or threonine residues after the removal of the initiating methionine

(Brown and Roberts, 1976; Driessen et al., 1985; Polevoda and Sherman, 2003b). In contrast,

N-terminal acetylation is presumed to be post-translational and rare in prokaryotes based primarily on studies of E. coli (Waller, 1963; Tanaka et al., 1989). Additionally, whether Nα-

acetylation occurs co- and/or post-translationally in Hfx. volcanii or other archaea remains to be determined.

In yeast, all seven of the CP α subunits are Nα-acetylated by NatA (NAT1 catalytic subunit) or NatC (MAK3 catalytic subunit) during translation (Kimura et al., 2000). Thus, similar to eukaryotic cells, it is possible that Hfx. volcanii uses a co-translational mechanism to acetylate the N-terminus of α1 and requires the N-terminal α-helix for this modification. If so, incomplete Nα-acetylation during translation of the Q2T, Q2D and Q2P variants could explain

why some of the α1 proteins in the whole population are acetylated and some are not. If the

acetylated forms of α1 have a higher affinity for incorporation into CPs, this would explain the

observed difference in the acetylated state of the assembled and unassembled populations of α1

protein. Alternatively, the observed unacetylated N1 and/or N2 forms that accumulated as α1

rings in these strains (expressing the α1 Q2T, Q2D and Q2P variants) may be degradation

intermediates in which the co-translationally added Nα-acetyl group was removed and the

resulting α1 protein has yet to be degraded. If so, modifying the N-terminal penultimate glutamine to an aspartate, threonine or proline residue may enhance the stability of the α1 protein. Although an archaeal N-end rule has yet to be described, N-terminal residues, including those in the penultimate position, are known to substantially influence protein stability in bacteria (Tobias et al., 1991) and eukaryotes (Varshavsky, 2000). Alternatively, the Nα-

acetylation of α1 is post-translational and requires CP structure including the N-terminal α-helix

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but not the initiator methionine. If so, it would be interesting to understand how the NAT

enzyme differentiates between α1 rings alone compared to α1 rings in the proteasomal CP

complex, since the site of N-terminal modification of α1 surrounds the openings on each end of

the CP cylinder.

Whether the acetylation is specific to mature Hfx. volcanii 20S proteasomes or lower

molecular mass complexes and what the function of this modification is remain to be

determined. In S. cerevisiae, which forms a single 20S proteasome of seven different α-type and

seven different β-type subunits, all seven of the α-type subunits and two of the β-type subunits

(β3 and β4) are modified by N-terminal acetylation (Kimura et al., 2000). This yeast 20S

proteasome purifies in a latent state that is activated by mild chaotropic agents such as SDS and

heat. Presumably, these agents selectively denature the N-termini of the α-type subunits which

form tightly closed constrictions on each end of the cylinder (Groll et al., 1997), resulting in an

opening of the channel of the 20S proteasome cavity. Although the role(s) of proteasomal

acetylation remains to be determined, 20S proteasomes purified in the latent state from a S.

cerevisiae nat1 mutant (which lacks the NatA activity required for acetylation of α1, α2, α3, α4,

α7 and β3) were slightly more active in catalyzing chymotrypsin-like peptidase activity than those purified from the parental strain (Kimura et al., 2000). Thus, the deletion of six of the nine

N-terminal acetyl groups of the yeast 20S proteasome (five of which are localized to the α-ring)

are speculated to change the higher order structure, possibly opening the central channel in the absence of mild chaotropic agents (Kimura et al., 2000) and/or regulatory ATPases.

Early studies suggested that archaeal 20S proteasomes are not gated. This was based on

the disordered configuration of the N-termini of the α subunits of the crystal structure of a

Thermoplasma acidophilum 20S proteasome purified from recombinant E. coli (Lowe et al.,

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1995) and the ability of archaeal 20S proteasomes to hydrolyze short peptides in the absence of

chaotrophic agents (Dahlmann et al., 1992; Maupin-Furlow et al., 1998). However, the activities

of most if not all archaeal 20S proteasomes are stimulated by heat and/or SDS (Dahlmann et al.,

1992; Maupin-Furlow et al., 1998), and deletion of residues 2 – 12 of the recombinant T. acidophilum α protein generates a central pore in the α rings evident by EM that increases the capacity of 20S proteasomes to degrade proteins with little tertiary structure (e.g., β-casein)

(Benaroudj et al., 2003; Smith et al., 2005). Based on this, the N-termini of archaeal α-type subunits do appear to function as a gate that prevents entry into 20S proteasomes of polypeptides seven-residues or larger. Thus, regulated acetylation of the Hfx. volcanii α1 and α2 subunits may control the configuration of the 20S gate and ultimately modulate substrate entry and/or the association of regulatory ATPases.

The α-type subunits from 20S proteasome enriched fractions in the haloarchaeon

Haloferax volcanii are acetylated. The acetylated (Ac1) and unacetylated (N2) form of the α1 protein were quantified and the acetylated form was shown to be in 100-fold excess. Using amino acid substitutions in the penultimate position of the α1 protein to probe whole cell N- terminal acetyltransferase and methionine aminopeptidase activity revealed that there is a more generalized acetylation pathway in Hfx. volcanii. The different α1 proteins (α1 Δ2-12, α1 Q2A,

α1 Q2D, α1 Q2P, α1 Q2S, α1 Q2T, α1 Q2V) have different acetylation and cleavage patterns.

The cells expressing the different α1 variants had a collection of phenotypes. Some of the variants hindered the growth of the organism, α1 Q2A and α1 Δ2-12, while other improved stress response to heat and hypo-osmotic stress, α1 Q2D and Q2T. Additional, while α1 Q2D, α1 Q2P, and α1 Q2T existed in whole cells as a mixture of acetylated and unacetylated forms, only acetylated proteins were found in complex with assembly 20S proteasomes. Taken together, this

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study shows that the α1 protein may play a role in stress response and acetylation could play a role in proper proteasome assembly.

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Table 3-1. Strains and plasmids used in this study Strain or Plasmid Genotype; oligonucleotide used for amplification Source Strain Escherichia coli - - + DH5α F recA1 endA1 hsdR17(rk mk ) supE44 thi-1 gyrA relA1 Life Technologies GM2163 F- ara-14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 New England galT22 mcrA dcm-6 hisG4 rfbD1 rpsL 136 dam13::Tn9 Biolabs xylA5 mtl-1 thi-1 mcrB1 hsdR2

Haloferax volcanii DS70 Wild-type isolate DS2 cured of plasmid pHV2 (Wendoloski et al., 2001) H26 DS70 ΔpyrE2 (Allers et al., 2004) GZ130 H26 ΔpsmA (Zhou et al., 2008)

Plasmid

pBAP5010 Apr Nvr; 11-kb shuttle expression vector derived from (Jolley et al., pHV2; includes P2rrn upstream of citrate synthase gene 1997) r r pJAM202 Ap Nv ; pBAP5010 containing P2rrn-psmB-his6; β-His6 (Kaczowka and expressed in Hfx. volcanii Maupin-Furlow, 2003) pJAM202c (Zhou et al., Apr; Nvr; pJAM202-derived control plasmid 2008) r r pJAM204 Ap Nv ; pBAP5010 containing P2rrn-psmA-his6; α1-His6 (Kaczowka and expressed in Hfx. volcanii Maupin-Furlow, 2003) r r pJAM809 Ap Nv ; pJAM202 containing P2rrn-hvo1862-strepII Maupin-Furlow, (KpnI site inserted upstream of StrepII coding sequence) unpublished pJAM816 Apr Nvr; pJAM809 containing psmB-strepII; β-StrepII This study expressed in Hfx. volcanii r r pJAM2510 Ap Nv ; pBAP5010 containing P2rrn-psmA-Q2P-his6; This study α1Q2P-His6 expressed in Hfx. volcanii r r pJAM2511 Ap Nv ; pBAP5010 containing P2rrn-psmA-Δ2-12-his6; This study α1Δ2-12-His6 expressed in Hfx. volcanii r r pJAM2512 Ap Nv ; pBAP5010 containing P2rrn-psmA-Q2S-his6; This study α1Q2S-His6 expressed in Hfx. volcanii r r pJAM2513 Ap Nv ; pBAP5010 containing P2rrn-psmA-Q2T-his6; This study α1Q2T-His6 expressed in Hfx. volcanii r r pJAM2514 Ap Nv ; pBAP5010 containing P2rrn-psmA-Q2Dhis6; This study α1Q2D-His6 expressed in Hfx. volcanii r r pJAM2515 Ap Nv ; pBAP5010 containing P2rrn-psmA-Q2A-his6; This study α1Q2A-His6 expressed in Hfx. volcanii pJAM2545 Apr Nvr; 797-bp PCR product blunt-end ligated into a This study

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Strain or Plasmid Genotype; oligonucleotide used for amplification Source BlpI cut and Vent treated pJAM816; β-StrepII and α1- His6 expressed in Hfx. volcanii pJAM2546 Apr Nvr; 797-bp PCR product blunt-end ligated into a This study BlpI cut and Vent treated pJAM816; β-StrepII and α1 Q2A-His6 expressed in Hfx. volcanii pJAM2547 Apr Nvr; 797-bp PCR product blunt-end ligated into a This study BlpI cut and Vent treated pJAM816; β-StrepII and α1 Q2D-His6 expressed in Hfx. volcanii pJAM2548 Apr Nvr; 797-bp PCR product blunt-end ligated into a This study BlpI cut and Vent treated pJAM816; β-StrepII and α1 Q2P-His6 expressed in Hfx. volcanii pJAM2549 Apr Nvr; 797-bp PCR product blunt-end ligated into a This study BlpI cut and Vent treated pJAM816; β-StrepII and α1 Q2S-His6 expressed in Hfx. volcanii pJAM2550 Apr Nvr; 797-bp PCR product blunt-end ligated into a This study BlpI cut and Vent treated pJAM816; β-StrepII and α1 Q2T-His6 expressed in Hfx. volcanii pJAM2551 Apr Nvr; 797-bp PCR product blunt-end ligated into a This study BlpI cut and Vent treated pJAM816; β-StrepII and α1 Q2V-His6 expressed in Hfx. volcanii pJAM2552 Apr Nvr; 764-bp PCR product blunt-end ligated into a This study BlpI cut and Vent treated pJAM816; β-StrepII and α1Δ2- 12-His6 expressed in Hfx. volcanii

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Table 3-2. Fragments of α1, α2 and β identified by QSTAR MS/MS from a trypsin digest (-T).

Mascot Proteina Res. no.b Δ Sequence Ion Score α1-T MQGQAQQQAYDR; N-acetyl; Oxidation 1 – 12 0.04 76 45.2 % (M) 13 – 22 0.01 48 GITIFSPDGR 23 – 30 0.03 44 LYQVEYAR 35 – 43 0.01 32 RGTASIGVR 44 – 55 0.03 32 TPEGVVLAADKR 56 – 68 0.03 38 SRSPLMEPTSVEK; Oxidation (M) 72 – 88 0.06 58 ADDHIGIASAGHVADAR 89 – 95 0.01 48 QLIDFAR 105 – 116 -0.02 34 YGEPIGIETLTK 150 – 163 -0.01 36 LYETDPSGTPYEWK

α2-T 12 – 22 0.02 59 GTSLFSPDGR 47.8 % 23 – 29 0.03 44 IYQVEYAR 43 – 53 0.03 61 TADGVVLAALR 54 – 67 0.04 34 STPSELMEAESIEK 104 – 115 0.03 49 YGEPIGVETLTK TITDNIQESTQSGGTRPYGASLLIGGVENGSGR 116 – 148 0.13 42 ; Deamidation (NQ) 149 – 162 0.00 45 LFATDPSGTPQEWK

β-T 21 – 36 -0.03 55 SNVFGPELGEFSNADR 37.9 % 57 – 68 0.02 45 TEEGVVLATDMR 69 – 78 0.01 47 ASMGYMVSSK; Oxidation (M) 83 – 109 0.05 65 VEEIHPTGALTIAGSVSAAQSLISSLR RGEDMSMQALSTLVGNFLR; 119 – 137 0.04 57 2 Oxidation (M) 236 – 243 0.02 44 HQNFEGLE

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Table 3-3. Fragments of α1, α2 and β identified by QSTAR MS/MS from a sequential trypsin and chymotrypsin digest (-TC).

Mascot Proteina Res. no.b Δ Sequence Ion Score α1-TC 13 – 22 -0.03 48 GITIFSPDGR 32.1 % 58 – 68 -0.06 33 SPLMEPTSVEK 72 – 88 -0.01 43 ADDHIGIASAGHVADAR 89 – 95 -0.04 43 QLIDFAR 105 – 116 -0.10 37 YGEPIGIETLTK 126 – 149 -0.06 62 TQVGGARPFGVALLIGGVENGTPR 135 – 149 -0.06 46 GVALLIGGVENGTPR

α2-TC 43 –53 -0.05 59 TADGVVLAALR 34 % 54 – 67 -0.05 48 STPSELMEAESIEK 71 – 87 -0.05 74 LDDALGAATAGHVADAR 104 – 115 -0.06 49 YGEPIGVETLTK 116 – 133 -0.05 69 TITDNIQESTSGGTRPY 134 – 148 -0.04 56 GASLLIGGVENGSGR

β-TC 57 – 68 -0.07 68 TEEGVVLATDMR 49.4 % 69 – 78 -0.05 38 ASMGYMVSSK 83 – 109 -0.04 98 VEEIHPTGALTIAGSVSAAQSLISSLR 119 – 128 -0.06 52 RGEDMSMQAL RGEDMSMQALSTL – Deamidation (Q); Phospho 119 – 131 0.06 57 (S) 129 – 137 -0.06 40 STLVGNFLR 138 – 159 -0.09 55 SGGFYVVQPILGGVDETGPHIY 214 – 235 -0.07 99 DLASGNGINIAVVTEDGVDIQR 236 – 243 -0.03 45 HQNFEGLE

a -T, trypsin digest; -TC, trypsin and chymotrypsin digested proteasomal α1, α2, and β proteins with the percent coverage of the deduced protein sequence.

bResidue number of deduced protein sequence, difference between the calculated and observed molecular mass in Da (Δ value), number of missed cleavages (Miss), Mascot ion score, and peptide sequence are reported.

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Table 3-4. Summary of MS/MS-spectral counting and integration with a calculated ratio of Ac1 to N2 forms of α1 purified from Hfx. volcanii ‘wild-type’ cells (DS70-pJAM204).

Peptide Sequence α1 form1 Mass Score E-value Integral MQGQAQQQAYDR + N-Acetyl Ac1 733.34 (2+) 87 2.4e-007 7.218 QGQAQQQAYDR N2 646.81 (2+) 69 1.3e-005 7.093 e-2 Ac1:N2 103:1

1α1 protein was purified from Hfx. volcanii cells expressing psmA-his6 (DS70-pJAM204) by Ni2+-affinity chromatography and, thus, is a survey of the population of α1 protein both unassembled and assembled into CPs.

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Table 3-5. N-terminal modifications of α1 alter its Nα-acetylation and MAP cleavage pattern in 20S proteasomes and whole cells and influence 20S proteasome-mediated peptidase activity. Relative abundance of α1 N-terminal formsa Specific activityb α1 Variant N1 N2 Ac1 Ac2 37 ˚C 65 ˚C

100 % (99 74 ± 1 363 ± 15 wild-type (1 %) %)

Δ2 – 12 (G13) 100 % 252 ± 7 1770 ± 70

Q2A 100 % 78 ± 3 621 ± 41

100 % (80 n.d. n.d. Q2D (20 %) %)

100 % (69 n.d. n.d. Q2P (18 %) (13 %) %)

Q2S 100 % 50 ± 4 421 ± 50

100 % (90 44 ± 13 370 ± 60 Q2T (10 %) %)

Q2V 100 % 16 ± 2 274 ± 25

aNα-acetylation and MAP cleavage pattern of α1 in CPs (and whole cells in parenthesis if different from CPs purified by the two-step affinity method) as determined by MS-integration and MS/MS-spectral counting of the N-terminal peptides specific for α1 protein. Spectral counting was validated by multiple reaction monitoring (MRM)-initiated detection of peptides.

bSpecific activity was determined for proteasomal CPs purified by the two-step affinity method using the chymotrypsin-like peptide substrate Suc-LLVY-AMC. Activity was monitored by the release of 7-amino-4-methylcoumarin. Assay conditions were in 20 mM Tris-HCl buffer at pH 7.2 with 2 M NaCl at the temperatures indicated. Activity was monitored in triplicate and is reported as nmols of product released ∙ min-1 ∙ mg-1 protein. n.d., not determined.

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Table 3-6. Three additional proteins identified by ESI-QTOF MS scans of proteasome-enriched samples. ORF no. pI / Amino acid position, Putative amino acid sequence of ORF a b Mr Pfam domain and description, E ( % His of total protein) (Da) value MSDHDQHYGADDHGRDQHHGEGDGDDDHGHHHHDVDDLEAAVL 4.49 / 41 – 184 aa, PF00994 molybdopterin binding, TISSTRDVDDDPAGDAIAELLHEAGHSVSVRRVVDDDYDEIQI HVO_2645 20752.38 Expect 1.3e-35 AVARMADRGDVDVTVTTGGTGVTPDDRTVEAATQLFEKTLPGF GELFRRLSYDDIGTKVVGTRATAGVVDGMPTFCLPGSENAARL

GTAEIIVPEAPHLTGLARRDAE (6.7 %) 54 – 101 aa, PF01022 HTH_5 bacterial MGSPFYNHRSDGVGGSLHCLTESNSCSARWNKLFTLPRRHAVM regulatory protein arsR family with helix-turn- TENAPNRIETSRKTIRVLDALREHGSSSVTFLARELGMNKSTV 5.96 / helix motif, Expect 1.1e-03 HNHLSTLEAEKFVVRDGTDYELSLRLLGFGGYVQHRHPLYQVA HVO_2108 32702.80 KREVHRLASQTGELANLMVEEYGQGVYLASEQGERAVDLDIYP GLRRPLHAIGLGKAILAHLPSERVDEIIEEHGLPAETDQTITD 167 – 292 aa, PF01614 IclR bacterial transcription regulator family, Expect 1.9e-39 RAELDAQLATVRDRGYAVDNEELIRGLRCVGAPIIAKDGTVLG AISVSQPVSRMNNERFTDEVPDIVQSAANVIELHTNH (4.7 %) 5 – 173 aa, PF03444 DUF293 Domain of unknown function may be distantly related to

119 HrcA heat-inducible transcriptional repressor, Expect 2.9e-67

MSSIELTSSQKTILTALINLYRDSEDAVKGEDIAAEVNRNPGT 13 – 68 aa, PF08279 HTH_11 HTH helix-turn- IRNQMQSLKALQLVEGVPGPKGGYKPTANAYEALDVDKMDEPA 4.60 / FVPLFHNDEEVEGVNVDEIDLSSVHHPELCRAEIHVQGSVREF HVO_1577 19377.70 helix domain in a wide variety of proteins, Expect 3.5e-06 HEGDKIRVGPTPLSKLVIDGTLDGKDDTSNILILRIDDMQAPV (3.4 %) GEPQH

13 – 126 aa, PF02082 Rrf2 Transcriptional regulator family related to Y_phosphatase2 and other transcription regulation families, Expect 5.1e-04 a For comparison, calculated pI and Mr of proteasomal proteins are: α1 (4.47 and 27594.16 Da), α2 (4.29 and 26727.17 Da), pre-β (4.56 and 25993.77 Da) and mature-β (4.52 and 20584.99 Da).

bDeduced amino acid sequence communicated by J. Eisen, TIGR with histidine residues underlined (April 2007 version, http://archaea.ucsc.edu/).

Table 3-7. MS/MS analysis of 20S proteasomes purified by two-step affinity purification.

Protein Residue no. MASCOT E value Sequence 1 – 12 73 3.4e-06 MQGQAQQQAYDR + Acetyl (N-term) 44 - 55 65 1.5e-05 TPEGVVLAADK α1 58 - 68 42 3.5e-03 SPLMEPTSVEK 58 - 68 46 1.4e-03 SPLMEPTSVEK + Oxidation (M) 72 - 88 83 3.5e-07 ADDHIGIASAGHVADAR 172 - 183 25 2.4e-01 GDHQEHLEENFR 12 - 22 59 5.8e-05 GTSLFSPDGR 23 - 29 35 1.5e-02 IYQVEYAR 54 - 67 64 2.6e-05 STPSELMEAESIEK α2 54 - 67 77 1.0e-06 STPSELMEAESIEK + Oxidation (M) 71 - 87 67 1.4e-05 LDDALGAATAGHVADAR 104 - 115 68 4.5e-06 YGEPIGVETLTK 149 - 162 68 7.5e-06 LFATDPSGTPQEWK β 57 - 68 48 7.9e-04 TEEGVVLATDMR 57 - 68 45 1.7e-03 TEEGVVLATDMR + Oxidation (M)

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Table 3-8. MS/MS-identification of the 30-kDa isoform chain predominant in Hfx. volcanii cells expressing the α1 Q2T variant as α1 protein. a.

Protein Residue no. MASCOT E value Sequence 13 - 24 44 1.3e-05 GITIFSPDGR α1 58 - 68 52 2.7e-03 SPLMEPTSVEK + Oxidation (M) 72 - 88 49 4.4e-03 ADDHIGIASAGHVADAR b.

Protein Residue no. MASCOT E value Sequence 1 - 12 33 2.0e-03 MTGQAQQQAYDR + Acetyl (N-term) α1 25 - 32 30 1.4e-03 LYQVEYAR 58 - 68 42 5.5e-03 SPLMEPTSVEK 72 - 88 49 1.7e-05 ADDHIGIASAGHVADAR c.

Protein Residue no. MASCOT E value Sequence 13 - 24 34 5.5e-03 GITIFSPDGR α1 25 - 32 61 3.7e-05 LYQVEYAR 58 - 68 57 2.2e-05 SPLMEPTSVEK 72 - 88 45 1.9e-06 ADDHIGIASAGHVADAR d.

Protein Residue no. MASCOT E value Sequence 25 - 32 61 4.1e-03 LYQVEYAR α1 58 - 68 37 1.7e-03 SPLMEPTSVEK + Oxidation (M) 72 - 88 60 2.2e-03 ADDHIGIASAGHVADAR

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Figure 3-1. Isoforms of 20S proteasomal proteins. 20S proteasomal proteins associated with α1- His were purified from recombinant Hfx. volcanii and separated by 2-DE (45 and 25 μg protein; A and B, respectively). Isoforms of α1 and/or α1-His (a to h), β (i to m) and α2 (n and o) proteins were identified by MS analysis of protein spots. A) Molecular mass standards on left and pI range of 3.9 to 5.1 on top are indicated. B) Magnified region of 2-DE gel.

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Figure 3-2. MS/MS Fragmentation of Acet-MoxQGQAQQQAYDR, an N-terminal fragment of the α1 protein of 20S proteasomes. The m/z of the doubly charged parental ion was 741.34 Da which corresponds to the mass of the defined fragment plus oxidation (16.00 Da for methionine sulfoxide; Mox) and acetylation (42.01 Da; Acet-) of the N- terminus with a delta value < 0.1 Da. The individual Mascot ion score was 76 with an E value of 5.5e-7. Labeled peaks include b- and y-ions as well as dominate internal ion fragments. Loss of ammonia (*, 17.03 Da) is indicated.

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Figure 3-3. MS/MS Fragmentation of Acet-MNRN(D)DKQAYDRGTSLFSPDGRIYQVE, an N- terminal fragment of the α2 protein of 20S proteasomes. The peptide was generated from a Glu-C endoprotease digest of α2 protein purified via association with α1-His. The peptide had an acetyl group at the N-terminus (Acet-) and a deamidation at the asparginine residue (N(D)) in the fourth position. The m/z of the parent ion was 776.62 Da. The individual Mascot ion score was 24 with an E value of 0.1. Labeled peaks include b- and y-ions.

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Figure 3-4. 20S proteasome CPs are rapidly isolated by two-step affinity purification from Hfx. volcanii. A) SDS-PAGE gel of cell lysate (lane 1) and chromatography fractions after purification by Ni2+-Sepharose (lane 2) and CPs (lane 3) isolated after the two- step affinity purification using Ni2+-Sepharose and StrepTactin chromatography. Molecular mass standards indicated on left. B) Size exclusion chromatogram of ‘two-step’ purified CPs. Proteasomal CPs were purified from Hfx. volcanii H26- pJAM2545 cells expressing the synthetic psmB-strepII psmA-his6 operon encoding β and α1 subunits of proteasomal CPs with C-terminal StrepII and His6 affinity tags, respectively.

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Figure 3-5. Quantitative mass spectrometry revealed α1 proteins with an acetylated N-terminal initiator methionine (Ac1) were dominant in ‘wild-type’ cells. MS-spectral integration was performed to quantify the various N-terminal forms of α1 protein after purification by Ni2+-Sepharose from Hfx. volcanii ‘wild-type’ cells (DS70- pJAM204). A) Isotopic envelope of the MS-scan for a 733.79, 2+ (m/z) peptide corresponding with the MS/MS of the Ac1 peptide. B) MS-scan for a 647.17, 2+ (m/z) peptide corresponding with the MS/MS of the N2 peptide. C) Total MS-scan for the MRM selecting for two peptides 733.79, 2+ for Ac1 and 647.17, 2+ for N2.

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Figure 3-6. Variant α1 Q2D, Q2P, and Q2T proteins accumulate in cells as heptameric rings. A) SDS-PAGE of cell lysate and Ni2+-Sepharose fractions of Hfx. volcanii GZ130 (∆psmA) cells expressing α1 wild-type (lanes 1 and 2), α1 Q2D (lanes 3 and 4) and Q2T (lanes 5 and 6), respectively. B) Size exclusion (Superose 6) chromatography of Ni2+-Sepharose fractions purified from GZ130 strains expressing α1 Q2P (―) and α1 wild-type (….). Results similar to α1 Q2P were observed for α1 Q2D and α1 Q2T proteins (data not shown). C) qRT-PCR analysis of the psmA-specific transcripts of Hfx. volcanii GZ130 cells expressing the psmA (α1)-his6 and psmA-Q2T (α1 Q2T)- his6 genes in trans (see methods for details).

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Figure 3-7. Variant α1 proteins either rescue the hypoosmotic stress phenotype of a chromosomal psmA deletion or exacerbate it. Hfx. volcanii GZ130 (∆psmA) with plasmids pJAM204 (α1) (●), pJAM202c (vector alone) (○), pJAM2514 (α1 Q2D) (■), pJAM2513 (α1 Q2T) (▲), pJAM2515 (α1 Q2A) (□) and pJAM2511 (α1 ∆2-12) (∆). For inoculums, cells were grown in ATCC 974 medium with novobiocin. Cells were streaked from -80 ºC glycerol stocks onto fresh plates, grown twice to log-phase in 2 ml medium and used as an inoculum for final analysis of growth in ATCC 974 medium with novobiocin and NaCl concentrations from 1 to 2.25 M as indicated. Each subculture was inoculated to a final OD600 of 0.01 to 0.02. Growth is represented as a percentage relative to growth for that strain at 2.25 M NaCl (100 % growth was ~1 × 109 CFU·ml-1). Experiments were performed in triplicate and the mean ± S.D. was calculated.

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Figure 3-8. Hfx. volcanii cells synthesizing the α1 Q2T variant displayed a thermotolerant phenotype and A) grew to higher cell density and B) at higher rates and that those expressing wild-type α1. Hfx. volcanii GZ130 (∆psmA) with plasmids pJAM204 (α1) (●) and pJAM2513 (α1 Q2T) (○).

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Figure 3-9. Two-dimensional gel electrophoresis of cell lysate of Hfx. volcanii GZ130 expressing α1 wild-type (upper) compared to α1 Q2T (lower). The 30-kDa isoform chain, predominant in the GZ130 strain expressing α1 Q2T (vs. wild-type) and identified as α1 protein by MS/MS is indicated (a, b, c, d).

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CHAPTER 4 N-TERMINAL PROTEOME AND ACETYLTRANSFERASES IN HALOFERAX VOLCANII

Introduction

In the yeast Saccharomyces cerevisiae, Nα-acetylation is catalyzed by at least three N- terminal acetyltransferases (NATs), NatA, NatB and NatC which contain Ard1p, Nat3p and

Mak3p catalytic subunits, respectively (Polevoda and Sherman, 2003a). These enzymes act co-

translationally on separate groups of substrates, each with degenerate motifs (Polevoda et al.,

1999; Polevoda and Sherman, 2000; Polevoda and Sherman, 2003b; Polevoda et al., 2003).

NatA typically acts on N-terminal residues such as Ser, Ala, Gly and Thr after the initiator Met is

removed co-translationally by methionine aminopeptidase (Moerschell et al., 1990). In contrast,

NatB and NatC often modify the N-terminal initiator Met when it precedes either Glu, Asp, Asn

or Met residues (NatB) or the bulky hydrophobic residues Ile, Leu, Trp or Phe (NatC).

In E. coli, three NATs have been identified (RimI, RimJ and RimL); however, these

enzymes are only known to modify the ribosomal proteins S18, S5 and L12 with N-terminal Ala-

Arg-, Ala-His- and Ser-Ile- residues, respectively (Yoshikawa et al., 1987; Tanaka et al., 1989).

There is no evidence that the Rim proteins act co-translationally. Instead, at least the RimL enzyme appears to act post-translational based on the finding that the ribosomal proteins L7 and

L12 have identical N-termini and yet only L12 is acetylated (Tanaka et al., 1989). Thus, RimL is thought to recognize a certain protein structure and not just the extreme N-terminus of the substrate protein.

Originally, only a few archaeal proteins were known to be modified by Nα-acetylation including: glutamate dehydrogenase-2 (Maras et al., 1992) and Alba1 chromatin proteins of

Sulfolobus solfataricus (Bell et al., 2002); ribosomal proteins S7P, L31e, and S19P of

Haloarcula marismortui (Kimura et al., 1989; Hatakeyama and Hatakeyama, 1990; Klussmann

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et al., 1993); and halocyanin of Natronobacterium pharaonis (Mattar et al., 1994). In addition

based on our studies the α1 and α2 subunits of 20S proteasomes from Hfx. volcanii as well as the

HrcA-like HVO_1577 were also acetylated (presented in Chapter 3) (Humbard et al., 2006).

Even with identification of three additional N-terminally acetylated proteins from Hfx. volcanii,

it was unclear whether this modification is common or rare in archaea or Hfx. volcanii.

Nothing is known regarding the mechanism or function of N-terminal acetylation in

archaea. The majority of these modifications are on N-terminal serine residues, exposed by a

presumed methionine aminopeptidase and followed by either Ser or Ala residues (Ser-Ser/Ala-).

Halocyanin represents an example for a relatively large group of predicted archaeal lipoproteins

in which an internal Cys appears to be modified by a diphytanyl (glycerol)diether, converted to

the N-terminal residue via cleavage by a signal peptidase II-like protease, and then Nα-acetylated

(Mattar et al., 1994). Glutamate dehydrogenase-2 (GDH-2) is the single example (prior to this study) of an archaeal protein acetylated directly on its initiating N-terminal methionine residue

(Maras et al., 1992). Although the presence of six internal N-epsilon-methyl-lysine residues is speculated to be responsible for the extreme thermostability of GDH-2 (Maras et al., 1992), the role of the N-acetyl group remains to be determined.

Regarding the archaeal enzymes which catalyze Nα-acetylation, recent phylogenetic

analysis suggests a family of uncharacterized bacteria and archaea acetyltransferases (BAAs)

may be responsible for this type of modification (Polevoda and Sherman, 2003a; Polevoda and

Sherman, 2003b). The proteasomal subunits α1 and α2 from Hfx. volcanii are both acetylated on

the N-terminal methionine and followed by bulky polar amino acids (glutamine and asparagine)

(Humbard et al., 2006). This motif is closer to the NatB motif of enzymatic activity found in

yeast cells (Polevoda and Sherman, 2003b). In contrast to Nα-acetylation in which the modifying

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enzymes and function(s) remain to be elucidated, acetylation of Alba1 on an internal lysine

residue (Lys16 of the mature protein) is known to be mediated by Sir2 (Bell et al., 2002) and alleviate repression of mini-chromosome maintenance activity in S. solfataricus (Marsh et al.,

2006).

Recently, a systematic study of the N-terminal peptides from the haloarchaea species

Halobacterium salinarum and Natronomonas pharaonis has demonstrated that there is likely a generalized Nα-acetylation pathway at work in the haloarchaea (Falb et al., 2006; Aivaliotis et

al., 2007).

To further investigate this, we examined the N-terminal proteome of Hfx. volcanii and measured the frequency of methionine aminopeptidase cleavage and Nα-acetylation. The

frequency of modifications were compared to the occurance in distantly related Hb. salinarum and N. pharaonis. In addition, genomic and phylogenetic studies were performed to examine the function different acetyltransferases may serve in Hfx. volcanii physiology.

Results and Discussion

Phylogenetic Distribution of GNAT Family in Haloferax volcanii

Phylogenetic analysis of acetyltransferase homologs from a variety of organisms reveals seven different groups: Ard1p, Nat3p, Mak3p, Nat5p, Camello, Bacterial and Archaeal acetyltransferases (BAA), and lysine acetyltransferases (Figure 4-1). Three of these families,

Ard1p, Nat3p, and Mak3p, are related to the dominant yeast N-terminal acetyltransferase activities (NatA, NatB, and NatC). The Camello family is most likely related, distantly, to the

Mak3p family, but examples of the Camello1 and Camello2 family are currently restricted to mouse, rats, Xenopus, and humans (Popsueva et al., 2001; Polevoda and Sherman, 2003b). Both the Nat5p and Bacterial and Archaeal acetyltransferase families have no known substrates or function and the lysine acetyltransferases represent a distant family of unrelated function.

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Interestingly, Nat5p is part of the NatA complex, comprised of Nat1p and the catalytic subunit

Ard1p (Gautschi et al., 2003).

Out of the three major families of N-terminal acetyltransferases that are responsible for the

majority of the activity in yeast, Ard1p, Nat3p, and Mak3p, Hfx. volcanii only encodes one clear

member of the Nat3p family, HVO_2320 (Figure 4-1). Interestingly, HVO_2320 is one of the

only acetyltransferase genes that could not be knocked out in Hfx. volcanii using standard

methods (Zhou et. al., unpublished). Cells with a conditional knock-down constructs of

HVO_2320 using a PtnaA promoter fusion had severely retarded growth and eventually lost the

fusion (Zhou et. al., unpublished).

The Nat5p family of acetyltransferases is a relatively new and mysterious family. Hfx.

volcanii encodes several putative acetyltransferases that belong to this family, HVO_1972,

HVO_0201, HVO_2930, HVO_2423, HVO_2804, HVO_2326, HVO_2795, and HVO_2734

(Figure 4-1)(Table 4-2). There is no current data on the substrate specificity of this family of acetyltransferases. However, Nat5p appears in complex with Ard1p and Nat1p of NatA. Nat5p subunits are archored to the ribosome through Nat1p, similar to Ard1p (Gautschi et al., 2003;

Arnesen et al., 2006). Homologues of Nat5p have been identified in yeast, humans, and fruit flies. Based on sequence similiarity, Nat5p is believed to have acetyltransferase activity

(Arnesen et al., 2006). Efforts to identify and isolate substrates in yeast have been unsuccessful.

In addition to Hfx. volcanii proteins and Nat5p from yeast, the Salmonella typhimurium YgjM was also present in this clad. It is interesting that Hfx. volcanii acetyltransferases are so heavily concentrated in this family. Perhaps the haloarchaea will be a good system to study the mechanism and effects these different genes have on acetylation in the cell. All eight of the

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acetyltransferases in this family encoded by Hfx. volcanii have been deleted individually from the chromosome (Zhou et. al., unpublished).

Members of the uncharacterized BAA N-terminal acetyltransferase family (e.g.

HVO_2709, HVO_1954) make up a well isolated branch of the phylogenetic tree (Figure 4-1).

There is currently no data that shows that these proteins have activity, acetyltransferase and otherwise. Therefore, there are no known substrates or any in vivo role purposed for these enzymes. These proteins are distantly related to the Rim proteins, specifically RimI, of

Escherichia coli. RimI acetylates a ribosomal protein in E. coli. Out of all the Rim proteins,

RimI shares the most similarity to eukaryotic N-terminal acetyltransferases (Polevoda and

Sherman, 2003b). Likewise, the Hfx. volcanii proteins HVO_2709 and HVO_1954 share the most homology with RimI and other Rim proteins.

Reversible post-translational modification of primary amines (lysine residues) by acetylation is common in eukaryotes, bacteria, and archaea. Hfx. volcanii encodes for three

separate protein acetyltransferases that act on lysine residues, HVO_1756, HVO_1821, and

HVO_2888. There is an additional putative Gcn5-family protein acetyltransferase, HVO_2886, that is proximal to HVO_2888 (Figure 4-1). All four of the different lysine acetyltransferases belong to the Gcn5 family but HVO_2888 also shares high-homology to the yeast Elp3 (Altman-

Price and Mevarech, 2009). Unlike N-terminal acetylation, acetylation on ε-amino groups of lysine residues is reversible. The reverse reaction, or deacetylation is carried out by Sir2-family

HVO_2194 and HdaI-family HVO_0522 deacetylases. The proposed target of these different acetyltransferases and deacetylases are DNA binding proteins such as Alba (Bell et al., 2002;

Wardleworth et al., 2002; Altman-Price and Mevarech, 2009). Acetylation and deacetylation of

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Alba affects its affinity for DNA. Parallels are drawn between histone acetylation, of the H3 /

H4 histone proteins, and Alba (Zhang et al., 2003b; Altman-Price and Mevarech, 2009).

There are two annotated ORFs in the Hfx. volcanii genome that did not fit into the different acetyltransferase families, HVO_2677 and HVO_2510. The N-terminal domain of HVO_2510 aligns well with the GNAT Pfam but the C-terminus does not show any significant homology to any other proteins. HVO_2677 is a short protein, only 82 amino acids in length, and shows only weak homology to the Gcn5-family of acetyltransferases (Table 4-2).

Other Nα-acetylated Proteins in Haloferax volcanii

Our initial observations indicated that the frequency of N-terminal acetylation in Hfx.

volcanii may be more common than is seen in bacterial species such as E. coli. As an outgrowth

of the study of 20S proteasomes in Hfx. volcanii, we discovered a contaminating protein

HVO_1577, a putative transcriptional regulator, was also N-terminally acetylated. Interestingly,

this protein was found to have an N-terminal serine residue modified by an acetyl group based on

the detection of a modified tryptic peptide spanning residues 2 to 11 (Acet-SSIELTSSQK)

(Figure 4-3). The N-terminal serine residue suggests the substrate is first cleaved by a methionine aminopeptidase and then acetylated at an N-terminal serine residue of a Ser-Ser/Ala- motif (similar to eukaryal NatA substrates).

In contrast to HVO_1577, acetylation at initiating N-terminal methionine, similar to the α1 and α2 proteins of Hfx. volcanii 20S proteasomes is less common among archaea, yet consistent with the S. solfataricus GDH-2. The consensus of these three archaeal proteins is Acetyl-Met-

(Glu/Gln/Asn)- and, thus, somewhat similar to eukaryal NatB substrates. Regarding the Hfx. volcanii α1, a mixed population of N-terminal acetylated and non-acetylated proteins (the latter of which appears to be cleaved by a methionine aminopeptidase) was detected, suggesting these modifications are regulated or vary between complexes of different quaternary structure.

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To generate a more comprehensive understanding of Nα-acetylation in Hfx. volcanii, a

variety of methods were used to separate cellular proteins and these fractions were analyzed by

MS (Kirkland et al., 2008b). Based on this shotgun proteomics approach, nearly 30 % of soluble

protein was found to be N-terminally acetylated. The Hfx. volcanii proteomic dataset was

systematically searched for N-terminal peptides and their derivatives including those modified

co- and/or post-translational. Overall, 297 unique peptides were identified that mapped to the N-

termini of 236 proteins, representing 18 % of the MS/MS-detected proteome (Table 4-3). None

of the MS/MS-detected peptides were formylated, consistent with previous findings that archaea

like eukaryotes initiate translation with methionine, in contrast to bacteria which use formyl-

methioinine (Ramesh and RajBhandary, 2001). Instead, the majority of N-terminal peptides

detected in the Hfx. volcanii proteome had patterns consistent with modification by methionine

aminopeptidase and/or N-terminal aminotransferase. In fact, over 70 % of the proteins with

MS/MS-detectable N-termini included peptides in which the initiator methionine was removed

(N2) and/or the initiator methionine and/or penultimate (second) residue of the deduced

polypeptide was Nα-acetylated (Ac1 and Ac2, respectively) (Table 4-3). The remaining ~30 % of

the proteins detected by MS/MS had retained their initiator methionine and were not modified

(N1). Multiple N-terminal peptide variants were detected for 22 % of the 236 total proteins. Most of these variants were apparent processing intermediates (i.e., mixtures of N1 and N2, N1 and

Ac1, N1 and Ac2, N2 and Ac2 or N1, N2 and Ac2). Only a small percentage (5.5 %) of the 236 total proteins with MS/MS-detected N-termini appeared to be protein isoforms including mixtures of N2 and Ac1 as well as Ac1 and Ac2 (Table 4-3).

Although Nα-acetylation is relatively rare in Bacteria with only a few protein

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examples (Isono and Isono, 1980; Cumberlidge and Isono, 1979; Arai et al., 1980), it is

becoming apparent that this type of modification is common among Archaea (at least

haloarchaea) (Aivaliotis et al., 2007; Falb et al., 2006) as well as Eukarya (Polevoda and

Sherman, 2003b). Over one-fourth (29 %) of the proteins with MS/MS-detectable N-termini

were Nα-acetylated in the Hfx. volcanii proteome (Table 4-3). Most of these Nα-acetylated proteins were singly modified with a relatively equal ratio (30:33) of Ac1 to Ac2 forms.

Interestingly, of the five proteins with both Ac1 and Ac2 isoforms, three were previously found to accumulate at high levels when Hfx. volcanii is grown in the presence of the irreversible proteasome inhibitor clasto-lactacystin-β-lactone (Kirkland et al., 2007). These included

HVO_0860, HVO_1545 and HVO_2784 annotated as a SufB-like FeS assembly protein, dihydroxyacetone kinase L subunit, and RpsM ribosomal protein S13p/S18e, respectively (Table

4-3). Although the N-end rule (in which the half-life of a protein is determined by its N-terminal

residue) is conserved in Bacteria and Eukarya (Mogk et al., 2007); its function including the

relationship of protein stability to Nα-acetylation is not known in Archaea. Thus, the relationship

of these Ac1 and Ac2 isoforms to protein stability and susceptibility to proteasome-mediated

degradation is highly speculative.

For the Nα-acetylated proteins, the majority (nearly 80 %) of those in an Ac2 form

appeared to products of a NatA-like acetyltransferase, which in yeast preferentially transfers

acetyl groups to the α amino group of small N-terminal residues after the initiator methionine has

been removed (Polevoda et al., 2003). In addition, a number of the Ac1 modified proteins

detected in the Hfx. volcanii proteome appeared to be a result of a yeast-like NatB

acetyltransferase activity in which the initiator methionine residue preceding a penultimate Asp,

Glu, Asn or Met was Nα-acetylated (Polevoda et al., 2003; Huang et al., 1987). However, none

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of Nα-acetylated peptides detected in the Hfx. volcanii proteome were related in N-terminal sequence to products of the yeast NatC acetyltransferase (i.e., Ac1 forms with penultimate Ile,

Leu, Trp or Phe residues) (Polevoda et al., 2003). Instead, the majority of Ac1 modified proteins

(over 80 %) had small penultimate residues suggesting an additional acetyltransferase activity distinct from the yeast-like NatA, NatB and NatC is present.

Occurrence of Different Amino Acids in the Penultimate and Antepenultimate Positions

The Hfx. volcanii genome does have coding capacity for a methionine aminopeptidase

(HVO_2600). The frequency of the second, penultimate, amino acid of the entire, deduced,

proteome favors small amino acids such as serine (21 %), threonine (14 %), alanine (10 %),

valine (5 %), but there is also a relatively frequent occurrence of larger, charged, amino acids

such as aspartic acid (6 %) and arginine (7 %). These proteins with small amino acids in the

second position are all potentially MAP substrates. In the baseline proteome data, the relative

abundance of Hfx. volcanii proteins with small residues (i.e., Gly, Ala, Pro, Val, Ser, Thr) in the penultimate position of their deduced primary sequence was significantly greater for N-terminal

MS/MS-detected peptides modified by methionine aminopeptidase and/or N-terminal aminotransferase (87 %) compared to those that were unmodified (N1 alone) (32 %) (Table 4-3).

In particular, the penultimate residue that was dominant for the modified proteins was serine (at

40 % of the total proteins modified) compared to its limited presence in this same position of unmodified proteins (16 % of total proteins unmodified). This bias, of small residues at the penultimate position, contrasts with the theoretical Hfx. volcanii proteome in which about 60 % of the deduced proteins have small residues and 21 % have serine residues at this position.

Based on analysis of the MS/MS-detected N-terminal peptides, initiator methionine removal occurred nearly exclusively when the penultimate residue of the protein was small, consistent with the substrate preferences of methionine aminopeptidases characterized from

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Bacteria, Archaea and Eukarya (Ben Bassat et al., 1987; Tsunasawa et al., 1985; Huang et al.,

1987; Miller et al., 1987) and similar to other archaeal proteomes (Falb et al., 2006). Of the 16 proteins detected in the Hfx. volcanii proteome that were exceptional to this rule (i.e., in which the initiator methionine was removed from a bulkier residue), most resulted in exposure of an acidic N-terminal residue (i.e., Asp or Glu) (Table 4-3) and, thus, may be a reflection of the increased number of acidic residues used for hydration of halophilic proteins in high salt

(Mevarech et al., 2000).

Conclusions

N-terminal processing involves multiple steps activities, most notably the activities of methionine aminopeptidases and N-terminal acetyltransferases. Following up on the observation that both α-type subunits from 20S proteasomes in Hfx. volcanii and HVO_1577 were N- terminally acetylated, a large proteomic survey of N-terminal peptides from Hfx. volcanii reveals evidence of a generalized protein acetylation pathway in the haloarchaeon. N-termini were identified from 236 different proteins. Over 70 % of those proteins identified were modified by a methionine aminopeptidase and 21 % were modified by an N-terminal acetyltransferase.

Several proteins had multiple N-terminal peptides identified, 297 peptides for 236 proteins.

These data are in good agreement with the recently published N-terminal proteomes of two other haloarchaea (Falb et al., 2006; Aivaliotis et al., 2007). Hfx. volcanii encodes for acetyltransferases that align with Nat3p, Nat5p, BAA, and lysine acetyltransferase families.

Although no activity has been demonstrated for any of these enzymes, HVO_2320, a member of the Nat3p family, may be essential based on the finding that a conditional knock-down displays severely inhibited growth under standard laboratory conditions (Zhou, unpublished). These data indicate that N-terminal acetylation of proteins could be an important cellular process in the haloarchaea.

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Table 4-1. Strains and plasmids used in this study

Strain or Plasmid Genotype; oligonucleotide used for amplification Source Strain Escherichia coli - - + DH5α F recA1 endA1 hsdR17(rk mk ) supE44 thi-1 gyrA Life Technologies relA1 GM2163 F- ara-14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 New England Biolabs galT22 mcrA dcm-6 hisG4 rfbD1 rpsL 136 dam13::Tn9 xylA5 mtl-1 thi-1 mcrB1 hsdR2 - - - BL21 (DE3) F ompT [lon] hsd SB (rB mB ) (an E. coli B strain) Novagen with DE3, a λ prophage carrying the T7 RNA polymerase gene

Haloferax volcanii H26 DS70 ΔpyrE2 (Allers et al., 2004) GZ130 H26 ΔpsmA (Zhou et al., 2008) GZ110 H26 ΔHVO_2677 (Zhou, unpublished) GZ111 H26 ΔHVO_2510 (Zhou, unpublished) GZ115 H26 ΔHVO_1756 (Zhou, unpublished) GZ116 H26 ΔHVO_2930 (Zhou, unpublished) GZ121 H26 ΔHVO_2623 (Zhou, unpublished) GZ122 H26 ΔHVO_2734 (Zhou, unpublished) GZ123 H26 ΔHVO_2795 (Zhou, unpublished) GZ124 H26 ΔHVO_2886 (Zhou, unpublished) GZ125 H26 ΔHVO_0201 (Zhou, unpublished) GZ127 H26 ΔHVO_1821 (Zhou, unpublished) GZ128 H26 ΔHVO_2804 (Zhou, unpublished) GZ129 H26 ΔHVO_2326 (Zhou, unpublished) GZ135 H26 ΔHVO_2423 (Zhou, unpublished) H26 PtnaA HVO_2320 (Zhou, unpublished) MH2085 H26 ΔHVO_2709 This study. MH2086 H26 ΔHVO_1954 This study.

Plasmid pJAM2018 Apr; pTA131 containing HVO_2677 with ~500 bp of (Zhou, unpublished) genomic DNA flanking 5’ and 3’ of the HVO_2677 coding region pJAM2019 Apr; pTA131-derived HVO_2677 suicide plasmid (Zhou, unpublished) pJAM2020 Apr; pTA131 containing HVO_2510 with ~500 bp of (Zhou, unpublished) genomic DNA flanking 5’ and 3’ of the HVO_2510 coding region pJAM2021 Apr; pTA131-derived HVO_2510 suicide plasmid (Zhou, unpublished) pJAM2014 Apr; pTA131 containing HVO_1756 with ~500 bp of (Zhou, unpublished) genomic DNA flanking 5’ and 3’ of the HVO_1756 coding region

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Strain or Plasmid Genotype; oligonucleotide used for amplification Source pJAM2015 Apr; pTA131-derived HVO_1756 suicide plasmid (Zhou, unpublished) pJAM2016 Apr; pTA131 containing HVO_2930 with ~500 bp of (Zhou, unpublished) genomic DNA flanking 5’ and 3’ of the HVO_2930 coding region pJAM2017 Apr; pTA131-derived HVO_2930 suicide plasmid (Zhou, unpublished) pJAM2034 Apr; pTA131 containing HVO_2623 with ~500 bp of (Zhou, unpublished) genomic DNA flanking 5’ and 3’ of the HVO_2623 coding pJAM2035 Apr; pTA131-derived HVO_2623 suicide plasmid (Zhou, unpublished) pJAM2036 Apr; pTA131-derived HVO_2795 suicide plasmid (Zhou, unpublished) pJAM2037 Apr; pTA131 containing HVO_2795 with ~500 bp of (Zhou, unpublished) genomic DNA flanking 5’ and 3’ of the HVO_2795 coding region pJAM2039 Apr; pTA131 containing HVO_2886 with ~500 bp of (Zhou, unpublished) genomic DNA flanking 5’ and 3’ of the HVO_2886 coding region pJAM2040 Apr; pTA131-derived HVO_2886 suicide plasmid (Zhou, unpublished) pJAM2048 Apr; pTA131 containing HVO_0201 with ~500 bp of (Zhou, unpublished) genomic DNA flanking 5’ and 3’ of the HVO_0201 coding region pJAM2049 Apr; pTA131-derived HVO_0201 suicide plasmid (Zhou, unpublished) pJAM2041 Apr; pTA131 containing HVO_1821 with ~500 bp of (Zhou, unpublished) genomic DNA flanking 5’ and 3’ of the HVO_1821 coding region pJAM2042 Apr; pTA131-derived HVO_1821 suicide plasmid (Zhou, unpublished) pJAM2043 Apr; pTA131 containing HVO_2804 with ~500 bp of (Zhou, unpublished) genomic DNA flanking 5’ and 3’ of the HVO_2804 coding region pJAM2044 Apr; pTA131-derived HVO_2804 suicide plasmid (Zhou, unpublished) pJAM2045 Apr; pTA131 containing HVO_2326 with ~500 bp of (Zhou, unpublished) genomic DNA flanking 5’ and 3’ of the HVO_2326 coding region pJAM2046 Apr; pTA131-derived HVO_2326 suicide plasmid (Zhou, unpublished) pJAM2050 Apr; pTA131-derived HVO_2423 suicide plasmid (Zhou, unpublished) pJAM2516 Apr; pTA131 containing HVO_2709 with ~500 bp of (Zhou, unpublished) genomic DNA flanking 5’ and 3’ of the HVO_2709 coding region pJAM2518 Apr; pTA131-derived HVO_2709 suicide plasmid (Zhou, unpublished) pJAM2519 Apr; pTA131 containing HVO_1954 with ~500 bp of (Zhou, unpublished) genomic DNA flanking 5’ and 3’ of the HVO_1954 coding region pJAM2520 Apr; pTA131-derived HVO_1954 suicide plasmid This study. pJAM2056 Apr, Nvr; Insert gene HVO_2320 into NdeI and KpnI This study. of pJAM809, Hvo2320-StrepII r pJAM2058 Ap ; pTA131 insert 323 bp PtnaA into XbaI and NdeI This study.

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Strain or Plasmid Genotype; oligonucleotide used for amplification Source site of pJAM2056, in front of hvo 2320 r pJAM2060 Ap ; pTA131 insert about 500 bp upstream PtnaA This study. HVO_2320

143

Figure 4-1. GNAT protein phylogenetic tree. Haloferax volcanii proteins compared to bacterial and eukaryotic homologues. Ap Aeropyrum pernix, Aa Aquifex aeolicus, At A. thaliana, Af Archaeoglobus fulgidus, Ce C. elegans, Cco Campylobacter coli, Cpn Chlamydia pneumoniae, Dr Deinococcus radiodurans, Dm D. melanogaster, Hae Haemophilus influenzae, Hn Halobacterium sp. NRC-1, Hv Haloferax volcanii, Hs H. sapiens, Mth Methanobacterium thermoautotrophicum, Mj Methanococcus jannaschii, Mm M. musculus, Pae Pseudomonas aeruginosa, Pa Pyrococcus abyssi, Ph Pyrococcus horikoshii, Rn Rattus norvegicus, Rr R. rattus, Sc S. cerevisiae, Sty Salmonella typhimurium, Sp S. pombe, Sty Streptomyces coelicolor, Ss S. solfataricus, Syn Synechocystis sp., Ta Thermoplasma acidophilum, Up Ureaplasma urealyticum.

144

145

Table 4-2. Methionine aminopeptidase and examples of acetyltransferase homologs proposed to play a role in N-terminal cleavage and acetylation of Hfx. volcanii proteins.

ORF no. Pfam domain, description, E value; Putative amino acid sequence of ORFa NCBI Conserved Domain Database or Clusters of Orthologous Groups, description, E value PF00583, acetyltransferase (GNAT) MRVLDAGLLEADAGEIRDRLAAGDCLVAEASGRVVGALALDGDYI family, Expect 1e-06 EAVAVSPSRRGRGVGTQLVRAALDRRGRLVVDFDGGVKPFYESLG HVO_0201- FDIEDRSDDEGRFRGVLRDE TG_gha_455 COG3153, Predicted acetyltransferase Expect 3e-04 PF00583, acetyltransferase (GNAT) MGETDVDRPNGASGSDGFWDASRCVGTPQCPPRCPRFVSKDGRGL family, Expect 4e-11 VVRPYDDADRDALAEMYRDYDPDARSMGLPPTDEAHITEWLSNLV HVO_1756- DRGRNFVAVADRGIVGHAAYVASDDPEPELAVFVHQDSHDRGIGT TG_gha_455 COG0456, RimI-type acetyltransferases, ELCRHVVADAADAGREALVLHVAPDNQRAVHVYRELGFETVSSDG

146 ADTKMRLDLTPPVPPAARPTTGTESRG Expect 5e-07 PF00583, acetyltransferase (GNAT) MSDRTFSDAVADPYDAPPLSFTDGEDRTIEVRAHDGSDEELEALV family, Expect 8e-15 EMYDAFDPSDRAQGIPPGREDRIRDWLENILDEDCLNVIAWDGDE HVO_1821- VAGHATLVPDGDAYELAIFVHQTYQRAGIGTRLIKALLGYGRVSG TG_gha_455 VEKVWLTVERWNRAAVGLYQTVGFETSDAESFELEMTIRLAEPPD COG0456, RimI-type acetyltransferases, DEGG Expect 4e-06 PF00583, acetyltransferase (GNAT) MVVRRVDRVDLLDVLRIERTCFSEPWPYSAFEMFVDEPAFLVAAR family, Expect 1.3e-20 GDEVLGYVVADVMPNHGNDIGHVKDLAVRPEARGNGLGRQLLVQS HVO_1954- LTAMAIAGATVVKLEVRVSNDPAIGLYRSLGFEPARRVPSYYGDG TG_gha_455 COG0456, RimI-type acetyltransferases, EDAYVMVLDVAEWQRA Expect 5e-18 PF00583, acetyltransferase (GNAT) MRVSSAAFSELDDLVELWVELAADQRASGSHLRAEANRTPIREAL family, Expect 3.9e-23 ARHVAADGVLVAREGDVSNGGTEVGSSAEDADADGLVGFVMFDIE ORF02176- AGAYEQDATRGMVRNLFVRPAYRDAGVGTRLLAAAEDALADAGVD TG_gha_446 AVALDVLADNEAARRFYRRHGYRPHRVELEKSMRSDTHSKEDG

ORF no. Pfam domain, description, E value; Putative amino acid sequence of ORFa NCBI Conserved Domain Database or Clusters of Orthologous Groups, description, E value PF00583, acetyltransferase (GNAT) MTGGHGRDGGDADVAGVRPARDTDLPAVESLQRFVSHPSPGLLDA family, Expect 2e-08 WPAVGTLLVAVDADDRPVGYLLGVGAHLTELAVSPDHRREGRATA HVO_2320- LVEAFRDAHRSGADAADAPLTLLVHPENDGARACYEALGFRRDAR TG_gha_455 COG0456, RimI-type acetyltransferases, VPDAFDGDDGLRLVLD Expect 3e-07 PF00583, acetyltransferase (GNAT) MGDIDDYDALAEDVDARAEALDYRGEQVDAEGVQTFVAVADAESA family, Expect 4e-12 AESGARPDAAAGSLRGYVTVAASESPPVFARGPVAKVKDLYVAPA HVO_2326- ARGEGVGTELLERAHEWGRDRGCERAALSVHADNDAARSCYESMG TG_gha_455 COG0456, RimI-type acetyltransferases, YETRYLKLDCPL Expect 5e-06 PF00583, acetyltransferase (GNAT) MSETPDGVEVRPYEPGDAAAFWELKRGFELGLGAGTGGDDKLSTY

147 family, Expect 3e-10 EAKLTDDYRERYLSWVERCATDDPGCVVVAEAVSDGGQSGKSEDA HVO_2423- TLVGYAFALPEEMTFIWDAAVLNELFLDADYRGSGAADELMDAVL

TG_gha_455 COG0456, RimI-type acetyltransferases, AHARSQDLPLDRIVLDVDEANGRARAFYERYGFDHWGELVARDL Expect 3e-07 PF00583, acetyltransferase (GNAT) MEIRPARPEDYDAVAAFTRDTWADRGGSDYIPDIYHDWISPGELA family, Expect 5e-8 QATLLVDMGEETPSDDVAAIAQVVMLSEYEGWAQGMRVAPDYREQ GLATRLSHALLDWARERGARRVRNMVYSWNVPSLANARTVGFDPG HVO_2510- IEFRWATPDPDATADPAAAIRDDADAAWTYWTGSEARTALSGLAL TG_gha_455 DPVETWALSELTRDRLHDAAADDRLFVVGDDRATGFTYRNRTYSR ERDDEGEGEETWAEYAVAAWDGASACESLLDAIRRDAASVGADRT RVLFPERIDWVTDAALARSDLAEEPTFVMEADLS PF00557, metallopeptidase family M24, MVEPGVTHLEVAEHAEARIRELADGCAFPVNISINEEASHASPGR Expect 1.6e-15; DDDTVFGEDMVCLDVGVHVDGYIADSAVTVDLSGNDELVESAEEA LDAALDMVEAGAHTGKIGAEIEDVIRGYGYTPVLNLSGHGVEQWD HVO_2600- AHTDPTIPNRGAERGVELEVGDVVAIEPFATDGTGKVTEGSKNEI TG_gha_455 cd01088, MetAP2, methionine YSLVNDRSVRDRMSRKLLDEVREEYKLLPFAARWFEGGRSEMAIR aminopeptidase 2. [E.C. 3.4.11.18], Expect RLEQQGILRGYPVLKEEDGAMVGQAEDTIIVTEDGYENLTR 3e-79

ORF no. Pfam domain, description, E value; Putative amino acid sequence of ORFa NCBI Conserved Domain Database or Clusters of Orthologous Groups, description, E value cd03357, LbH_MAT_GAT, Maltose O- MPSEKEKMLAGDSYDASDPELVAERKRARRLTRRFNDTDETRIER acetyltransferase (MAT) and Galactoside REELIRELFGAVGESFEIEPPFRCDYGYNVRVGEEFFANFDCVFL HVO_2623- O-acetyltransferase (GAT) Expect 1e-55 DVCPITIGDNCQLGPGVHVYTATHPVDAAERIKGPESGEPVTIGD TG_gha_455 NAWLGGRAVVNPGVTIGDDAVVASGAVVTDDVPDSVVVGGNPARV VKELD

GCN5-related N-acetyltransferase Expect MAELQTQTVSSGKTVFVATDEPERGSKGPFYVVYSTEDAENRWGY 8e-20 LCGNCDSFDTAMDTMARIECNNCGNVRKPEEWDAAHE HVO_2677- TG_gha_455

PF00583, acetyltransferase (GNAT) MNPIPKGRTRPTLVLSVGDGFASRTPRVRASFVSVSYPVCRRRLR

148 family, Expect 2.6e-16 SDLQTDGGVIFSLRLRRDVSVNVEMRVDAPGQREYAEAAWDLKER HVO_2709- IRVEEGLLKQRRGFFMDAYRRSNTYLLYENDALVAFASTRRDGYI

TG_gha_455 LFLAVSPDARGEGYGKRLVAEVAREHPVVTCHARTTNTNALDFYE COG0456, RimI-type acetyltransferases, RMGFEVRRRIDNYYEDGGAAFYLRLGEEAGLREKLSEFLRR Expect 2e-11 (only 75 % aligned) PF00583, acetyltransferase (GNAT) MEVTDSLNFGHKDRKDIYEYIERHGTVREDEVRRALNFEPAALGA family, Expect 6e-11 HLTVLRRDGYIRKVGNHVEVAYAPGEAETVELGDLDVTIRMAQNV HVO_2734- DLESLVDAIHEVADEGRYIEAETVADLLDHEEVVLRHNEVSSRMV TG_gha_455 FVATVGDELAGWVHLDLPEAEKLSHTAVLTVGTRPEFRGHGIGSK COG0456, RimI-type acetyltransferases, LLDRGVDWARERGFEKLYNSVPATNEEAIAFLEGHGWETEAVRDG Expect 2e-04 HYKIDGDYVDEVMMAVSLR PF00583, acetyltransferase (GNAT) MRATERPTFESEASKVIYEYVERHGTAKRNVLLDVAPVPSDEFRT family, Expect 3e-6 ELDRLKSRGYLEEDGGTLQLALDVGTIEEHDTDAGIVTIRPGRQR HVO_2795- DFEGLIDAIRDVTSEETYVVAETIAEQLLYDEAVTRHNRVESRVF TG_gha_455 FVATLGEEVVGWTHLDLPQVSRIRETAQQTVGVKEDYRGAGIGSL COG0456, RimI-type acetyltransferases, LLRRGLDWAEANGFRKVYNSVPMTNDRALEFLGDHDWETEAIRRD Expect 7e-04 HYTIGDEHVDEVMMAYRF

ORF no. Pfam domain, description, E value; Putative amino acid sequence of ORFa NCBI Conserved Domain Database or Clusters of Orthologous Groups, description, E value COG2153, ElaA, Predicted acyltransferase MTNSDVRVATGDAERDDAFGVRKAVFVDEQGVDEELEWDEHDDPD Expect 9e-10 AEAVHFVASRDGDAVGAARLREYEPGVGKVERVAVLESARGEGWG HVO_2886- RRLMEALEAEARERGFDSLLLHGQTTAEGFYRGLGYETKSDEFDE TG_gha_455 AGIPHVEMRKSL

PF00583, acetyltransferase (GNAT) MTVRLRAATPSDLPAIREIYAPFVENTAISFAYDPPSVADLETKL family, Expect 7e-12 EQKTDYPWLVCELDGEVAGYAYAGAIRERIAYRWAVETSIYVRPE HVO_2930- FQRRGVARGLYTALLDLLERQGYVSAVAVITTPNPASIAFHESFG TG_gha_455 FERVGRFERVGYKGDAWHDVEWWSLDLDDRPDDPDAPLSVADARD COG1247, Sortase and related RDWWDDALTRGAALVENSA acyltransferases Expect 7e-36

149 Deduced amino acid sequence communicated by J. Eisen, TIGR, April 2007 version (http://archaea.ucsc.edu/).

Frequency of Amino Acids in Penultimate Position Y 2% W 0% V A 10% 5% C 1% D T 6% 14% E 4% F 2% G 4% H 2% I S K 2% 21% 4% L 4% M 1% R Q P 7% 2% 5% N 5%

Figure 4-2. Frequency of amino acids in the penultimate position of the N-termini of proteins from the deduced proteome of Hfx. volcanii.

150

Figure 4-3. MS/MS Fragmentation of Acet-SSIELTSSQK, an N-terminal fragment of HVO_1577. This peptide had a Mascot individual ion score of 39, an E value of 3.1e-3, and an MS/MS spectra that had a complete y-ion series and 4 of 9 b-ions. N- terminal acetyl group (Acet-).

151

Table 4-3. Summary of N-terminal peptides and associated proteins of Hfx. volcanii detected by MS/MS.

Protein Accession Number and Name N-terminal Form Peptide Sequence Group I. N1 alone Penultimate residue small (e.g., Gly, Ala, Pro, Val, Ser, Thr, Cys) HVO_0028 hypothetical protein N1 MTRAELER HVO_0041 argF ornithine carbamoyltransferase N1 MLETTHFTDIDDISASELDR HVO_0062 dipeptide ABC transporter dipeptide-binding N1 MPDTNK HVO_0196 conserved hypothetical protein N1 MADLIVK HVO_0203 rfcS replication factor C small subunit N1 MSEAAESGDAPAGR HVO_0274 conserved hypothetical protein N1 MSGRKQGDSAVYAAAQGR HVO_0455 cctB Thermosome subunit 2 N1 MSQRMQQGQPMIIMGEDAQR HVO_0874 epf mRNA 3''-end processing factor homolog N1 MSSVDKQLENLK HVO_0887 porB 2-oxoglutarate Fd oxidoreductase beta N1 MSSNVR HVO_0888 porA 2-oxoglutarate Fd oxidoreductase N1 MPADFNWAIGGEAGDGIDSTGK HVO_1009 aad oxidoreductase N1 MSLDYRR 152 HVO_1054 gatA Glutamyl-tRNA(Gln) amidotransferase N1 MSLNAFITK

HVO_1145 RPS1A 30S ribosomal protein S3Ae N1 MSERSVSKQKR HVO_1684 thrS threonyl-tRNA synthetase N1 MSDIAVILPDGTELSVEEGATVR HVO_1799 FAD dependent oxidoreductase putative N1 MTDNYDVIIAGAGPAGGQAAR HVO_1914 3-ketoacyl-CoA thiolase N1 MPVPVIAAAYR HVO_2110 arcR transcription regulator N1 MATNKPR HVO_2158 mdh nadp-dependent malic enzyme N1 MTLGDDAR HVO_2222 ppiA peptidyl-prolyl cis-trans isomerase N1 MSDNPTATLHTNKGDITVELFEDK HVO_2564 rplC ribosomal protein L3 N1 MPQPSRPRKGSMGFSPR HVO_2740 ndk Nucleoside diphosphate kinase N1 MSDAERTFVMVKPDGVQR HVO_A0266 transcriptional regulator putative N1 MVNSVLRLAMNER

Penultimate residue other HVO_0138 tyrS tyrosyl-tRNA synthetase N1 MDAYDLITR HVO_0199 pmm phosphoglucomutase/phosphomannomutase N1 MDEISFGTDGWR HVO_0435 phosphoribosyl-ATP pyrophosphohydrolase N1 MDADTPTDEVLDELFATIESR HVO_0452 leuS leucyl-tRNA synthetase N1 MDYDPQELEAR HVO_0826 TET aminopeptidase homolog N1 MEFDFDR HVO_1381 mdmC caffeoyl-CoA O-methyltransferase N1 MDILTDETR

Protein Accession Number and Name N-terminal Form Peptide Sequence HVO_1402 pmu phosphomannomutase N1 MELFGTAGIR HVO_1568 HydD putative N1 MDLDYGMLGGR HVO_1585 acs Acetyl-coenzyme A synthetase N1 MDVPDTDTVVHEPDPAFVEDANVTR HVO_1678 eIF-2 Translation initiation factor N1 MDYDDQLDR HVO_1896 Ribosomal protein S24e N1 MDIEIISEEENPMLHR HVO_2091 gabT 4-aminobutyrate aminotransferase N1 MDRDSVEPTVTSLPGPK HVO_2554 Chain J Trigger Factor Ribosome Binding N1 MEALKADITKGVAR HVO_2601 hit histidine triad protein N1 MDQLFAPWR HVO_2712 RtcB RNA terminal phosphate cyclase oper N1 METREFGDIELR HVO_2716 acd Acyl-CoA dehydrogenase N1 METVGSATGLTDEQR HVO_2790 mrp Mrp protein homolog N1 MDEADVR HVO_2796 conserved hypothetical protein N1 MDWPHDPDGEEGSEGGR HVO_2899 conserved domain protein N1 MELAAIEDLIESHIEDADATVSRPR HVO_A0286 unknown N1 MEFAIWAYPWDVLDAGPR HVO_B0268 alkanal monooxygenase-like N1 MDFSIVDLSPVPK HVO_0387 conserved hypothetical protein N1 MNDEELDELR

153 HVO_0581 cell division protein FtsZ N1 MQDIVREAMER HVO_0790 conserved hypothetical protein N1 MNTDVGLSAR

HVO_0806 pyk pyruvate kinase N1 MRNAKIVCTLGPASFDR HVO_0819 sirR transcription repressor N1 MLSDVMEDYLK HVO_0973 aspB aspartate aminotransferase N1 MNFDFSDR HVO_0999 cobyrinic acid ac-diamide synthase N1 MNTVLVTSTGESTGK HVO_1082 orotate phosphoribosyltransferase N1 MKNVDDLIASAAELADR HVO_1099 dapD 23 4 5-tetrahydropyridine-2-carboxy N1 MNLESDVR HVO_1198 syrB universal stress protein N1 MYSHILFPTDGSDCADAALDHAIEHAR HVO_1344 conserved hypothetical protein TIGR00291 N1 MISLDEAVTAR HVO_1377 Hypothetical UPF0145 protein N1 MIVTTTETVIGR HVO_1380 mcmA methylmalonyl-CoA mutase subunit alpha N1 MFDPDELEEIR HVO_1382 alpha-NAC homolog N1 MFGGGGMNPR HVO_1527 glucose-1-phosphate thymidylyltransferase N1 MQAVVLAAGK HVO_1830 gndA 6-phosphogluconate dehydrogenase N1 MQLGVIGLGR HVO_2057 glucose-1-phosphate thymidylyltransferase N1 MKGVLLSGGTGSR HVO_2068 ftsZ cell division protein N1 MNVFCFGAGK HVO_2313 nirH heme biosynthesis protein N1 MIQADADLSR HVO_2373 ribosomal protein S8.e N1 MKDQGRSK HVO_2543 rpmD ribosomal protein L30P N1 MQAIVQLR

Protein Accession Number and Name N-terminal Form Peptide Sequence HVO_2699 bcp Bacterioferritin comigratory protein N1 MLEPGDDAPDFELPDQHGDLVSLSDFR HVO_2706 eif2bd translation initiation factor eIF N1 MIDETIEEIR HVO_2869 DNA binding domain similar to hmvng1864 N1 MYDLTGFQR HVO_A0586 rffH1 glucose-1-P thymidylyltransferase N1 MQTVILAAGR HVO_B0064 unknown function N1 MFGEYLTR

Group II. N2 and intermediates Penultimate residue small (e.g., Gly, Ala, Pro, Val, Ser, Thr, Cys) HVO_0024 tssB thiosulfate sulfurtransferase N2 VDVVSPTWLADR HVO_0027 transcription anti-termination factor N2 SELLDTLRDDHETPLSR HVO_0069 arylsulfatase N2 TAESPENVLFVVMDTVR HVO_0104 radA DNA repair and recombination protein N2 AEDDLESLPGVGPATADK HVO_0107 SUF system FeS assembly protein NifU family N2 GIGSDMYR HVO_0118 Ribosomal LX protein N2 SQFIITGSFTSR HVO_0123 srp Signal recognition 54 kDa protein N2 VLDNLGSSLRGSLDK HVO_0167 ahcY adenosylhomocysteinase N2 SEHYAPVSEHLDDVEAAR

154 HVO_0177 arsC arsC protein N2 TDAADVDVDADTDATTTR HVO_0304 etfA Electron transfer flavoprotein alpha N2 SDVLAVVEHR

HVO_0329 ilvE branched-chain amino acid aminotran N2 GFDEMDVSTIWQSGEYK HVO_0353 ribosomal protein S23 (S12) N2 ANGKYAAR HVO_0491 Pyridoxamine 5'-phosphate oxidase family N2 SLDQQTELSPDEIR HVO_0541 acnA aconitate hydratase 1 N2 SDTETLDAIR HVO_0704 dgs dolichol-P-glucose transferase N2 SQSVGVVVPAYRPDPER HVO_0736 Domain of unknown function DUF302 superf N2 ALPIDPSAIKPEDIGEER HVO_0738 conserved hypothetical protein N2 ADEEADEAPAVELGTGASVEGAPLAR HVO_0870 proS prolyl-tRNA synthetase N2 SDEQELGITESK HVO_0880 coiled-coil protein of COG 134 N2 VTKQEVLSEFDVQELDEAR HVO_1000 acetyl-CoA synthetase N2 GELSELFAPNR HVO_1020 PBS lyase HEAT-like repeat domain protein N2 SDGDDDTTELSPESFDER HVO_1022 nadh-dependent flavin oxidoreductase N2 TDSLFTPLSLR HVO_1053 gatC glutamyl-tRNA(Gln) amidotransferase N2 SDTPVDADEVR HVO_1077 conserved protein N2 TSEWVSLFSGGK HVO_1132 purA adenylosuccinate synthetase N2 TVTIVGSQLGDEGK HVO_1174 TFIIE alpha subunit N2 AFEELLNDPVIQK HVO_1257 moxR methanol dehydrogenase regulatory N2 SDNPDRFGDDGQR

Protein Accession Number and Name N-terminal Form Peptide Sequence HVO_1311 dehydrogenase putative (TBD) N2 AFDDVDFEGR HVO_1451 Glutamate dehydrogenase N2 ASAAPSTATDDEPTEETALETAR HVO_1453 gdhA Glutamate dehydrogenase N2 AQEANPFESLQEQIDDAATYLDVR HVO_1492 conserved hypothetical protein N2 AHWTDSIVGDRMTVDR HVO_1493 conserved hypothetical protein N2 PQIHLDDETVAR HVO_1506 ilvC ketol-acid reductoisomerase N2 TELTTEVYYDDDADR HVO_1541 glpK glycerol kinase N2 SGETYVGAIDQGTTGTR HVO_1595 conserved hypothetical protein N2 SDDTAPDENWGQQFDR HVO_1626 metallo-beta-lactamase N2 TVSDWSDWLPR HVO_1745 hypothetical protein N2 VAPISHADELDPFIALELPPGWVR HVO_1857 conserved protein N2 SGSPDDERLEELR HVO_1871 Chlorite dismutase family N2 VEAPQTDEGWFALHDFR HVO_1946 translation initation factor SUI1 putative N2 SEVCSTCGLPEELCVCEDVAK HVO_1973 conserved hypothetical protein N2 TESESDTEAEAESER HVO_2029 trh transcription regulator AsnC family N2 TYENLDSDLINALLEDGR HVO_2256 Protein of unknown function DUF262 family N2 AIYVDDPLNSNDEDWDISK

155 HVO_2265 hypothetical protein N2 TDDPEDIPDPVKPTTLQK HVO_2300 eif5A translation initiation factor eIF-5A N2 AKEQKQVR

HVO_2371 conserved hypothetical protein N2 VVDSLSDGTR HVO_2400 conserved hypothetical protein N2 SDLEIER HVO_2413 tuf translation elongation factor EF-1 s... N2 ADKPHQNLAIIGHVDHGK HVO_2436 tme nadp-dependent malic enzyme N2 GLDDDSRKYHR HVO_2452 ribonucleoside-diphosphate reductase ade N2 SDANLSTDELVLPVK HVO_2557 rpmC ribosomal protein L29 N2 AILYTEEIR HVO_2558 30S ribosomal protein S3P N2 ADEHQFIENGLQR HVO_2559 rplV ribosomal protein L22 N2 GINYSVEADPDTTAK HVO_2624 pyrG CTP synthase N2 PTDEYDPEMGRK HVO_2677 acetyltransferase (gnat) family N2 AELQTQTVSSGK HVO_2723 snp snRNP homolog N2 SGRPLDVLEASLDEPVTVLLK HVO_2737 RPL8A 50S ribosomal protein L7Ae N2 AVYVDFDVPADLADSAVEALEVAR HVO_2743 methionine synthase vitamin-B12 independent N2 TELVSTTPGLYPLPDWAK HVO_2753 Domain of unknown function N2 SESEQR HVO_2758 ribosomal protein L11 N2 AGTIEVLVPGGK HVO_2774 eno phosphopyruvate hydratase N2 TRITSVALR HVO_2783 rpsD ribosomal protein S4 N2 TTGNNTKFYETPNHPFQGER HVO_2945 valS valyl-tRNA synthetase N2 PSGEYDPETVEAK

Protein Accession Number and Name N-terminal Form Peptide Sequence HVO_2961 lpdA dihydrolipoamide dehydrogenase N2 VVGDIATGTELLVIGAGPGGYVAAIR HVO_2981 upp uracil phosphoribosyltransferase N2 PIEDRDDAYLITHALAK HVO_A0406 hypothetical protein N2 GLFDSGDVNPEAQR HVO_A0472 trxB2 thioredoxin reductase N2 ADVIVIGGGPAGLTTALFAAK HVO_A0503 amidohydrolase N2 TVLDWVDEPR HVO_B0113 Luciferase-like monooxygenase superfam. N2 SDISFEYNVPVFAGAPDEGTEPTHR HVO_B0295 ugpC sn-glycerol-3-phosphate transport N2 SDITIQNLR HVO_B0376 oxidoreductase N2 TTLEDIDLDFVPFGQTGLQTSELQFGTWR HVO_C0038 hypothetical protein N2 TEYDEDSIPSHTLESNGR HVO_0213 Ferritin-like domain subfamily N1/N2 MSEDVTALLK / SEDVTALLK HVO_0313 ATP synthase (E/31 kDa) subunit N1/N2 MSLDNVVEDIR / SLDNVVEDIRDEAR HVO_0354 rpsG ribosomal protein S7 N1/N2 MSESDAPEPESPASSEEAK / SESDAPEPESPASSEEAK HVO_0359 tuf translation elongation factor EF-1 s... N1/N2 MSDKPHQNLAIIGHVDHGK / SDKPHQNLAIIGHVDHGK HVO_0711 conserved hypothetical protein N1/N2 MGLLDTISSLWK / GLLDTISSLWK HVO_0812 ppsA phosphoenolpyruvate synthase N1/N2 MAVVWLDDVR / AVVWLDDVR HVO_0964 trxC thioredoxin N1/N2 MSDDELTDIR / SDDELTDIRK

156 HVO_1577 imd inosine-5''-monophosphate dehydrogenase N1/N2 MSSIELTSSQKTILTALINLYRDSEDAVK / SSIELTSSQK HVO_1699 aminotransferase classes I and II superf N1/N2 MTATFPGIPYLEWIVDR / TATFPGIPYLEWIVDR

HVO_1858 Ribosomal protein S19e N1/N2 MVTIYDVPADALIEEVAGR / VTIYDVPADALIEEVAGR HVO_1869 hypothetical protein N1/N2 MSLIDFIR / SLIDFIR HVO_2361 carB carbamoyl-phosphate synthase large N1/N2 MTDEDTSTGPQEGTEDR / TDEDTSTGPQEGTEDR HVO_2464 sucD succinyl-CoA synthase alpha subunit N1/N2 MSIFVDDDTRVVVQGITGGEGK / SIFVDDDTR HVO_2547 rpl32e ribosomal protein N1/N2 MSEEITELEDISGVGPSK / SEEITELEDISGVGPSK HVO_2577 pyrF orotidine 5'-phosphate decarboxylase N1/N2 MGFFDDLR / GFFDDLR HVO_2625 guaA GMP synthase C-terminal domain N1/N2 MVNVDEFIEDATASIR / VNVDEFIEDATASIR HVO_A0377 hydantoin racemase putative N1/N2 MTEILWVDPVGHDDFSGDIEALLQNAAR / TEILWVDPVGHDDFSGDIEALLQNAAR HVO_A0488 cobO cob(I)alamin adenosyltransferase N1/N2 MTDAPDGTPETGDETTPDSVR / TDAPDGTPETGDETTPDSVR

Penultimate residue other HVO_1238 oxidoreductase aldo/keto reductase family N2 EYTTLGDTGMEVSR HVO_1824 RecJ-like exonuclease N2 EDELIDSSQLSLPR HVO_B0167 narJ chaperonin-like protein N2 DTNTNAETRTDDTNRDTDR HVO_1654 cysK Cysteine synthase N1/N2 MDDSILDAIGSPLVR / DDSILDAIGSPLVR HVO_1443 ABC transporter ATP-binding protein N2 ITVENLR HVO_2621 ppc phosphoenolpyruvate carboxylase N2 WYSYSSRMR

Protein Accession Number and Name N-terminal Form Peptide Sequence HVO_A0529 ilvB3 acetolactate synthase large N2 NTSERLVESLERLGVER

Group III. Ac1 and intermediates

Penultimate residue small (e.g., Gly, Ala, Pro, Val, Ser, Thr, Cys) HVO_0133 cctA Thermosome subunit 1 Ac1 Ac-MSQRMQQGQPMIILGEDSQR HVO_0424 rli RNase L inhibitor homolog Ac1 Ac-MADDSIAVVDLDR HVO_0481 gap glyceraldehyde-3-phosphate dehydrogenase Ac1 Ac-MSEKSYLSAGENVDESDVVR HVO_0827 conserved protein Ac1 Ac-MSDTESQLR HVO_1164 HD domain protein Ac1 Ac-MSDTEDTLNGGR HVO_1716 exsB protein Ac1 Ac-MTTEPTSDDK HVO_2598 ppk polyphosphate kinase Ac1 Ac-MSDDEVRGGDSQGR HVO_2779 rpl18e Chain O Ac1 Ac-MSKTNPRLNSLIAELK HVO_2902 gatE glutamyl-tRNA(Gln) amidotransferase Ac1 Ac-MTEYDYDYEDLGLVAGLEIHQQLDTETK HVO_A0021 unknown Ac1 Ac-MANYEVIERVVVDRWGDEEGR HVO_B0053 hypothetical protein Ac1 Ac-MACAELEALR

157 HVO_C0043 hypothetical protein Ac1 Ac-MTDDGLVMER HVO_0536 Nutrient-stress induced DNA binding prot Ac1/N1 Ac-MSTQKSVLKEAGSVGDNPVRLDTEK / MSTQKSVLKEAGSVGDNPVR

HVO_2336 pyridoxine biosynthesis protein Ac1/N1 Ac-MPEETDLEELR / MPEETDLEELR HVO_B0117 unknown Ac1/N1 Ac-MGTDQSTNRR / MGTDQSTNR HVO_B0217 livK-2 branched-chain amino acid ABC Ac1/N1 Ac-MTDQPALTDESRR / MTDQPALTDESR

Penultimate residue NatB-like (e.g., Asp, Glu, Asn, Met) HVO_1120 conserved hypothetical protein Ac1 Ac-MDDDDPRRRPR HVO_2923 psmC Proteasome subunit alpha2 Ac1 Ac-MNRNDKQAYDR HVO_B0045 L-2,4-diaminobutyrate decarboxylase Ac1 Ac-MNGVGDVDGERR HVO_0572 lpl lipoate protein ligase Ac1/N1 Ac-MNDSQGSIADREWRVIR / MNDSQGSIADR

Penultimate residue other HVO_1967 pgi glucose-6-phosphate isomerase Ac1 Ac-MQVDLGNVLDTAPAHGVSR

Group IV. Ac1 and N2 isoforms and intermediates Penultimate residue small (e.g., Gly, Ala, Pro, Val, Ser, Thr, Cys) HVO_0291 conserved protein Ac1/N2 MSSNEIPTREVAR / SSNEIPTREVAR HVO_1250 thiol-disulfide isomerase/thioredoxin Ac1/N2 Ac-MVLLESDSELER / VLLESDSELER

Protein Accession Number and Name N-terminal Form Peptide Sequence HVO_1560 Uncharacterized protein conserved Ac1/N2 MSNYLVAMEAAWLVR / SNYLVAMEAAWLVR HVO_2056 spsK spore coat polysaccharide synthesis Ac1/N2 Ac-MPNIHDVEVR / PNIHDVEVRDLQVNADQR HVO_2809 sdhB succinate dehydrogenase chain B hom Ac1/N2 Ac-MSTQVPETVEEESATEAASAGK / STQVPETVEEESATEAASAGK HVO_0628 dppD dipeptide ABC transporter ATP-binding Ac1/N1/N2 Ac-MSQDGNDVSRR / MSQDGNDVSRR / SQDGNDVSR HVO_0783 ATP-dependent protease Lon protease Ac1/N1/N2 Ac-MSNDTNTDDSLHER / MSNDTNTDDSLHER / SNDTNTDDSLHER HVO_2959 2-oxoacid decarboxylase E1 beta chain Ac1/N1/N2 Ac-MSSQNLTIVQAVR / MSSQNLTIVQAVR / SSQNLTIVQAVR

Penultimate residue other HVO_1091 psmA proteasome subunit alpha1 Ac1/N2 Ac-MQGQAQQQAYDR / QGQAQQQAYDRGITIFSPDGR

Group V. Ac2 and intermediates Penultimate residue small/NatA-like (e.g., Gly, Ala, Pro, Val, Ser, Thr, Cys) HVO_0070 NifU-like domain protein Ac2 Ac-STETQDGEDDLKER HVO_0116 Ribosomal protein L31e Ac2 Ac-SANDFEER HVO_0778 cctA Thermosome subunit 3 Ac2 Ac-ASRMQQPLYILAEGTNRTHGR HVO_0862 conserved hypothetical protein Ac2 Ac-SVGYQVSSDHQLAR

158 HVO_1804 nadC nicotinate phosphoribosyltransferas Ac2 Ac-SDFDIVGPEAIR HVO_2226 trpD anthranilate phosphoribosyltransferase Ac2 Ac-AVVESEFGEWPLKR

HVO_2348 conserved hypothetical protein TIGR00294 Ac2 Ac-SHQLPDVQASQPDVTVGLSQVGVTGVEK HVO_2506 Isopentenyl-diphosphate delta-isomerase Ac2 Ac-SNAAADEATELHK HVO_2550 rpsN ribosomal protein S14p/S29e Ac2 Ac-SDSETEQTGEHASR HVO_2551 rpl5p ribosomal protein L5. Ac2 Ac-SEADFHEMR HVO_2562 rplW ribosomal protein L23 Ac2 Ac-SIIEHPLVTEKAMNQMDFDNK HVO_A0520-TG_gha_452 uncharacterized domain 1 putative Ac2 Ac-SEQDDYAEIRER HVO_C0074-TG_gha_454 dppF dipeptide ABC transp. ATP-binding Ac2 Ac-SVVSETHTSQDGR HVO_0316 ATP synthase archaeal A subuni Ac2/N1 Ac-SQATQDSVREDGVIASVSGPVVTAR / MSQATQDSVR HVO_0551 mutL-2 DNA mismatch repair protein mutL Ac2/N1 Ac-SDIKQLDEK / MSDIKQLDEKTVQR HVO_0979 ndhG nadh dehydrogenase/oxidoreductase Ac2/N1 Ac-SSEQKPFVTDDTQVQTETR / MSSEQKPFVTDDTQVQTETR HVO_0729 ppa inorganic pyrophosphatase Ac2/N2 Ac-VNLWEDMETGPNAPDEIYAVVECLK / VNLWEDMETGPNAPDEIYAVVECLK HVO_0861 sufB/sufD domain protein Ac2/N2 Ac-SAQLPANLSAETVR / SAQLPANLSAETVR HVO_0884 aldehyde reductase Ac2/N2 Ac-TENLASADDAPR / TENLASADDAPR HVO_1637 slyD peptidyl-prolyl cis-trans isomerase Ac2/N1/N2 Ac-SDEQQAEAEQVDEEVESGIQDGDFVR / MSDEQQAEAEQVDEEVESGIQDGDFVR / SDEQQAEAEQVDEEVESGIQDGDFVR HVO_2126 oligopeptide ABC transporter oligopeptid Ac2/N1 Ac-SDDNITEVNMDR / MSDDNITEVNMDR HVO_2600 map methionine aminopeptidase type II Ac2/N2 Ac-SIGPLDDETVEK / SIGPLDDETVEK

Protein Accession Number and Name N-terminal Form Peptide Sequence HVO_2620 rieske 2Fe-2S domain protein Ac2/N1 Ac-SDSDKYPADSGR / MSDSDKYPADSGR HVO_2757 L1P family of ribosomal proteins Ac2/N1/N2 Ac-ADTIVDAVSR / MADTIVDAVSR / ADTIVDAVSR HVO_A0378 hydantoin utilization protein B Ac2/N2 Ac-STHDVDPATVEVIR / STHDVDPATVEVIR

Penultimate residue other HVO_0662 DNA binding protein Ac2 Ac-KFIEEIVVDAFLPTFRALLAEDLRDR HVO_1308 aroA 3-phosphoshikimate 1-carboxyvinyltr Ac2 Ac-DAHVTPSR HVO_1497 FruB phosphocarrier protein Hpr Ac2 Ac-ERTVTVVPEDGLHARPASK HVO_2486 bccA biotin carboxylase Ac2 Ac-FSKVLVANR HVO_C0075-TG_gha_454 oligopeptide/dipeptide ABC transporter Ac2 Ac-QYELAMDSDTALDRR HVO_2242 eif probable translation initiation fact Ac2/N1 Ac-EYQTALDR / MEYQTALDR HVO_A0571 hydrolase isochorismatase family Ac2/N1 Ac-LDATASQAK / MLDATASQAK HVO_B0112 mandelate racemase/muconate lactonizi Ac2/N1 Ac-EITDISATK / MEITDISATK

Group V. Ac1 and Ac2 isoforms and intermediates Penultimate residue small (e.g., Gly, Ala, Pro, Val, Ser, Thr, Cys) includes NatA-like Ac2 forms

159 HVO_0136 translation initiation factor eIF-1A Ac1/Ac2 Ac-MSDDENESR / Ac-SDDENESRR

HVO_0860 sufB FeS assembly protein SufB Ac1/Ac2/N1 Ac-MSSDQDHLKETDTEAR / Ac-SSDQDHLKETDTEAR/MSSDQDHLK HVO_1396 P-hydroxybenzoate hydroxylase Ac1/Ac2/N1 Ac-MSTDENDTAEDSSASPR / Ac-STDENDTAEDSSASPR / MSTDENDTAEDSSASPR HVO_1545 dihydroxyacetone kinase L subunit Ac1/Ac2/N1 Ac-MADAETQREAVLDALDNVAER / Ac-ADAETQR / ADAETQREAVLDALDNVAER HVO_2784 rpsM ribosomal protein S13p/S18e Ac1/Ac2/N1/N2 Ac-MSAEEPQDSSPEEEEDLRYFIR / Ac-SAEEPQDSSPEEEEDLR / MSAEEPQDSSPEEEEDLR /SAEEPQDSSPEEEEDLR

CHAPTER 5 PHOSPHORYLATION AND METHYLATION OF SUBUNITS FROM 20S PROTEASOMES AND PROTEASOME ACTIVATING NUCLEOTIDASE A IN HALOFERAX VOLCANII

Introduction

Phosphorylation is one of the most common post-translational modifications found in all three domains of life. Several studies have demonstrated that archaeal proteins are phosphorylated; however, relatively little is known regarding the identities of the proteins that are modified or the sites of modification (Kennelly, 2003; Eichler and Adams, 2005; Kirkland et al., 2008a). Furthermore, the impact phosphorylation has on the functional properties of the archaeal proteins that are modified or the kinases/phosphatases that regulate these events are poorly understood.

Proteasomes are multicatalytic proteases found in all three domains of life. In many organisms, including eukaryotes and haloarchaea, the catalytic 20S core particle of proteasomes is essential for growth (Lee et al., 1992; Lee et al., 1991; Georgatsou et al., 1992; Fujiwara et al.,

1990; Zhou et al., 2008). Although it is known that eukaryotic proteasomes are regulated through interactions with accessory proteins, such as the 19S cap, and the ubiquitin-targeting pathway, several subunits of the 20S and 19S complex are also subject to various forms of co-

and post-translational modification including N-terminal acetylation, phosphorylation, S- glutathionation, N-myristoylation, and O-linked glycosylation (for details see Table 1-1).

Several different kinases have been used to study the in vitro phosphorylation of different 26S proteasome subunits including Polo-kinases and casein kinase II (Bose et al., 2004; Bose et al.,

1999; Castano et al., 1996; Feng et al., 2001; Fernandez et al., 2002; Horiguchi et al., 2005;

Iwafune et al., 2004; Mason et al., 1996; Zhang et al., 2007). Additionally, there is evidence that some of the post-translational modifications, such as phosphorylation, are regulated by growth

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phase or signaling molecules such as γ-interferon (Bose et al., 2004; Bose et al., 2001; Horiguchi

et al., 2005; Rivett et al., 2001; Tokumoto et al., 2000; Wakata et al., 2004).

Subunit composition of Hfx. volcanii proteasomes also changes as a function of growth

phase. 20S core particles from Hfx. volcanii are made up of two different α-type subunits and one β. Hfx. volcanii synthesizes different 20S core particles made up of three different subunits:

α1, α2, and β (Kaczowka and Maupin-Furlow, 2003). In addition to the three different subunits of 20S core particles, Hfx. volcanii also synthesizes two different proteasome activating nucleotidases, PanA and PanB. While the transcripts of all five genes increase as cells transition into stationary phase, the levels of both α2 and PanB increase severalfold over α1, β, and PanA

(Reuter et al., 2004). As α2 levels increase in the cell, asymmetric proteasomes (α1ββα2) may become more prevalent or proteasomes that contain only α2 (α2ββα2).

This study elaborates on the phosphorylation state of different 20S core particle subunits and the proteasome activating nucleotidase PanA and connects the phosphorylation states of the

α1 protein to the phase of growth of Hfx. volcanii. The ability of variant α1 and PanA proteins to complement hypo-osmotic stress was examined as well as whole-cell chymotrypsin-like peptidase activity of cells expressing variant subunits. A candidate Hfx. volcanii Rio1-type kinase was purified and used to study the in vitro transfer of γ-32P-ATP to different,

recombinantly purified proteasome subunits. In addition, a new post-translational modification,

methyl-esterification, was identified on several residues of the α1 protein from in vivo isolated samples.

Results and Discussion

Isoforms of α1 Modulate During Growth-phase Transitions

Post-transcriptional modification of the predominant proteasomal proteins of Hfx. volcanii (α1 and PanA) was analyzed during various stages of growth from log- to late-stationary

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phase. 2-DE immunoblot of cell lysate from this haloarchaeon revealed two α1-specific

isoforms of pI 3.0 and 4.4, both of which migrated at 37.5 kDa consistent with the mobility of

the α1 protein in reducing SDS-PAGE gels (Wilson et al., 1999). The α1-isoform of pI 4.4 was

present at all stages of growth while the second more acidic α1-isoform of pI 3.0 disappeared in

late-stationary phase (Figure 5-1A).

Affinity purification of 20S proteasomes from early stationary-phase cells expressing a hexahistidine tagged (-His6) derivative of the catalytic β protein revealed that both acidic

isoforms of α1 (pI 3.0 and 4.4) associated with β as peptidase-active (Suc-LLVY-AMC

hydrolyzing) 20S proteasomes in Hfx. volcanii (Figure 5-1B). The α1 isoform of pI 4.4 was also

detected in non-20S proteasomal fractions. Treatment of these purified 20S proteasomes with

phosphatase resulted in a basic shift in pI of both α1 and α2 signifying both α-type subunits were phosphorylated (Figure 5-1C).

Together these results suggest that 20S proteasomal α-type proteins of Hfx. volcanii are decorated by phosphorylation. Based on the IEF migration and phosphatase sensitivity of both

α1 isoforms, we propose that α1 harbors at least two phosphosites. The least phosphorylated form of α1 (pI 4.4) was detected at all stages of growth and occurred in both free and CP- associated forms. In contrast, the more extensively phosphorylated form of α1 (pI 3.0) was only detected in association with CPs and was not present in late-stationary phase. In addition to these modified forms of α1, CP subtypes appear to incorporate a phosphorylated form of α2.

Thus, Hfx. volcanii not only modulates the types of subunits present in the CP and PAN subtypes during the transition from log- to stationary-phase growth (Reuter et al., 2004), but appears also to post-translationally modify the proteasomal proteins during this transition (Humbard and

Zuobi-Hasona, unpublished).

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β Subunit is Phosphorylated

MS analysis of the β subunit of proteasomes purified by Ni2+ affinity chromatography of

α1His6 proteins revealed a phosphopeptide that contained S129 of the β subunit with an individual

MOWSE score of 57 and an Expect value of 2.5e-4. It was 13 amino acids in length and spanned

residues 119 to 131 (RGEDMSMQALS TL) (numbering includes the 49 amino acid fragment at

the N-terminus that is cleaved during maturation) and had a monoisotopic mass of 1518.66 Da.

It eluted by RP-HPLC between 69.69 minutes and 70.27 minutes as a doubly charged ion

(760.340, 2+). The peptide contained a deamidation on the eighth residue (glutamine 126) and a

phosphorylation on the eleventh residue (serine 129). The MS/MS fragmentation contained

primarily b- and a-ions instead of y (Figure 5-2). The loss of phosphate on a b11 indicates that the

phosphorylation site was on serine 129 and not threonine 130. The mass defect between b10 and

b11 – 98 is consistent with dehydroalanine, the β-eliminated form of phosphoserine. There is also

a b12 – 98 ion. The predominance of a b-ion series (and corresponding lack of a y-ion series) can be attributed to the presence of an arginine at the N-terminus of a peptide. It has been previously observed that the presence of basic amino acids (arginine, lysine, and histidine) can alter the normal fragmentation patterns seen in mass spectrometry (van Dongen et al., 1996). Specifically the presence of an N-terminal arginine supports the formation of a-ions (and d-ions) over y-ions.

To bolster the validity of the spectrum missing y-ion series, several a-ions as well as several ions

resulting from internal fragmentation were identified (inset Figure 5-2). An additional

phosphopeptide was detected. That spectrum was unable to differentiate between a phosphate

group on S129 and T130. The actual site of phosphorylation was determined to be S129 based on

the spectrum shown in Figure 5-2. To validate the spectrum and phosphorylation site, a

synthetic peptide (RGEDMSMQALpSTL) was synthesized and analyzed by direct-infusion on

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the QSTAR. Again, the b-ion series was favored over the y-ion series and the characteristic b11

and b12 neutral-loss sequence ions (-98 Da) were observed (Figure 5-3).

NetPhos (Blom et al., 1999) did not predict S129 or T130 to be phosphorylation sites.

S129 had a NetPhos score of 0.020 and T130 had a score of 0.095. NetPhos predictions are based on eukaryotic phosphorylation motifs so perhaps its not surprising that this algorithm did not identify the site. Currently, little is known about the specificity or phosphorylation motifs of archaeal kinases.

The phosphorylated form of the β subunit was isolated in complex with a hexahistidine tagged α1 subunit. Both phosphorylated and unphosphorylated forms of the β subunit copurified with the α1. Since this phosphopeptide was isolated in a Ni2+ affinity column using a hexahistidine tagged α1 protein, it would have been in complex with α1, meaning it was formed into an active 20S proteasome along with unphosphorylated subunits. Using the crystal structure of the 20S proteasome from Thermoplasma acidophilum, S129 was modeled to be at the interface between two β subunits. It is unlikely in that position that it could interact with the other β ring or the α rings. This phosphorylation event may have something to do with the assembly of the ring itself.

S129 is a partially conserved residue in archaea. The β subunit for Hfx. volcanii was aligned against the 20 closest homologues in archaea. Out of the 20 closest homologues, 11 have a serine in that position, the remaining 9 have alanine. Two of the closest related proteins, β subunits (Q18GX3 and B9LTS6) from Haloquadratum walsbyi and Halorubrum lacusprofundi, have a serine at the corresponding position. If the modification is conserved on that residue, and the predicted location of the phosphorylation site is at the interface between two β subunits, it may play a role in the assembly of the different β-rings in Haloquadratum walsbyi and

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Halorubrum lacusprofundi. Broadening the BLAST results shows that S129 is also conserved in

eukaryotes. The serine was conserved in β5 subunits in several eukaryotic organisms. β1, β2, β5

all contain proteolytic function. Currently there are no phenotypes associated with various

phosphorylation events on 20S proteasome subunits. It has been demonstrated that

phosphorylation events on the 19S cap in yeast can promote the assembly of the 20S proteasome

and 19S cap into 26S proteasomes (Satoh et al., 2001).

PanA is Phosphorylated

A phosphopeptide specific to PanA was detected in several ESI-Q-TOF experiments of tryptic digestions of hexahistidine tagged PanA chromatography fractions. The PanA phosphopeptide eluted from RP-HPLC at 69.05 minutes as a quadrupoly charged ion (m/z

684.8). The peptide has a Mascot score of 70 and an expect (E) value of 1.4e-7. The phosphopeptide is composed of amino acid residues 337 – 361

(MNVpSDDVDFVELAEMADNASGADIK). There is only one serine or threonine in the peptide, S340 (Figure 5-4). MS/MS fragmention confirmed that S340 was the phosphorylated amino acid. Despite the large, 25 amino acid, peptide. A high Mascot score and low expect value were obtained due to the assignment of 9 y-ions and 6 b-ions in the MS/MS fragmentation.

Additional peaks were assigned that increased the confidence of this peptide including a neutral loss peak (–98 for phosphorylation) and several internal fragmentation ions. The non- phosphorylated peptide was detected in the same preparations as well. It is important to note, that the phosphopeptide for S340 in PanA was detected in fractions from both hexahistidine tagged

PanA and hexahistidine tagged PanB fractions. This indicates that the phosphorylated PanA is in complex with PanB in Hfx. volcanii cells. Interestingly, PanA and PanB differ in this residue.

While PanA has a serine in position 340, the corresponding residue in this position in PanB is an alanine.

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α2 Subunit is Phosphorylated

An α2-specific phosphopeptide was detected by precursor ion scanning of a tryptic digest of 20S proteasomes purified by nickel chromatography of α1His6. The phosphopeptide eluted

from RP-HPLC at 37.9 minutes as a doubly charged ion (m/z 558.2). The peptide has a Mascot

score of 35 and an expect (E) value of 1.4e-3. The peptide is composed of amino acid residues

12 – 21 (GTSLFSPDGR). Unfortunately, the MS/MS could not distinguish between the T13 and

S14 as the phosphosite (Figure 5-5). The MS/MS fragmentation contains 6 unique y-ions and 3

unique b-ions. The proximity of the phosphosite to the N-terminus of the peptide made the

assignment more difficult. The precursor ion scan was performed on the QqQ instrument

(QTRAP). It was set to detect the neutral loss of a phosphate group. Several of the ion b-ions on

the MS/MS fragmentation show the characteristic neutral loss (–98) for a phosphate, but the

differientiating ions, b2 or y8 are both missing in the spectrum. Since this phosphopeptide was

detected by a precursor ion scan, the unphosphorylated peptide was not dectected in the same

run. Subsequent experiments have found the unphosphorylated form of the peptide in

chromatography fractions.

α1 Subunit is Phosphorylated

An α1-specific phosphopeptide was detected by an ESI-QTOF run of a tryptic digest of

20S proteasomes purified by nickel affinity chromatography of α1His6. It was a doubly charged

ion (m/z 479.3) with a Mascot score of 30 and an expect (E) value of 0.001. It was composed of

amino acid residues 137 – 149 (ALLIGGVENGpTPR) of the α1 protein. Only one amino acid,

T147, is predicted to be phosphorylated in that peptide, and the MS/MS annotation confirmed

that T147 was the site of phosphorylation (Figure 5-6). The MS/MS fragmentation contained 7 y-ions and 6 out of the 7 showed the characteristic neutral loss (–98) indicative of a phosphorylation. Several other fragments ions could be assigned to internal sequencing ions of

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the peptide. These peak assignments bolster the MS/MS assignment. The peptide, although

generated by a tryptic digest of a 20S proteasome preparation, was a semi-tryptic fragment, an internal chymotryptic-like cut on the N-terminus of the peptide. An additional scan using an error-tolerant algorithm for this phosphorylation site hit on two additional peptides;

LIGGVENGpTP + 22 Da and LIGGVENGpT + 155 Da. The Mascot score for these two peptides were 50 and 58 and the E-values were 0.0006 and 0.001, respectively. The unphosphorylated form of the peptide was also detected in the same fraction.

Even though NetPhos predicted that Thr147 of α1 is likely phosphorylated (0.851), it was not the most likely residue. There are several other predicted phosphorylation sites that were more likely than Thr147. For example, Ser58 was predicted (0.996) to be phosphorylated as well as Thr158 (0.981). Based on the 2D-GE data, the α1 protein is likely phosphorylated on more than one residue. Therefore, site-directed mutations were made in the psmA gene that resulted in amino acid substitutions Tyr28Phe, Ser58Ala, Thr147Ala, and Thr158Ala.

Variant α1 Proteins Affect Carotenoid Pigmentation and Cell Viability

Knock-out strains of both psmA (α1) and panA (PanA) have a dimished capacity to adapt to low salt environments (Zhou et al., 2008). This phenotype can be complemented by providing a copy of the gene in trans. Cells (H26 ΔpanA) expressing either panB, panA, or panAt1018g

(PanA Ser340Ala) were grown in varying concentrations of NaCl (Figure 5-7). Interestingly,

over-expression of panB did not rescue the sensitivity of the panA mutant to low salt conditions,

but expression of PanA Ser340Ala restored growth to wild-type levels. This indicates that the

phosphorylation event on Ser340 is not important for hypo-osmotic stress response. Substitution

mutations in the psmA gene that introduced amino acid substitutions Ser58Ala, Thr147Ala,

Thr158Ala, and Tyr28Phe were also tested with their ability to complement the hypo-osmotic stress response phenotype of the ΔpsmA strain along with over-expression of psmC (α2) from a

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plasmid. Over-expression of psmC (α2) and psmA-a83t (α1 Tyr28Phe) had no effect on stress

response to low salt; however, other substitutions had severly inhibited and highly variable

growth (data not shown). While each of the constructs was successfully transformed into ΔpsmA

cells, after being transferred to liquid media or streaked onto a fresh medium, the cells began to

die (Figure 5-8A). Some of the substitutions appeared to affect carotenoid biosynthesis as cells

expressing α1 Ser58Ala and α1 Thr158Ala were whiter than cells expressing wild-type α1 or no

α1 at all (Figure 5-8A). Whole cell chymotrypsin-like activity was measured for the different substitution mutants. There were only modest differences in activity between the strains with the exception of cells expressing α1 Thr158Ala, which had a nearly 2-fold increase in activity compared to wild-type (data not shown). It is unclear at this time if differences in activity can account for the phenotypes seen. Both the hypo-osmotic stress and pigmentation phenotypes are recessive. Expression of variant α1 proteins in wild-type Hfx. volcanii cells does not result in the same growth and pigmentation phenotypes (data not shown). Mutations and phenotypes are summarized in Table 5-2.

Haloferax volcanii Encodes a Number of Putative Protein Kinases

Based on genome sequence, Hfx. volcanii encodes for a number of putative

serine/threonine kinases including two different Rio-type protein kinases, type-1 (Hvo_0135) and a type-2-like kinase (Hvo_0569) (Figure 5-9A) as well as PrkA1 and PrkA2. Rio-type kinases are a relatively new atypical protein kinase family with four unique types, Rio1, Rio2,

Rio3, and RioB. Rio-type kinases are widespread throughout eukaryotes and archaea. Archaea typically have two different Rio-type kinases, Rio1 and Rio2 (LaRonde-LeBlanc et al., 2005).

The Rio2 kinases can vary somewhat in their different domains. Rio1 kinases are characterized by a consensus sequence in the N-terminal domain of the protein, STGKEA, and a second region of homology in the C-terminal domain, IDxxQ. Rio2 kinases are characterized by a helix-turn-

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helix motif in the N-terminal domain followed by the amino acid sequence GxGKES and the C-

terminal sequence IDFPQ (LaRonde-LeBlanc and Wlodawer, 2005a). The Rio1p kinase from

Hfx. volcanii contains the signature domain sequence but the Rio2p does not (Figure 5-9A).

Rio1p kinase was cloned and purified for kinase studies.

Rio type-I Kinase from Hfx. volcanii Catalyzes Autophosphorylation in vitro

To investigate the activity of Rio1, the kinase was fused to StrepII tag and purified from

recombinant Hfx. volcanii (H26 pJAM2558) using Strep-Tactin chromatography. Rio1p was

electrophoretically pure after passage through the Strep-Tactin column (Figure 5-9B) and mass

spectrometry confirmed that only Rio1p could be detected in elution fractions (Table 5-3).

Rio1p ran as a monomer on a gel filtration column at the molecular weight of 50 kDa (data not

shown). Rio1p can autophosphorylate using either Mg2+ or Mn2+ as the divalent cation (Figure

5-9C). The ideal concentration of cation was between 10 and 50 mM Mg2+ with 2 M NaCl.

Rio1p chromatography fractions were dialyzed into 20 mM Tris buffer (pH 7.2) with either 2 M

KCl or 2 M NaCl. There was no significant difference in autokinase activity (data not shown).

All subsequent kinase reactions were carried out in 2 M NaCl 50 mM MgCl2 20 mM Tris buffer

(pH 7.2). Multiple bands were visible on the autoradiograph after the phosphorylation reaction.

There is a doublet at the apparent molecular weight of Rio1p (monomer) and there is another

band running at approximately twice the mass. The MS results did not identify any

contaminating proteins in the fraction. These other bands may represent a mixture of tagged and

untagged Rio1p or a transient dimer that forms between two Rio1p proteins. It is also possible

that there is a contaminating protein that is not detected by SYPRO staining or mass spectrometry.

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Rio type-I Kinase Phosphorylates α1 in vitro

Purified Rio1p was used to phosphorylate α1His6 subunits purified from E. coli, 20S core

particles purified from Hfx. volcanii, and casein (boiled). The Rio1p preparation was able

phosphorylate the unboiled α1 preparation but was unable to phosphorylate 20S proteasomes or

casein (Figure 5-10A). When equal amounts of α1 subunits and variants all purified from E. coli were used in the kinase assay, unequal amounts of phosphorylation took place. Wild-type α1 was the most heavily phosphorylated under these conditions. Both α1 S58A and α1 T147A were phosphorylated but to a lesser extent than wild-type α1 (2.5 fold lower based on densitometry of autoradiograph). The variant α1 T158A was not phosphorylated under these conditions (Figure

5-10B). Based on these results, coupled with our earlier findings by 2DGE, the α1 protein is most likely phosphorylated on 2 or 3 different sites. It is unclear at this time why α1 T158A could not be phoshporylated by Rio1p in vitro. There may be drastic structural changes in this

α1 protein that prevents meaningful interaction with the kinase.

α1 Subunit is Methylesterified

Five unique methylated peptides were identified in several ESI-Q-TOF experiments of

tryptic digests of 20S proteasomes purified by nickel affinity chromatography with α1His6 (Figure

5-11 – Figure 5-15). Each of the methylated peptides are derived from the α1 protein. The peptides are composed of amino acid residues 13 – 22 (GITIFSPDmethylGR), 23 – 30

(LYQVEmethylYAR), 58 – 68 (SPLMEmethylPTSVEK), 105 – 116 (YGEPIGIEmethylTLTK), 150 –

163 (LYETDPSGTPYEmethylWK). All five peptides eluted from the RP-HPLC as doubly charged

ions: 538.77 (m/z), 528.23 (m/z), 616.28 (m/z), 667.82 (m/z) and 567.23 (m/z). The Mascot

scores for the 5 peptides were 42, 33, 38, 66, and 38, respectively, and the expect values were

0.0044, 0.032, 0.017, 2.5e-5, 0.026. The high Mascot scores and corresponding low E-values

validate the assignment of the peptides and the methylation sites. In addition to high Mascot

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scores and low E-values, several of the MS/MS fragmentations showed neutral losses of methyl groups (–14). Specifically, peptide 23 – 30 had three neutral loss peaks, 58 – 68 had one neutral loss peak, and 105 – 116 had three neutral loss peaks. The MS/MS fragmentation was complete enough in all 5 spectra to definitively assign the location of the methylation. Although each of the methylated peptides also were detected in the same fractions as unmodified peptides, only α1 peptides had high enough Mascot scores and were repeatable enough to report as methylated.

Conclusions

Phosphorylation. Due to the relatively low number of confirmed phosphorylation sites on archaeal proteins, the identification of a phosphorylation sites for the α1, α2, β subunit of 20S proteasomes and PanA is a significant advance in our understanding of archaeal protein phosphorylation and the proteasome system in Hfx. volcanii.

The Ser129 residue of the Hfx. volcanii β protein that is phosphorylated is not highly conserved among archaea. Of 20 archaeal β-type proteins analyzed, a slight majority (11 / 20) had a serine residue at the analogous position. Broadening the BLAST search revealed that the

Ser129 residue is widespread in eukaryotes and was conserved in all eukaryal β5 and β5i

(LMP7i) proteasomal subunits examined, ranging from yeast to humans. β5 is responsible for the chymotrypsin-like peptidase activity of the housekeeping 20S proteasomes (Orlowski and

Wilk, 2000), and β5i replaces β5 to form immunoproteasomes after γ-interferon induction

(Kloetzel, 2004).

In this study, the phosphorylated form of the Hfx. volcanii β protein was isolated in complex with the His-tagged α1 subunit. Unphosphorylated peptides, which spanned this same phosphosite (β Ser129) and copurified with α1, were also detected. Since all of the β proteins present in the α1-His enriched samples were in complex with α1 and appeared similar to mature

(processed) β subunits, the results suggest that both phosphorylated and unphosphorylated forms

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of β are present in 20S proteasomes. Automated modeling (Schwede et al., 2003) of the β

Ser129 to crystal structures of analogous 20S proteasomes suggests this residue is located at the

interface of the β subunits responsible for association into heptameric rings and distant from the

interface of the two β rings and the β-α ring interface. Thus, phosphorylation of β Ser129 may

influence formation of the heptameric β -ring as it assembles on the α ring scaffold.

Although phosphorylation of archaeal 20S proteasomes has not been previously described,

a number of phosphosites of the eukaryotic counterpart have been mapped including: α3 Ser248 of Candida albicans (Fernandez et al., 2002); α7 Ser243 and Ser250 of monkey (Bose et al.,

2004); α7 Ser243, α7 Ser250 (Castano et al., 1996), α2 Tyr120 of rat (Benedict and Clawson,

1996); and α5 Ser56 (Beausoleil et al., 2004), α7 Ser249 (Claverol et al., 2002; Beausoleil et al.,

2004), α2 Tyr23, α2 Tyr97, α7 Tyr160, β2 Tyr154, and β7 Tyr102 (Rush et al., 2005) of human

(Figures 5-16 and 5-17). Several of these phosphorylation sites are conserved in Hfx. volcanii

20S proteasome subunits including the human α2 Tyr23, α5 Ser56, and β2 Tyr154 residues, rat

α2 Tyr120 residue and C-terminal Ser/Thr residues of a number of α-type subunits. Based on in vitro phosphorylation data with Rio1p, it seems that the residue corresponding to the Ser56 in humans, Ser58 in Hfx. volcanii, may very well be phosphorylated. It is also possible that these modifications do occur in Hfx. volcanii and may account, in part, for the additional proteasomal isoforms detected by 2D-SDS PAGE (Figure 3-1 and Figure 5-1A).

In analogy to eukaryotic cells, it is quite likely that the phosphorylation site of the Hfx. volcanii 20S proteasome subunits detected in this study regulates biological function. A number of studies in eukaryotes suggest that phosphorylation regulates the subcellular distribution and assembly of 26S proteasomes. For example, phosphorylation of regulatory particle Rpt6 subunit appears to promote its association with the α2 subunit of 20S proteasomes to form the 26S

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proteasome (Satoh et al., 2001). Likewise, the levels of phosphorylated α7 and α3 are higher in

26S proteasomes than free 20S proteasomes (Mason et al., 1996). Phosphorylation at either one of the two α7 sites (Ser243 and Ser250) is essential for association with 19S regulatory complexes, and the ability to undergo phosphorylation at both sites gives the most efficient incorporation of α7 into 26S proteasomes (Rivett et al., 2001). In addition, γ-interferon treatment decreases the level of phosphorylation of proteasomes, which coincides with a decrease in the levels of 26S proteasomes and increase in PA28-containing proteasomes, suggesting phosphorylation may play role in regulating formation of proteasomal complexes in animal cells (Bose et al., 2004). Thus, the assembly and disassembly of 26S proteasomes appear regulated by kinase(s) and/or phosphatase(s). Proteasomal phosphorylation also appears to be important in development and subcellular distribution. For example, the α4 subunit of proteasomes is preferentially phosphorylated in immature vs. mature oocytes of goldfish

(Horiguchi et al., 2005). Futhermore, phosphorylation of the α2 Tyr120 residue of rat is important in nuclear localization of proteasomes (Benedict and Clawson, 1996). Perturbation of putative sites of modification in the α1 protein in Hfx. volcanii resulted in drastic changes in cell viability and growth. These data indicate that phosphorylation of the α1 subunit has immediate effects on cellular processes. Future studies are needed to elucidate the role of the phosphorylation of the Hfx. volcanii proteasomal proteins observed in this study. The identification of several of these modification sites now lays a foundation for these studies.

Methylesterification. Although methylation has not been previously reported as a post- translational modification of a 20S core particle subunit in any organism, there are examples of reversible O-linked methylation in other bacteria and archaea. The haloarchaea have several examples of O-linked modification of taxis proteins in response to external stimuli (Brooun et

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al., 1997; Hildebrand and Schimz, 1990; Hou et al., 1998). Haloarchaea may regulate the activity of other systems using this strategy.

The α1 subunit from 20S core particles in Hfx. volcanii is heavily methylated. The lower pI of halophilic proteins is due to the prevalence of aspartic and glutamic acids and the lack of positively charged residues such as arginine and lysine. The acidic nature of proteins in the halophile is a strategy called “salting in”. The negative charge on the surface of the protein aids in the solubility in high salt solutions by creating a hydration shell around the proteins.

Methylation of the aspartic acids could be a mechanism of buffering the protein with the solution. Although the presence of the different methylation sites was demonstrated repeatably, the unmodified peptide was almost always present in the same preparation, suggesting that the modified and unmodified proteins existed simultaneously in the cell. O-linked methylesterification is a reversible post-translational modification, and therefore probably regulated. There is no clear correlation to growth phase or environmental conditions that increase or decrease the frequency of modification. Future work is needed to better understand how and why this modification takes place.

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Table 5-1. Strains and plasmids used in this study Strain or Plasmid Genotype; oligonucleotide used for amplification Source Strain Escherichia coli F- recA1 endA1 hsdR17(r - m +) supE44 thi-1 gyrA relA1 Life DH5α k k Technologies F- ara-14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 New England GM2163 galT22 mcrA dcm-6 hisG4 rfbD1 rpsL 136 dam13::Tn9 Biolabs xylA5 mtl-1 thi-1 mcrB1 hsdR2 - - - F ompT [lon] hsd SB (rB mB ) (an E. coli B strain) with Novagen BL21 (DE3) DE3, a λ prophage carrying the T7 RNA polymerase gene

Haloferax volcanii DS70 ΔpyrE2 (Allers et al., H26 2004) H26 ΔpanA (Zhou et al., GZ109 2008) H26 ΔpsmC (Zhou et al., GZ114 2008) H26 ΔpsmA (Zhou et al., GZ130 2008) H26 P -psmB (Zhou et al., GZ138 tnaA 2008)

Plasmids pET24b Kmr; E. coli expression vector Novagen r Km ; pET24b psmB-his6 (β-His6) expressed in E. coli (Kaczowka and pJAM621 Maupin-Furlow, 2003) r Km ; pET24b psmA-his6 (α1-His6) expressed in E. coli (Kaczowka and pJAM622 Maupin-Furlow, 2003) r Km ; pET24b psmC-his6 (α2-His6) expressed in E. coli (Kaczowka and pJAM623 Maupin-Furlow, 2003) Kmr; pET24b psmA-t172g-his6 (α1-S58A-His ) This study pJAM2521 6 expressed in E. coli Kmr; pET24b psmA-a471g-his6 (α1-T158A-His ) This study pJAM2523 6 expressed in E. coli Kmr; pET24b psmA-a439g-his6 (α1-T147A-His ) This study pJAM2554 6 expressed in E. coli Kmr; pET24b psmA-a83t-his6 (α1-Y28F-His ) expressed This study pJAM2533 6 in E. coli Kmr; pET24b psmB-t385g-his6 (β-S129A-His ) This study pJAM2529 6 expressed in E. coli

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Strain or Plasmid Genotype; oligonucleotide used for amplification Source Kmr; pET24b psmB-a388g-his6 (β- T130A-His ) This study pJAM2531 6 expressed in E. coli Kmr; pET24b psmC-a37g-his6 (α2-T13A-His ) This study pJAM2537 6 expressed in E. coli Kmr; pET24b psmC-t40g-his6 (α2-S14A-His ) This study pJAM2525 6 expressed in E. coli Kmr; pET24b psmC-a235g-his6 (α2-T79A-His ) This study pJAM2527 6 expressed in E. coli Apr; Nvr; pBAP5010 P2 -panA (PanA) expressed in (Zhou et al., pJAM648 rrn Hfx. volcanii 2008) r r Ap ; Nv ; pBAP5010 P2rrn-psmB-his6 (β-His6) expressed (Kaczowka and pJAM202 in Hfx. volcanii Maupin-Furlow, 2003) r r Ap ; Nv ; pBAP5010 P2rrn-psmA-his6 (α1-His6) (Kaczowka and pJAM204 expressed in Hfx. volcanii Maupin-Furlow, 2003) r r Ap ; Nv ; pBAP5010 P2rrn-psmC-his6 (α2-His6) (Kaczowka and pJAM205 expressed in Hfx. volcanii Maupin-Furlow, 2003) Apr; Nvr; pJAM809 P2 -psmB-strepII (β-StrepII) (Humbard et al., pJAM816 rrn expressed in Hfx. volcanii 2009) Apr; Nvr; pJAM816 psmB-strepII psmA-his (β-StrepII, (Humbard et al., pJAM2545 6 α1-His6) expressed in Hfx. volcanii 2009) Apr; Nvr; pBAP5010 P2 -psmA-t172g-his6 (α1-S58A- This study pJAM2522 rrn His6) expressed in Hfx. volcanii Apr; Nvr; pBAP5010 P2 - psmA-a471g-his6 (α1- This study pJAM2524 rrn T158A-His6) expressed in Hfx. volcanii Apr; Nvr; pBAP5010 P2 - psmA-a439g-his6 (α1- This study pJAM2555 rrn T147A-His6) expressed in Hfx. volcanii Apr; Nvr; pBAP5010 P2 -psmA-a83t-his6 (α1-Y28F- This study pJAM2534 rrn His6) expressed in Hfx. volcanii Apr; Nvr; pBAP5010 P2 -psmB-t385g-his6 (β-S129A- This study pJAM2530 rrn His6) expressed in Hfx. volcanii Apr; Nvr; pBAP5010 P2 - psmB-a388g-his6 (β-T130A- This study pJAM2532 rrn His6) expressed in Hfx. volcanii Apr; Nvr; pBAP5010 P2 -psmC-a37g-his6 (α2-T13A- This study pJAM2538 rrn His6) expressed in Hfx. volcanii Apr; Nvr; pBAP5010 P2 -psmC-t40g-his6 (α2-S14A- This study pJAM2526 rrn His6) expressed in Hfx. volcanii Apr; Nvr; pBAP5010 P2 -psmC-a235g-his6 (α2-T79A- This study pJAM2528 rrn His6) expressed in Hfx. volcanii Apr; Nvr; pBAP5010 P2 -panA-t1018g (PanA-S340A) This study pJAM2553 rrn expressed in Hfx. volcanii Apr; Nvr; pJAM816 P2 -Hvo0135-strepII (Rio1- This study pJAM2558 rrn StrepII) expressed in Hfx. volcanii

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Strain or Plasmid Genotype; oligonucleotide used for amplification Source Apr; Nvr; pJAM816 P2 -Hvo0569-strepII (Rio2- This study pJAM2559 rrn StrepII) expressed in Hfx. volcanii

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Table 5-2. Site-directed mutants and phenotypes.

Mutation Substitution Experiment Phenotype psmA-a83t α1 Y28F Homology None psmA-t172g α1 S58A NetPhos Reduced pigmentation psmA-a439g α1 T147A MS / MS Slow growth, reduced pigmentation psmA-a471g α1 T158A NetPhos Unstable cells, low growth psmC-a37g α2 T13A MS / MS Not examined psmC-t40g α2 S14A MS / MS Not examined panA-t1018g PanA S340A MS / MS None

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Table 5-3. MS/MS results of Rio1p-Strep fractions. Protein Residue no. MASCOT E value Sequence 185 – 192 23 0.033 DYLEGDPR Rio1p 221 – 233 40 0.00072 AGVRVPKPIAVQR (HVO_0135) 193 – 205 23 0.034 FENIGHDKGQVVR 234 – 249 17 0.15 NVLVMELVGVVDDRAR

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Figure 5-1. Phosphatase-sensitive isoforms of α-type 20S proteasomal proteins as a function of Hfx. volcanii growth. A) An α1 isoform of pI 4.4 was present at all stages of growth with a more acidic isoform of pI 3.0 present in log- and early-stationary phase, but absent in late-stationary phase. Cell lyase was prepared from various stages of 9 -1 growth as indicated (1 OD600 unit ~ 1 × 10 CFU∙ml ), separated by 2-DE and analyzed by immunoblot using anti-α1 antibody (α-α1). B) The two α1 isoforms of pI 3.0 and 4.4 are associated in 20S proteasomes. 20S proteasomes were purified by Ni-NTA chromatography from early stationary-phase cells expressing β-His6. Proteins that flowed through the Ni-NTA column (5 mM imidazole) as well as those that bound the column (500 mM imidazole) were separated by 2-DE and analyzed by immunoblot using anti-α1 antibody (α-α1). Fractions were also assayed for peptidase activity using the fluorogenic endopeptidase substrate succinyl-Leu-Leu-Val-Tyr-7- amido-4-methylcoumarin (Suc-LLVY-AMC), as previously described (Wilson et al., 1999). u.d., undetectable. C) Both α1 and α2 isoforms are sensitive to phosphatase treatment. 20S proteasomes (purified as above) were treated with (red) and without (green) phosphatase, separated by 2-DE, and probed by immunoblot with anti-α1 and anti-α2 antibodies as indicated (α-α1 and α-α2, respectively). Data generated by Kheir Zuobi-Hasona.

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Figure 5-2. MS/MS Fragmentation of RGEDMSMQALpSTL, an internal phosphopeptide of the β subunit of 20S proteasomes. The fragment spans residues 119 to 131 of the deduced β protein and was generated by a sequential trypsin and chymotrypsin digest of the β subunit purified in complex with α1-His. The mass of the parent ion (1518.66 Da) corresponds to the mass of the peptide (1438.63 Da), deamination (1 Da) of glutamine (Q(E)), and phosphorylation (79.99 Da) of serine (pS). The individual Mascot ion score was 62 with an E value of 6.8e-4. A predominant b-ion series was detected due to the N-terminal arginine of the peptide. Characteristic CID neutral loss of H3PO4 and subsequent formation of dehydroalanine at residue 11 verified the phosphorylation site at Ser129. The b(11)- and b(12)-H3PO4 are b(11) - and b(12) - 97.99 Da, respectively. Loss of ammonia (*, 17.03 Da) and H2O (º, 18.01 Da) are indicated.

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Figure 5-3. MS/MS fragmentation of the de novo synthesized peptide RGEDMSMQALpSTL. Region corresponding to residues 119 – 131 of the deduced β protein. The synthetic peptide was analyzed by direct infusion into the QSTAR.

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Figure 5-4. A PanA-specific phosphopeptide was reproducibly detected by ESI-QTOF analysis of tryptic digestions of PanA purified from Hfx. volcanii DS70 cells. The phosphopeptide eluted from RP-HPLC at 69.05 min as a quadrupoly charged ion (m/z 684.8, 4+) and was composed of amino acid residues 337 to 361 of PanA (MNVpSDDVDFVELAEMADNASGADIK). MS/MS fragmentation confirmed that Ser340 was the phosphorylated amino acid. Despite the large, 25-amino acid peptide, a high Mascot score (70) and low expect value (1.4e-7) were obtained due to the assignment of 9 y-ions and 6 b-ions in the MS/MS fragmentation. Additional peaks were assigned that increased the confidence of this peptide including a neutral loss peak (–98 for phosphorylation) and several internal fragmentation ions. The non- phosphorylated peptide was detected in the same preparations.

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Figure 5-5. An α2-specific phosphopeptide detected in purified 20S proteasomes by MS/MS. The phosphopeptide, which eluted from RP-HPLC at 37.9 min, was detected as a doubly charged ion (m/z 558.2) by ESI-Q-ToF. The peptide had a Mascot score of 35 and an expect value (E) of 1.4e-3. The peptide was composed of amino acid residues 12 – 21 (GTSLFSPDGR) of α2. Unfortunately, MS/MS could not distinguish between Thr13 and Ser14 as the phosphosite. Although the MS/MS-fragmentation contained 6 unique y-ions and 3 unique b-ions, the close proximity of the phosphosite to the N-terminus of the peptide made the assignment difficult. A precursor ion scan was performed using a QQQ instrument (ABI QTRAP 4000) set to detect the neutral loss of a phosphate group. Several of the ion b-ions on the MS/MS fragmentation displayed the characteristic neutral loss (–98) for a phosphate, but the differentiating ions, b2 vs. y8 were missing in the spectrum. Since this phosphopeptide was detected by a precursor ion scan, the unphosphorylated peptide was not detected in the same run. However, subsequent experiments have detected the unphosphorylated form of this peptide.

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Figure 5-6. An α1-specific phosphopeptide was detected in purified 20S proteasomes by ESI-Q- ToF with precursor ion scanning. The peptide had a Mascot score of 30 and an expect (E) of 0.001. It was a doubly charged ion (m/z 479.3) mapping to a semi- tryptic fragment of α1 spanning amino acid residues 137 to 149 (ALLIGGVENGpTPR). Only one amino acid, Thr147, can be phosphorylated in that peptide, and MS/MS-annotation confirmed that Thr147 was the site of phosphorylation. The MS/MS fragmentation contained 7 y-ions and 6 out of the 7 showed the characteristic neutral loss (–98) indicative of a phosphorylation. Several other fragments ions could be assigned to internal sequencing ions of the peptide. These peak assignments bolster the MS/MS assignment. The peptide, although generated by a tryptic digest of a 20S proteasome preparation, was a semi-tryptic fragment, an internal chymotryptic-like cut on the N-terminus of the peptide. An additional scan using an error-tolerant algorithm for this phosphorylation site hit on two additional peptides; LIGGVENGpTP + 22 Da and LIGGVENGpT + 155 Da. The Mascot score for these two peptides were 50 and 58 and the E-values were 0.0006 and 0.001, respectively. The unphosphorylated form of the peptide was also detected in the same fraction.

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160

140

120

100 202c PanA 80 PanB

% survival % 60 S340A

0 1 1.5 2 2.5 Conc. NaCl (molar)

Figure 5-7. Hypo-osmotic stress response of H26 ΔpanA cells expressing pJAM202c, panA, panA t1018g (PanA S340A) and panB at different NaCl concentrations.

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Figure 5-8. Phenotype of GZ130 (H26 ΔpsmA) cells expressing various α-type subuntis from Hfx. volcanii. ATCC novobiocin plate with cells expressing different α-type subunits streaked out. Both α1 T147A and α1 T158A inhibit growth of Hfx. volcanii cells and α1 S58A and α1 T147A alter the pigmentation of Hfx. volcanii ΔpsmA cells.

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Figure 5-9. Rio-kinases in Hfx. volcanii. A) Rio-type kinase sequence explanation. Conserved domains in bold and underlined. Rio2p kinase does not have a complete C-terminal domain but contains three of the five residue. B) Purification / gel filtration of Rio- type kinases. Lane 1 is cell lysate and lane two is a electrophoretical pure Rio1p running at approximately 50 kDa. C) Autoradiographs of Rio in vitro phosphorylation with varying levels of divalent cations Mg2+ and Mn2+. Optimal autophosphorylation happened in the presence of Mg2+ between 10 – 50 mM final concentration. The band labeled with the “*” has not been identified.

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Figure 5-10. Strep_II tagged Rio1p purified from Hfx. volcanii phosphorylation of α1His6 subunits purified from E. coli. A) Rio1p used for in vitro phosphorylation of recombinantly purified α1 (boiled and unboiled), 20S core particles purified from Hfx. volcanii (boiled and unboiled) and casein (boiled). B) Rio1p used for in vitro phosphorylation of recombinantly purified α1 proteins. The α1 proteins have not been boiled.

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Figure 5-11. MS/MS spectra of a methylesterified peptide fragment of α1 corresponding to residues 105 – 116, YGEPIGIEmethylTLTK. Several neutral loss peaks indicating a loss of a methyl group were identified.

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Figure 5-12. MS/MS spectra of a methylesterified peptide fragment of α1 corresponding to residues 58 – 68, SPLMEmethylPTSVEK.

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Figure 5-13. MS/MS spectra of a methylesterified peptide fragment of α1 corresponding to residues 23 – 30, LYQVEmethylYAR.

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Figure 5-14. MS/MS spectra of a methylesterified peptide fragment of α1 corresponding to residues 13 – 22, GITIFSPDmethylGR.

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Figure 5-15. MS/MS spectra of a methylesterified peptide fragment of α1 corresponding to residues 150 – 163, LYETDPSGTPYEmethylWK.

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Figure 5-16. Multiple sequence alignment of select α-type 20S proteasome proteins. Phopshorylation sites determined by biochemical studies are boxed and indicated by ● below the alignment. These include: Hs_α7 Ser249 (Claverol et al., 2002; Beausoleil et al., 2004); Hs_α7 Tyr160, Hs_α2 Tyr23 and Hs_α2 Tyr97 (Rush et al., 2005); Rat_α2 Tyr120 (Benedict and Clawson, 1996); Hs_α5 Ser56 (Beausoleil et al., 2004); Rat_α7 and monkey Ser243 and Ser250 (Castano et al., 1996; Bose et al., 2004); and Cal_α3 Ser248 (Fernandez et al., 2002). Abbreviations: Hs, Homo sapiens; Cal, Candida albicans; Rat, Rattus norvegicus; Hvo, Haloferax volcanii. Swiss-Prot or GenBank accession numbers are as follows: Hs_α1, P60900; Hs_α2, P25787; Rat_α2, P17220; Hs_α3, P25789; Cal_α3, 46441895; Hs_α4, O14818; Hs_α4-like, Q8TAA3; Hs_α5, P28066; Hs_α6, P25786; Hs_α7, P25788; Rat_α7, 203207; Hvo_α1, Q9V2V6; Hvo_α2, Q9V2V5. Conserved residues are highlighted in grey and black with amino acid position numbers indicated on the right and left.

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Figure 5-17. Multiple sequence alignment of the central region of select β-type 20S proteasome proteins. Phosphorylation sites determined by biochemical studies are boxed and indicated by ● below the alignment. These include: Hs_β7 Tyr102 and Hs_β2 Tyr154 (Rush et al., 2005); Hvo_β Ser129 (This study). Abbreviations: Hs, Homo sapiens; Hvo, Haloferax volcanii. Swiss-Prot or GenBank accession numbers are as follows: Hs_β1, P28072; Hs_β1i, P28065; Hs_β2, Q99436; Hs_β2i, P40306; Hs_β3, P49720; Hs_β4, P49721; Hs_β5, P28074; Hs_β5i, P28062; Hs_β6, P20618; Hs_β7,P28070; Hvo_β, Q9V2V4. Conserved residues are highlighted in grey and black with amino acid position numbers indicated on the right and left.

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CHAPTER 6 SUMMARY AND CONCLUSIONS

Summary of Findings

The goals of this study were to advance our understanding of how the different proteins of

the proteasome system in Haloferax volcanii are modified post-translationally and to understand

the effect those modifications had on the function of the proteasome and cellular biology of Hfx.

volcanii. Much has been learned about proteasome systems in the past five years including

advances in proteasome assembly (Hirano et al., 2008; Rosenzweig and Glickman, 2008a;

Sharon et al., 2007), structure of regulatory ATPases and core particles (Medalia et al., 2009;

Djuranovic et al., 2009), and new targeting mechanisms for degradation by prokaryotic proteasomes (Mukherjee and Orth, 2008). Specifically, the components of the proteasome system are essential for cell viability under standard laboratory conditions and are important in hypo-osmotic stress response and heat shock in Hfx. volcanii (Zhou et al., 2008).

This study demonstrated that all three of the 20S proteasomal proteins of Hfx. volcanii (α1,

α2, and β) are modified either post- or co-translationally. In addition, the proteasome-activating nucleotidase PanA is also post-translationally modified. Both α-type subunits, α1 and α2 contained N-terminal acetyl groups. Closer examination of the α1 protein revealed that there was a mixture of acetylated methionine (Ac1) and unacetylated glutamine (the second amino acid of the deduced protein sequence, N2) as the N-terminal residue. Quantitative mass spectrometry revealed the acetylated form of α1 (Ac1) was in 100-fold excess of unacetylated and cleaved forms (N2). To further examine this, α1 proteins with penultimate (second) residue substitutions as well as an α1 protein with a deletion in the α-helical ‘gate’ (residues 2-12) were synthesized in Hfx. volcanii cells devoid of the chromosomal copy of the psmA (α1) gene. These

α1 variants were analyzed for acetylation as a whole population as well as those assembled into

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20S core particles. While α1 ΔGln2-Arg12 was not modified, α1 Gln2Ala was cleaved and

acetylated on its penultimate residue (alanine), and α1 Gln2Ser and α1 Gln2Val were acetylated

on their initiating methionine residues 100 % of the time. The α1 Gln2Asp, α1 Gln2Pro, and

α1Gln2Thr were a mixture of unmodified, acetylated and/or cleaved forms; however, only forms acetylated on the initiating methionine were observed in 20S core particles. Interestingly, cells expressing α1 Gln2Asp and α1 Gln2Thr variants were more tolerant of hypo-osmotic stress and grew at higher temperatures than cells expressing unmodified α1. These results suggest acetylation of α1 is important for and/or requires 20S proteasome assembly and bolster our earlier findings that α1 plays an important role in the stress response of Hfx. volcanii.

In addition to acetylation, each of the proteins of 20S core particles and PanA are modified by phosphorylation. To investigate the sites and role of post-translational modifications on proteasomal proteins, Haloferax volcanii cell lysate was separated by 2-DE and analyzed by immunoblot. Using this approach, phosphatase sensitive isoforms of α1 were detected in lag- and log-phase cells. Further analysis by tandem mass spectrometry (MS/MS) of proteasomal proteins purified from Hfx. volcanii revealed β Ser129, α1 Thr147, α2 Thr13 or Ser14 and PanA

Ser340 phosphosites. To further investigate these and other putative phosphosites, a variety of protein variants were generated by site-directed mutagenesis including α1 Ser58Ala, α1

Thr158Ala, α1 Tyr28Phe, α1 Thr147Ala, α2 Ser13Ala, α2 Thr14Ala, α2 Thr79Ala, β Ser129Ala,

β Thr130Ala, and PanA Ser340Ala. Of these, the α1 and PanA variants were examined for their ability to complement the hypoosmotic-sensitive phenotype of ∆psmA (α1) and ∆panA (PanA) strains. While the PanA Ser340Ala and the α1 Tyr28Phe variants were able to complement the mutant strains to wild-type, the α1 Ser58Ala, α1 Thr147Ala, and α1 Thr158Ala variants did not.

Whether the failure to complement was caused by an inability to phosphorylate those individual

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residues is unclear. A RIO-type kinase (Hvo_0135) purified from the native host Hfx. volcanii

was able to catalyze autophosphorylation as well as the phosphorylation of wild-type α1, α1

Ser58Ala, α1 Thr147Ala purified from recombinant E. coli with γ-32P-ATP. Stoichiometry of

phosphotransfer was less for both of the variants α1 Ser58Ala and α1 Thr147Ala (about 40% wild-type). RIO did not transfer any phosphate to α1 Thr158Ala. This could indicate that perturbing residue 158 could change the structure of α1 such that it can no longer be phosphorylated by Rio1p or that the phosphorylation events are sequential and the phosphorylation of Thr158 is the first site of modification. In addition to phosphorylation,

MS/MS-scans revealed multiple methylation sites on the α1 protein that were highly reproducible. The exact role and the enzyme responsible for this latter modification remain to be seen.

In addition to specific work done on the proteasome system in Hfx. volcanii, advances in the understanding of phosphorylation and acetylation as global processes in the cell have also been made (Kirkland et al., 2008b; Kirkland et al., 2008a). A large subset of the proteome was found to N-terminally acetylated. This is evidence of a generalized acetylation pathway in Hfx. volcanii. Several putative N-terminal acetyltransferases genes were identified in the genome sequence and some of them clustered with known acetyltransferases from yeast.

All together, the proteins of the proteasomal system in Hfx. volcanii are heavily modified co- and / or post-translationally. Altering some of these modifications has drastic effects on cell viability and proteasome function. Further study of the mechanisms and phenotypes of these modifications will give researchers greater insight into proteasome systems in the haloarchaea as well as basic biology of the cell.

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This work has resulted in the publication of three peer reviewed primary author papers with a fourth in preparation as well as three secondary author papers, one patent application, and

three review articles.

Future Directions

Future investigation as a continuation of this work will focus on further characterizing the

post-translational modifications by examining the enzymology of the separate modifications.

Hfx. volcanii synthesizes several putative N-terminal acetyltransferases, but no activity has been

assigned to any of them. Biochemical characterization and establishing substrate specificity as

well as identification of in vivo substrates is required to better understand this process. The

mechanism of N-terminal acetylation is still unknown, whether it is co-translational or post-

translational. Great advances have taken place over the past few years with respect to the

identification of sites of phosphorylation on archaeal proteins. In vivo work with different

kinases, analyzing different substrates to assign substrate specificity to different kinases may

help to better understand the targeting pathway proteins take to the proteasome or how the

proteasome is specifically regulated. Finally, identifying the environmental or physiological

signals that trigger changes in modification, specifically with respect to phosphorylation and

methylation of proteasomes, is important in understand the role these modifications are playing

in the cell.

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APPENDIX A PRIMERS USED IN THIS STUDY

Table A-1. Oligonucleotide primers used in this study.

Mutation (protein a variant) Primer Sequence psmA (wild-type) 5’-GCTCTAGACATATGCAGGGACAAGCG-3’ psmA* (α1 Q2A) 5’-GCTCTAGACATATGGCGGGACAAGCG-3’ psmA* (α1 Q2D) 5’-GCTCTAGACATATGGACGGACAAGCG-3’ psmA* (α1 Q2P) 5’-GCTCTAGACATATGCCGGGACAAGCG-3’ psmA* (α1 Q2S) 5’-GCTCTAGACATATGTCGGGACAAGCG-3’ psmA* (α1 Q2T) 5’-GCTCTAGACATATGACGGGACAAGCG-3’ psmA* (α1 Q2V) 5’-GCTCTAGACATATGGTGGGACAAGCG-3’ psmA*(α1 Δ2–12) 5’-CGGGATCCCATATGGGGATTACGATCTTCTCGCCGGAT-3’ psmA-his6 rev 5’-CCGATGCTCAGCTTAGTGGTGGTGGTGGTGCTCTTCGGTCTGTTCGTCGTCCTCGG-3’ psmC* (α2 N2T) 5’-GCTCTAGACATATGACCCGAAACGACAAGC-3’ psmC-his6 rev 5’-CCGATGCTGAGCTTAGTGGTGGTGGTGGTGCTCCTCGCGTTCGTCCGTCTCGTCGGAα-3’ psmB-strepII for 5’-CTTACCTCATATGCGTACCCCGACTC-3’ psmB-strepII rev 5’-TGGTACCTTCAAGGCCTTCGAAGTTC-3’

aNdeI, BlpI and KpnI restriction sites used for cloning are underlined. The start and stop codons of psmA and psmB are highlighted in grey.

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Table A-2. Oligonucleotide primer pairs for site-directed mutagenesis.

Mutation Primer Sequence (5'  3') psmA-t172g (α1 S58A) 5’-CGGACAAGCGCTCTCGCGCGCCGCTGATGGAAC-3’ 5’-GTTCCATCAGCGGCGCGCGAGAGCGCTTGTCCG-3’ psmA-a471g (α1 T158A) 5’-GACCGACCCCTCGGGCGCCCCCTACGAGTGGAAGGC-3’ 5’-GCCTTCCACTCGTAGGGGGCGCCCGAGGGGTCGGTC-3’ psmA-a439g (α1 T147A) 5’-GGCGGCGTCGAAAACGGTGCGCCGCGCCTCTACGAG-3’ 5’-CTCGTAGAGGCGCGGCGCACCGTTTTCGACGCCGCC-3’ psmA-a83t (α1 Y28F) 5’-CTCTATCAGGTCGAATTCGCTCGTGAGGCCGTC-3’ 5’-GACGGCCTCACGAGCGAATTCGACCTGATAGAG-3’ psmB-t385g (β S129A) 5’-AGCATGCAGGCGCTGGCGACGCTCGTCGGCAACTTC-3’ 5’-GAAGTTGCCGACGAGCGTCGCCAGCGCCTGCATGCT-3’ psmB-a388g (β T130A) 5’-ATGCAGGCGCTGTCGGCGCTCGTCGGCAACTTCCTC-3’ 5’-GAGGAAGTTGCCGACGAGCGCCGACAGCGCCTGCAT-3’ psmC-a37g (α2 T13A) 5’-GCCTACGACCGCGGAGCGTCGCTTTTCTCCC-3’ 5’-GGGAGAAAAGCGACGCTCCGCGGTCGTAGGC-3’ psmC-t40g (α2 S14A) 5’-GCCTACGACCGCGGAACGGCGCTTTTCTCCCCCGAC-3’ 5’-GTCGGGGGAGAAAAGCGCCGTTCCGCGGTCGTAGGC-3’ psmC-a235g (α2 T79A) 5’-ACGCGCTCGGCGCGGCCGCGGCCGGCCACGTCGCC-3’ 5’-GGCGACGTGGCCGGCCGCGGCCGCGCCGAGCGCGT-3’ panA-t1018g (PanA S340A) 5’-ACCCGGAAGATGAACGTCGCCGACGACGTGGACTTCGTC-3’ 5’-GACGAAGTCCACGTCGTCGGCGACGTTCATCTTCCGGGT-3’

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Table A-3. Oligonucleotide primer pairs for cloning of kinase genes from Hfx. volcanii.

Gene Number Primer Sequence (5'  3') HVO_0135 (Rio1p) 5’-CGTAGCCCTAGTCATATGACTGACGAG-3’ 5’-GGGTTGGGTACCCTCGTCTCCGTCTCCGTCC-3’ HVO_0569 (Rio2p) 5’-CGGACGGACGCATATGGTACGGAACGTCGCC-3’ 5’-CGGCGGTACCGACGGCGAACTCGTCGACGCTC-3’ HVO_2849 (PrkA1) 5’-GGCTCCACGAACATATGACCGGCG-3’ 5’-CATGGTCAGGTACCCCCTTCGAGTTCGGTC-3’

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Table A-4. Oligonucleotide primer pairs for N-terminal acetyltransferase knockouts.

Construct Description Primer Sequence (5'  3') HVO_1954 ± 500 bp 5’-CGCCTCTAGAACTTCACGCTC-3’ 5’-CGAGGCCCTCGAGAATCGTG-3’ HVO_2709 ± 500 bp 5’-CGAGGACGTCTAGATTGAACGGG-3’ 5’-GTGCATCTCGATGGCGATGTCCAC-3’ ΔHVO_1954 (inverse) 5’-TCGACGTGGCGGAGTGG-3’ 5’-CGACTCGCCTGACGACC-3’ ΔHVO_2709 (inverse) 5’-GCTCTCGGAGTTCCTCC-3’ 5’-CTTCGGTATGGGATTCACG-3’

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BIOGRAPHICAL SKETCH

Matthew Adam Humbard was born in February of 1980 in Pittsburg, Kansas. He received a Bachelor of Science in Microbiology from Arizona State University in August of 2003 in

Tempe, Arizona. He worked as an undergraduate research assistant for Dr. Rajeev Misra on protein-protein interaction of the multidrug resistance efflux pump AcrAB-TolC of Escherichia coli and toxin colicin E1 import mechanism across the outer membrane of E. coli until he joined the graduate program at the Department of Microbiology and Cell Science at the University of

Florida in August of 2004. He began working with Dr. Julie Maupin-Furlow on post- translational modification of 20S proteasomes from Haloferax volcanii. He married Kristina

Marie Palmer on October 14th, 2006 in Gainesville, Florida. He attended several national and international scientific meetings and was a member of United States delegation to the 57th

Meeting of Nobel Laureates in Lindau, Germany in 2007. Matthew Humbard plans on continuing his work in proteolysis at the National Cancer Institute at the National Institutes of

Health campus in Bethesda, Maryland.

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