POLYGLUTAMINE TRACT EXPANSION INCREASES PROTEIN S-NITROSYLATION and the BUDDING YEAST ZYGOTE TRANSCRIPTOME by CHUN-LUN NI

POLYGLUTAMINE TRACT EXPANSION INCREASES PROTEIN

S-NITROSYLATION AND

THE BUDDING YEAST ZYGOTE TRANSCRIPTOME

CHUN-LUN NI

Submitted in partial fulfillment of the requirements

For the degree of Doctor of Philosophy

Dissertation Advisor: Dr. Alan M. Tartakoff

Cell Biology Program,

Department of Molecular Biology and Microbiology

CASE WESTERN RESERVE UNIVERSITY

January, 2017

CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the dissertation of

CHUN-LUN NI

Candidate for the Doctor of Philosophy degree.

(signer) Piet A. J. de Boer, Ph.D.

(chair of the committee)

Alan M. Tartakoff, Ph.D.

Cathleen R. Carlin, Ph.D.

Thomas T. Egelhoff, Ph.D.

Xin Qi, Ph.D

Man-Sun Sy, Ph.D.

(Date) November, 2016

Table of Contents

List of Tables vii

List of Figures and Supplementary Figures viii

Acknowledgements xii

List of Abbreviations xiv

Abstract xvi

Chapter 1 “Introduction of Huntington’s disease and Huntingtin regulation.” 1

1-1. Genetics of Huntington’s disease (HD) 1

1-1-1. CAG trinucleotide expansion links to HD 2

1-1-2. Polyglutamine expansion and Huntingtin aggregation 2

1-1-3. Evolutionary conservation and the HEAT repeat motif 3

1-2. Huntingtin and cellular physiology 4

1-2-1. Embryogenesis 5

1-2-2. BDNF transport and expression 6

1-2-3. Mitochondrial fragmentation 7

1-2-4. Protein-protein interaction 8

1-3. Huntingtin is regulated by posttranslational modifications 10

1-3-1. Proteolysis 11

1-3-2. Ubiquitination and SUMOylation 13

1-3-3. Phosphorylation 15

1-3-4. Acetylation 19

1-3-5. Myristoylation 19

1-3-6. S-palmitoylation 20

Chapter 2 “Introduction of cysteine modifications” 28

2-1. Overview of cysteine modifications 28

2-2. S-nitrosylation 29

2-3. S-acylation 34

2-4. S-nitrosylation and other posttranslational modifications 36

2-5. Cysteine modifications of the HEAT repeat motif 37

Chapter 3 “Polyglutamine expansion increases protein S-nitrosylation” 40

3-1. Discovery of Huntingtin S-nitrosylation 40

3-2. PolyQ expansion increases protein S-nitrosylation 41

3-2-1. PolyQ expansion increases Htt S-nitrosylation 41

3-2-2. PolyQ expansion increases Ataxin-1 S-nitrosylation 42

iii

3-2-3. PolyQ-dependent high molecular weight species are highly 43 cysteine-modified

3-3. S-nitrosylation and S-acylation occur at multiple sites of Htt 44

3-3-1. Htt N548 fragment S-nitrosylation and S-acylation 44

3-3-2. Full-length Htt S-nitrosylation and S-acylation 45

3-3-3. Identification of a major site of S-nitrosylation and S-acylation 46

3-3-4. Htt Phosphorylation in response to nitric oxide donor treatment 46

3-4. Polyglutamine-induced S-nitrosylation is not a global effect 47

3-4-1. PolyQ-expanded Htt does not change global S-nitrosylation and 47

S-acylation

3-4-2. PolyQ-expanded Htt does not affect gross S-nitrosylation and 47

S-acylation of normal Htt

Chapter 4 “Interaction of Huntingtin and nitric oxide synthases (NOS)” 66

4-1. NOS expression increases Htt inclusions 66

4-2. PolyQ-expansion does not markedly increase NOS-Htt interaction 67

Chapter 5 ” Discussion: Polyglutamine expansion and protein S-nitrosylation” 73

5-1. PolyQ modulates specificity of S-nitrosylation and S-acylation 73

5-2. S-Acylation and S-palmitoylation 73

5-3. Inspecting a S-nitrosylated HEAT repeat motif of Htt 74

5-4. Significance of S-nitrosylation of Htt 75

5-5. Future directions 79

Chapter 6 “Zygote formation in S. cerevisiae” 85

6-1. Cell-cell fusion and budding yeast zygote formation 85

6-1-1. Two mating types of budding yeast and the pheromone response 86

6-1-2. Cell membrane fusion and nuclear envelope fusion 87

6-1-3. The yeast spindle pole body 88

6-1-4. Karyogamy deficiency 89

6-1-5. Ploidy and chromosome tethering 90

6-2. Transcriptome profiles of yeast zygotes 92

6-2-1. Budding yeast zygote purification and transcriptome analysis 92

6-2-2. Genetic determinants of budding yeast cell types 92

6-2-3. The pheromone response transcriptome 94

6-2-4. Zygote-specific transcriptome 97

6-3. Discussion 101

6-4. Future directions 102

Appendix “Materials & Methods” 130

A1. Reagents 130

A2. Mammalian cell culture and recombinant protein expression 131

A3. Mouse tissues 132

A4. Plasmids used in this study 132

A5. Site-directed mutagenesis 133

A6. Detection of S-nitrosylation and S-acylation by resin-assisted capture 133

A7. Detection of S-nitrosylation and S-acylation sites by LC-MS/MS 134

A8. MS data analysis 136

A9. Fluorescent microscopy study of EGFP-tagged Htt N548 inclusions 137

A10. Htt HEAT repeat motif simulation 137

A11. Yeast strain growth and zygote purification 138

A12. RNA purification and microarray analysis 139

A13. Microarray data analysis 140

A14. Gene Ontology (GO) analysis 141

Bibliography 151

List of Tables

Table 1-1 Htt-interacting proteins 25

Table 1-2 Age- and cerebellum-specific interactomes in HD mouse model 26

Table 1-3 Htt posttranslational modifications (PTMs) 27

Table 3-1 Summary of SNO and S-acylation sites in Htt N548Q15 and 63

N548Q128

Table 3-2 Summary of increased SNO and phosphorylation of Htt 65

N548Q15 by the nitric oxide donor eCysNO

Table 6-1 Spindle pole body genes 115

Table 6-2 Pheromone-responsive genes (top 100) 116

Table 6-3 A subset of pheromone-responsive genes 119

Table 6-4 Zygote-specific genes 120

Table 6-5 Zygote-specific genes (ordered by type) 125

Table 6-6 Additional Type I zygote-specific genes 126

Table 6-7 Type II-IV zygote-specific genes with manually-clustered 127

functions

Table 6-8 Peak expression of zygote-specific genes in haploid cells 128

vii

List of Figures and Supplementary Figures

Figure 1-1 Htt is a conserved protein 22

Figure 1-2 Full-length Htt, Htt N-terminal fragment and posttranslational 24

modifications

Figure 2-1 Reduced form of glutathione (GSH), thioredoxin (Trx), or 39

coenzyme A (CoA) denitrosylates SNO modifications via

transnitrosylation

Figure 3-1 Htt constructs 49

Figure 3-2 PolyQ expansion increases S-nitrosylation of Htt 50

Figure 3-3 PolyQ expansion increases S-nitrosylation of Ataxin-1 52

Figure 3-4 PolyQ expansion in Ataxin-1 and Htt N548 proteins increases 54

S-nitrosylated and S-acylated high molecular weight (HMW)

species

Figure 3-5 The high molecular weight (HMW) species of Ataxin-1Q85 do 56

not disappear at 37ºC incubation with sample buffer

Figure 3-6 Summary of the SNO and S-acylation sites identified by 57

LC-MS/MS

viii

Figure 3-7 The fragment beyond the N-terminal region is S-nitrosylated and 58

S-acylated

Figure 3-8 The C214S mutation reduces Htt S-nitrosylation and S-acylation 59

in the context of the normal polyQ tract

Figure 3-9 Expression of polyQ-expanded Htt does not significantly increase 60

S-nitrosylation and S-acylation of global proteins

Figure 3-10 Expression of polyQ-expanded Htt does not significantly increase 61

S-nitrosylation and S-acylation of wild-type Htt

Figure 4-1 Co-expression of nitric oxide synthase (NOS) promotes Htt N548 68

inclusion formation

Figure 4-2 Inclusions of N548-EGFP in cells expressing nitric oxide 70

synthase

Figure 4-3 NOS co-expansion increases S-nitrosylation of Htt N548Q15 and 71

NOS-N548 interaction is not significantly affected by polyQ

expansion

Figure 5-1 Local environment of S-nitrosylated cysteine residues of Htt 80

Figure 5-2 Flanking sequences of S-nitrosylated cysteine residues of Htt 81

Figure 5-3 Full-length Htt is S-nitrosylated in mouse tissues and polyQ 82

expansion increases S-nitrosylation of full-length Htt expressed in

neuron-like PC12 cells

Figure 5-4 Cellular dysfunctions result from Htt oligomers 84

Figure 6-1 Life cycle of budding yeast (S. cerevisiae) 103

Figure 6-2 Structure and protein components of budding yeast spindle pole 105

body

Figure 6-3 Yeast cell-type specificity regulation 106

Figure 6-4 Upregulated haploid MATa- and MATα-specific genes that are 107

identified in both previous studies and in this study

Figure 6-5 Downregulated genes in diploid cells that are identified in both 108

previous studies and in this study

Figure 6-6 Pheromone-responsive genes identified in both studies and their 109

expression profiles

Figure 6-7 Zygote-specific genes and clustered gene functions 111

Figure 6-8 Identified genes whose products are involved in the chromosome 113

organization and segregation

Figure 6-9 Identified genes whose products are involved in the respiratory 114

chain of mitochondria

Figure S1 Detection of protein S-nitrosylation by SNO-RAC and protein 142

S-acylation by acyl-RAC

Figure S2 Determination of S-nitrosylated and S-acylated cysteine residues 144

by Mass spectrometry (MS)

Figure S3 MS/MS analysis 146

Figure S4 Separation of yeast zygote cells and pheromone-stimulated 150

haploid cells by flow cytometry

Acknowledgements

First of all, I would like to thank Dr. Tartakoff for supporting me through these years to develop this thesis. I appreciate the committee members (Dr. de Boer, Dr. Carlin, Dr.

Egelhoff, Dr. Qi, and Dr. Sy), who have made great efforts to help me improve the organization of this thesis. For the S-nitrosylation study, I would like to thank Dr. Stamler and his team, especially Dr. Seth, Dr. Fonseca, and Dr. Hayashi. As to the mass spectrometry, I would like to thank Dr. Wang in the Center for Proteomics and

Bioinformatics, CWRU. I also thank Dr. Xiao in the Department of Pathology for the structure simulation and suggestions. I feel grateful to Dr. Leahy and Dr. Bai for inspecting the yeast transcriptome data analysis.

People who have worked in Dr. Tartakoff’s lab, especially Serendipity, Rose, Vipul,

Eric, Krysta, Purnima, Kyle, Phillip, and David, have helped me establish lots of works in the lab. Dr. Monnier and the people in his lab, especially Dr. Fan and Jeremy, are generous and share their experiences with me. Dr. Singh and the people in her lab, especially Ajai and Juan, also help me a lot. My classmates, especially Urvashi, Kien,

Ryan, Dan, Bobby, Jen, Marissa, Hieu, Jaffre, and James, are always willing to discuss with me when I have questions. I am also grateful to all my friends who have helped me in life and work for these years, especially Dr. Y.S. Chen, Dr. H.C. Lin, Dr. K.S. Hsu, Dr. xii

Racca, Dr. Y.C. Lin, Dr. I.J. Yeh, Dr. T.F. Kuo, Dr. Balow, Janice, John, Johnnie, Brinn,

Christine, Corrie, Donna Mary, Lorrie, Laura, Luke, Liang, and Annie.

Last, but certainly not least, I would like to thank my family for their understanding and support.

xiii

List of Abbreviations

FC Fold change

GO Gene ontology

HD Huntington’s disease

HEAT Huntingtin, Elongation factor 3, protein phosphatase 2A, and the

yeast kinase TOR1

HMW High molecular weight

HTT Huntingtin gene

Htt Huntingtin protein

IT15 Interesting transcript number 15 gene (HTT)

LC-MS/MS Liquid chromatography coupled with tandem mass spectrometry

MATa Yeast haploid cell mating type “a”

MATα Yeast haploid cell mating type “α”

MT Microtubule

MTOC Microtubule organizing center

N548 Htt N-terminal 548 a.a. fragment

NO Nitric oxide

xiv

NOS Nitric oxide synthase

NOS1/nNOS Neuronal NOS

NOS2/iNOS Inducible NOS

NOS3/eNOS Endothelial NOS

PolyQ Polyglutamine tract

PTM Posttranslational modification

RAC Resin-assisted capture

S-acyl S-acylation

SCA1 Spinocerebellar ataxia type 1

SNO S-nitrosylation

SPB Spindle pole body

Polyglutamine Tract Expansion Increases Protein S-Nitrosylation and

the Budding Yeast Zygote Transcriptome

Abstract

CHUN-LUN NI

This thesis describes two distinct projects in my graduate study:

(A) The relationship between polyglutamine (polyQ) expansion and protein

S-nitrosylation (Chapter 1-5).

(B) Transcriptome remodeling during yeast zygote formation (Chapter 6).

Chapter 1 describes the general background of Huntington’s diseases (HD), mutations of the Huntingtin protein (Htt), and the regulatory post-translational modifications (PTMs) of Htt. Chapter 2 specifically describes the current knowledge about regulations of S-nitrosylation and S-acylation on cysteine residues and their effects.

Chapter 3 discusses our discovery that Htt S-nitrosylation is polyQ-dependent. We also observe a comparable polyQ expansion effect for Ataxin-1, another polyQ-containing protein whose polyQ expansion is pathogenic. Additionally, neither global S-nitrosylation nor normal Htt S-nitrosylation is increased in cells expressing polyQ-expanded Htt. Thus, polyQ expansion may regulate intramolecular S-nitrosylation. xvi

Mass spectrometry demonstrates that multiple sites are both S-nitrosylated and S-acylated in Htt. We test the effect of the C214S mutation on Htt S-nitrosylation and S-acylation because C214 was demonstrated to be a S-palmitoylation site. We find that this mutation significantly reduces S-nitrosylation and S-acylation in the context of normal polyQ but not for the expanded form. Therefore, polyQ expansion may regulate the profile of cysteine modifications within a protein molecule.

In the Chapter 4, I discuss the interaction between nitric oxide synthases (NOSs) and

Htt. We show that co-expression of Htt and NOS increases Htt inclusions in cultured cells.

Nevertheless, co-immunoprecipitation experiments do not show markedly polyQ length-dependent Htt-NOS physical association.

Chapter 5 first summarizes the results of this study, describes the computational simulation of HTT HEAT repeat motifs in cluster 1, and discusses the possible biological significance of S-nitrosylation regulated by polyglutamine tracts. We propose a simulated structure of the N-terminal fragment, based on the studies of other HEAT repeat structures and find that several S-nitrosylated and S-acylated cysteine residues are spatially clustered. Modifications of these cysteine residues therefore may be important for protein conformational changes and/or protein-protein interactions.

In Chapter 6, I describe transcriptome analysis of budding yeast (S. cerevisiae) xvii zygotes. For this purpose, we isolated zygotes and compared their transcriptomes to those of haploid cells, pheromone-stimulated haploid cells, and to diploid cells. We identified groups of genes that coincidentally increase or decrease in zygote via microarray analysis.

These genes may imply the regulation of chromosome modulation and mitochondrial respiratory chain relevant to zygote physiology. This chapter is ended with the discussion and future directions for yeast zygote transcriptome study.

Parts of the content of this thesis have been published:

Polyglutamine Tract Expansion Increases S-Nitrosylation of Huntingtin and Ataxin-1

Chun-Lun Ni, Divya Seth, Fabio Vasconcelos Fonseca, Liwen Wang, Tsan Sam Xiao,

Phillip Gruber, Man-Sun Sy, Jonathan S. Stamler, and Alan M. Tartakoff. PLoS One.

2016

Flow cytometry-based purification of S. cerevisiae zygotes.

Serendipity Zapanta Rinonos, Jeremy Saks, Jonida Toska, Chun-Lun Ni, and Alan M.

Tartakoff. J Vis Exp. 2012

xviii

Chapter 1

Introduction of Huntington’s disease and Huntingtin regulation

Summary

Huntington’s disease (HD) is an autosomal dominant disease of progressive neurodegeneration. This Chapter starts at the discovery of HTT gene, the genetic cause of

HD, the encoding protein Huntingtin (Htt), and the translation of mutation at protein level: expansion of polyglutamine tract (polyQ). Then it describes Htt pathogenic characteristics, physiological functions, sequence conservation among species, and the regulation by posttranslational modifications.

1-1. Genetics of Huntington’s disease (HD)

HD was first clinically characterized by George Huntington, an American physician, in 1872 [1]. A recent systematic review suggests the prevalence of HD is 5.7 per 100,000 people in North America, Europe, and Australia whereas 0.40 per 100,000 in Asia [2].

HD is an inherited movement disorder characterized by progressive neurodegeneration that primarily affects medium spiny neurons of the striatum, as well as other neuronal cell types [3].

Known as an autosomal dominant disease via pedigree studies, the HD-linked locus was identified by Gusella and his colleagues in 1993. It was called IT15 gene for

“Interesting Transcript number 15” and later renamed as HTT, which is located on the short arm of chromosome 4, spans ~180 kb, and encodes a protein (Huntingtin; Htt) ~350 kDa [4].

1-1-1. CAG trinucleotide expansion links to HD

In the same study, Gusella and colleagues identified a novel trinucleotide (CAG) repeat in HTT exon-1 [4]. The number of CAG repeats in this region is variable among individuals [5-8]. Normal people have 18 CAG repeats, on average. In contrast, HD patients have at least one HTT allele with a CAG repeat to 48 on average. Later research subdivided CAG repeat number into four intervals: (1) Normal alleles have a repeat number less than 26. (2) Repeat numbers between 27 and 35 are normal but mutable since de novo mutations in meiosis during germ cell formation can lead to CAG repeat expansion, which subsequently results in HD pathogenesis in offspring [6]. (3) Repeats from 36 to 39 result in HD with low penetrance. A recent cohort study suggests that in the range of 36-39 repeats, ~40% of individuals will be asymptomatic at the age of 65 and

~30% at the age of 75 [9]. (4) Repeats of greater than 40 will absolutely progress to HD

(100% penetrance).

1-1-2. Polyglutamine expansion and Huntingtin aggregation

The (CAG) repeats encode a polyglutamine tract (polyQ). PolyQ-expanded forms of Htt, as well as many other proteins implicated in neurodegeneration, accumulates in the cytoplasm, where they form dynamic “aggregates” [10]. The in vitro and in vivo aggregates of Htt aggregates exhibit birefringence upon Congo red staining, which is the character of β-amyloid like structures. Additionally, Htt aggregates appear to form fibrils, as detected by transmission electron microscopy [11, 12]. It remains controversial as to whether aggregation contributes to cellular toxicity; however, it is known that the

2 aggregation and possible toxicity are modulated by sequences that flank the polyQ tract

[13-15]. For example, yeast cells expressing recombinant Htt polyQ with various flanking sequences show that a FLAG tag fused at both N- and C-terminal regions modulate Htt cellular toxicity and aggregate morphology [14]. On the other hand, deletion of the short poly proline tract (polyPro), which is located after polyQ, augments polyQ-induced toxicity in yeast [13, 14]. Additionally, a comprehensive in vitro study also demonstrated that the intrinsically unfolded N-terminal 17 residues (N17) modulate aggregation of expanded polyQ [16]. Furthermore, the N17 region undergoes conformational extension due to the polyQ expansion and consequently mediates nonfibrillar oligomerization prior to amyloid fiber formation [16].

Emerging evidences showed that prefibrillar oligomer but not insoluble aggregate of Htt and other proteins relevant to neurodegenerative disease are cellular toxicity

[17-19]. Oligomers derived from misfolded proteins are the intermediate species during the fibril formation. Studies in these oligomeric species provide a new angle inspecting the cellular toxicity [20, 21]. However, the underlying mechanism that directs the effect of Htt oligomers on cellular toxicity is unclear.

1-1-3. Evolutionary conservation and the HEAT repeat motif

The human HTT exon-1 CAG repeats encode a polyglutamine tract (polyQ). This region is located toward the N-terminus of Htt and generally increases during chordate evolution (Fig 1-1A). For example, there are 4 uninterrupted glutamine residues in zebrafish, 7 in mouse and 18~25 in human. In contrast, the polyglutamine tract in Htt of insects such as Drosophila melanogaster is located in the middle of the protein, not near

3 the N-terminus [22].

Htt is an evolutionarily conserved protein (Fig 1-1B). An ancient prototype Htt is found in Dictyostelium discoideum (a slime mold) but the homologs are absent from plants and fungi. Htt knockout in D. discoideum is not lethal but leads to morphological changes and cytoskeletal disorganization under starvation condition, as well as delayed spore production [23].

The genomic structure of HTT is conserved in chordates. The HTT genes in fish and human both have 67 exons [24]. Furthermore, the sequence preceding the polyglutamine tract is also highly conserved in chordates, as 16 of 17 residues (N17 region) are identical in fish and human (Fig 1-1A). N17 is critical for defining polyQ-expanded Htt pathogenicity in mammals. Phosphorylation, ubiquitination, and SUMOylation of this region affect Htt turnover and aggregation (details are described in 1-3).

Although the primary sequence of Htt does not define its function, Htt is composed of four clustered HEAT repeat motifs (Htt, Elongation factor 3, protein phosphatase 2A, and the yeast kinase TOR1) that forms a solenoid-like structure [25, 26]. HEAT is a helix-turn-helix motif composed of a pair of short helices with a Leu-rich hydrophobic core [27]. HEAT motifs often mediate protein-protein interactions; for example, the

HEAT repeats of CRM1/XPO1 (exportin 1) interact with many export cargos [28-30].

1-2. Huntingtin and cellular physiology

Htt is expressed in multiple mammal tissues such as lung, liver, and kidney whereas the expression level is higher in brain and testis [31-34]. Htt has been reported to associate with vesicles; however, the general function of Htt remains unclear. The

4 following paragraphs describe several intensively studied events which are regulated by

Htt. Taken together, these lines of evidence suggest that Htt is a versatile scaffold protein that regulates multiple aspects of cell physiology.

1-2-1. Embryogenesis

Since knockout of the Htt homolog in a slime mold (Dictyostelium discoideum) is not lethal [23], Htt is not required in dividing cells. Indeed, Htt is not required in self-renewing mammalian neuronal stem (NS) cells [35]. However, in vitro differentiation study indicates that NS cells lacking Htt differentiate more glial than neuronal cells [35]. Interestingly, polyQ-knockin stem cells undergo neuronal cell death during differentiation in vitro [35].

An interesting set of observations links evolution to possible function of Htt [36]. In brief, during in vitro differentiation of mouse embryonic stem (ES) cells, Htt promotes neuroepithelial cell-cell interaction by inhibiting Ncadherin cleavage and metalloprotease activity of ADAM10. Lack of endogenous Htt impairs this interaction. Complementary expression of recombinant mouse or human Htt in these ES cells lacking endogenous Htt remarkably rescues the defect. In contrast, zebrafish Htt partially restores the function but fruit fly Htt does not.

Thus, Htt is relevant to the embryogenesis. A previous study found that Htt protein expression in extra-embryonic tissues is essential for early development of mice [37].

HTT null embryos manifest a higher incidence of apoptosis than normal littermate embryos. Further study found that transplantation of HTT-/- ES cells into wild-type blastocysts (HTT+/+) can rescue this lethal defect of null cells. Conversely, wild-type ES

5 cells injected into null blastocysts have resemble defect of null embryos and die at an early stage [37]. These studies show that extra-embryonic expression of Htt is essential for embryogenesis and have an anti-apoptotic effect.

Htt deprivation also affects zebrafish embryogenesis [38, 39]. HTT-knockdown by morpholino injection into one-cell stage of the zebrafish embryo has demonstrated that

Htt is required for developing the olfactory and lateral line sensory systems as well as for anterior neural plate formation [40].

1-2-2. BDNF transport and expression

It is well known that transport of BDNF (brain-derived neurotrophic factor) from cortex to striatum is essential for striatal neuron survival [41]. Studies of HD mouse models expressing Htt conditionally indicate that polyQ-expanded Htt expression in pyramidal or striatal neurons alone is not sufficient to result in a movement disorder. In contrast, polyQ-expanded Htt expression in cortical neurons other than pyramidal neurons is required for the pathogenesis [42, 43].

These lines of evidence suggest cell-cell interaction and BDNF transport are required for striatal neuron survival and that polyQ-expanded Htt expression interferes with these processes. It has been shown that transport of vesicles containing BDNF is facilitated by wild-type Htt and is disrupted by polyQ-expanded Htt [44].

BDNF-containing vesicles are transported via microtubule. Wild-type Htt expression promotes association of microtubule and motor proteins via HAP1 (Htt-associated protein

1) and dynactin. PolyQ expansion increases interaction between Htt, HAP1, and dynactin.

However, polyQ-expanded Htt decreases association between motor proteins and

6 microtubule, and therefore it reduces the efficiency of BDNF transport [44].

BDNF de novo expression is also modulated by Htt. In brief, recombinant expression of wild-type Htt in the neuronal cell lines increases the production of the

BDNF transcript and BDNF protein. In contrast, Htt with polyQ-expansion reduces

BDNF gene products at both the mRNA and protein levels [45]. Consistently, BDNF transcripts and BDNF proteins are also reduced in brain tissues of HD model mice [45].

It is also known that wild-type Htt promotes BDNF transcription by inhibiting the transcription repressor of BDNF [45, 46]. The neuron restrictive silencer element (NRSE) upstream to the BDNF gene is recognized by the transcription repressor REST/NRSF

(repressor element-1 transcription factor/neuron restrictive silencer factor). Wild-type Htt can associates with REST/NRSF and localizes it to the cytoplasm. In contrast, polyQ-expanded Htt is unable to sequester REST/NRSF, leading to reduced BDNF expression.

In support of BDNF being the central for Htt-mediated neuron survival, studies of zebrafish embryos have found that morphological defects in head development due to

HTT- and BDNF-knockdown are similar [38-40]. Moreover, adding recombinant human

BDNF protein into the culture media can rescue this defect due to Htt or BDNF deficiency.

1-2-3. Mitochondrial fragmentation

Aberrant mitochondrial fragmentation and loss in neurons has been observed in HD patients and in HD model mice [47, 48]. Primary neurons from HD model mice are more susceptible than control groups to apoptosis that is induced by staurosporine (a kinases

7 inhibitor). Promotion of mitochondrial fusion by expressing pro-fusion mitochondrial proteins such as Opa1 and Mfn1, which against pro-fission proteins such as Drp1, reduced neuronal cell death [47].

Further studies in dysfunctional mitochondria in HD patients and model mice have described the aberrant interaction of polyQ-expanded Htt and Drp1 (dynamin-related protein-1), a GTPase involved in mitochondrial fission [49, 50]. This interaction (gain of function) due to polyQ expansion in Htt increases Drp1 activity and its mitochondrial localization. Subsequently, this results in mitochondrial fission and dysfunction.

Conversely, diminishing mitochondrial localization of Drp1 by a short peptide

P110-TAT, which blocks interaction between Drp1 and Fis1, a mitochondrial outer membrane protein required for mitochondrial fission, ameliorates polyQ-expanded

Htt-induced mitochondrial dysfunction [51, 52].

A recent study has found that treating HD model mice with bezafibrate ameliorated movement disorder, reduced oxidative stress, and increased de novo biogenesis of mitochondria. This result confirms the relevance of mitochondrial dysfunction to HD pathogenesis [53].

1-2-4. Protein-protein interaction

Sine no enzyme activity is annotated with Htt, identification of its protein binding partners could elucidate its biological significance [54]. The summary of these binding proteins are listed in Table 1-1.

A pioneering yeast two-hybrid (Y2H) screening identified a Htt-associated protein

(HAP1/E2-25K) [55]. The first 230 amino acids of Htt with polyQ 23 (normal) or 44

(mutant) was used as bait in Y2H and it was found that the HAP1-Htt (1-230 a.a.) interaction increases when polyQ is expanded. Consistently, in the same study using mammalian cells, Htt (1-930 a.a.) co-precipitation with GST-HAP1 was increased in a polyQ-dependent manner. Since HAP1 mRNA is enriched in brain especially in subthalamic nucleus, cortex, and caudate (part of striatum) [56], this interaction suggests that HAP1 modulates HD pathogenesis.

Another Y2H screening of Htt interacting proteins used polyQ-expanded Htt fragment as bait. Screening cDNA libraries from human fetal brain frontal cortex and human testis yielded 13 Huntingtin Yeast Partners (HYPs; HYP-A to HYP-M) [57]. All these interactors bind to HttQ62 (1-425 a.a.) or HttQ58 (1-550 a.a.). The same study found three of these proteins containing WW domains, which bind to the proline-rich region. Annotated functions of these interactors include mRNA processing

(HYPA/FBP11, HYPC/PR40B, and HYPI/Symplekin), ubiquitin-proteasome system

(HYPD/MAGA3, HYPF/PSMD8, and HYPG/E2-25K), vesicle trafficking

(HYPJ/α-adaptin C and HYPL/Optineurin), histone methylation (HYPB/SETD2),

AMPylation (HYPE/FICD), S-palmitoylation (HYPH/HIP14/ZDHHC17), and chaperone-like activity (HYPK).

Recently, Yang and his colleagues reported an in vivo interactome of full-length wild-type Htt/polyQ-expanded Htt that highlighted functions including 14-3-3 signaling, microtubule-based transport, proteostasis, and ATP biosynthesis [58]. They studied Htt interactomes in cortex, striatum, and cerebellum from mice of different ages (2 or 12 months). Both wild-type and transgenic (BACHD) mice were studied. The BACHD model mice which express additional copy of human Htt with polyQ-expansion (Q97)

9 exhibited a mild behavioral deficit at 2 months and severe deficit at 12 months [58]. This data set included 747 candidate proteins such as FBP11 and α-adaptin C, which were already identified in the initial yeast two-hybrid screening [57]. Expressing several candidate proteins in a fly model of HD demonstrated that proteins identified in this research could modify HD pathogenesis [58]. Specifically, two interacting proteins of high confidence are striatum-specific: voltage-gated potassium channel subunit β-1

(encoded by Kcnab1) and calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1B (encoded by Pde1b) [58]. In addition to region-specific groups

(striatum, cortex, and cerebellum), they also classified age-dependent groups (2 or 12 months) [58]. Since HD is age- and corticostriatal circuitry-dependent [59], they suggest that interactomes specific to 12 months, striatum, or cortex are putative mediator of HD pathogenesis; however, interactomes specific to 2 months or cerebellum may be neuroprotective. Intriguingly, their lists of cerebellum- and 12 months-specific interactomes have 10 overlapping proteins, involved in mitochondrial respiratory chain

(Ndufv1, Sdhb, and Uqcrc1), cell signaling (Ndrg2, Rps6ka1, and Sash1), mitochondrial fusion (Mfn2), neuronal nitric oxide production (Nos1/NOS), vesicular transport (Gbas), and transmethylation regulation (Ahcy) (Table 1-2).

In summary, Htt interactome studies have unveiled an array of modulators of Htt physiology and pathogenesis. For example, E2-25K and HIP14 reduce polyQ-expanded

Htt toxicity by regulating Htt ubiquitination and S-palmitoylation, respectively (details are described in 1-3).

1-3. Huntingtin is regulated by posttranslational modifications

A summary of Htt posttranslational modifications (PTMs) described in the following sections is listed in Fig 1-2 and Table 1-3.

Htt undergoes multiple PTMs and these modifications modulate Htt physiology and/or pathogenesis [60]. Most identified PTMs of Htt are located at the N-terminal region in the vicinity of the polyQ tract [60]. The following sections will introduce different PTMs of wild-type and polyQ-expanded Htt proteins. These PTMs include proteolysis, ubiquitination, SUMOylation, phosphorylation, acetylation, myristoylation, and finally S-palmitoylation. S-palmitoylation is a kind of modifications on cysteine residues. The other modifications of cysteine residues will be discussed in Chapter 2 and the focus will be on S-nitrosylation and S-acylation.

1-3-1. Proteolysis

Proteolysis produces small Htt fragments. Formation of polyQ-expanded Htt fragments is relevant to its cellular toxicity. The following paragraphs focus on Htt fragment formation mediated by caspase, calpain, and matrix metalloproteinase.

PolyQ expansion in Htt leads to intranuclear accumulation of Htt N-terminal fragments in post-mortem brains from patients. Western blotting analysis indicates that these fragments account for 5~20% of total Htt [61]. Thus proteolysis of Htt is relevant to

HD pathogenesis.

The most comprehensively studied proteolytic site of Htt is D586 (Fig 1-2A, inset).

Caspase-6 has been proposed to be the protease that cleaves at D586 because the flanking region shows a consensus sequence recognized by caspase-6, and because the polyQ-expanded Htt N-terminal fragment colocalizes with caspase-6 in the nucleus [62].

Proteolysis of pro-caspase is required to activate caspase. Active form of caspase-6 but not pro-caspase-6 is increased in aging mice or mice expressing polyQ-expanded Htt

[63].

Additionally, HD model mice expressing caspase-6-resistant polyQ-expanded Htt, in which the cleavage site has been mutated, don’t exhibit abnormal motor activity or striatal neuron degeneration [64]. Furthermore, protein quantity of activated caspase-6 in mice expressing caspase-6-resistant polyQ-expanded Htt is comparable to those expressing polyQ-expanded Htt with the normal cleavage site [63]. Strikingly, administration of an inhibitory peptide that contains the sequence around D586 protects a

HD mouse model from pathogenesis [65]. Nevertheless, gene knockout that eliminates caspase-6 production does not abolish Htt fragment formation and HD pathogenesis in

HD model mice [66, 67]. Therefore, Htt cleavage at D586 is relevant to HD pathogenesis and can be accomplished by multiple proteases.

In addition to D586, other caspase cleavage sites include D513 (caspase-3) and

D552 (caspase-2 and caspase-3) (Fig 1-2A, inset) [68, 69]. Enzymatic analysis in vitro did not show different efficiency of substrate cleavage in comparing wild-type and polyQ-expanded Htt proteins [68, 69]. Because it has been found that cleavage at D513 and D552 are preceded by the cleavage at D586, these cleavage events may contribute to

HD pathogenesis after D586 cleavage [62, 70].

It has been shown that calpain, which is a calcium (Ca2+)-dependent cysteine protease, is elevated in the brains of HD patients and model mice [71, 72]. Expression of polyQ-expanded Htt elevates cytoplasmic Ca2+ concentration, suggesting that calpain participates in fragmentation of polyQ-expanded Htt [73]. Kinetic analysis of calpain

12 cleavage in vitro demonstrated that polyQ-expanded Htt is cleaved faster than wild-type

Htt [71]. Motif prediction and deletion analysis on recombinant Htt identified calpain cleavage sites at T469 and S536 (Fig 1-2A) [72]. Additionally, expression of calpain-resistant (cleavage site deletion) polyQ-expanded Htt reduced cellular toxicity, confirming the relevance of polyQ-expanded Htt fragmentation for HD pathogenesis

[72].

Matrix metalloproteinase 10 (MMP-10) was recently reported a protease to contribute to Htt proteolysis at G402 (Fig 1-2B) [74]. It was identified by first screening for protease-specific siRNAs that reduced Htt N-terminal fragment formation in HEK293 cells. The active form and activity of MMP-10 are elevated in striatal cells from HD model mice. Cell culture and in vitro protease tests verified that MMP-10 bound and cleaved Htt directly. Thus, intracellular MMP-10 can cleave Htt. Furthermore, MMP10 gene knockdown reduces caspase activation, which is mediated by polyQ-expanded Htt, and ameliorates motor dysfunction in HD model flies [74].

Since the activities of these proteases are elevated in polyQ-expanded

Htt-expressing cells as described above and since they subsequently promote toxic polyQ-expanded Htt fragment formation, they are likely to be interconnected to contribute to HD pathogenesis.

1-3-2. Ubiquitination and SUMOylation

Ubiquitination and SUMOylation are the PTMs of lysine residues. They regulate protein stability and trafficking in cells. The following paragraphs describe that these two modifications regulates Htt turnover and subcellular distribution.

Accumulation of toxic full-length polyQ-expanded Htt protein and its N-terminal fragments implies that proteostasis is disrupted and that the mechanisms regulating protein turnover are impaired, such as ubiquitin-proteasome system. Protein ubiquitination requires three enzymatic activities for ubiquitin activation (E1), conjugation (E2), and ligation (E3) to the lysine residues of target proteins. A study of the immortalized lymphoblasts from heterozygous HD patients showed that 5~10% of Htt is ubiquitinated [75]. Coexpressing recombinant E3 ligase increased 2~3 fold ubiquitination of a recombinant N-terminal Htt fragment (NtHtt) expressed in mouse neuro2a cells, suggesting that Htt is normally a target of cellular ubiquitination machinery [76].

Previous yeast two hybrid studies have shown that E2-25K (conjugating enzyme) is a

Htt-interacting protein [55, 57]. PolyQ expansion in Htt increased Htt ubiquitination.

Coexpressing E3 ligase promoted NtHtt degradation, reduced NtHtt aggregation, and partially rescued cell viability [76]. Immunostaining has shown that the intranuclear Htt aggregates in patients’ brains are ubiquitinated. However, it is unclear whether these ubiquitinated aggregates are processed for degradation or are the cause of cellular toxicity

[61].

Later studies identified SUMOylation on the same lysine residues (K6, K9, and K15) that are sites for ubiquitination (Fig 1-2B) [77]. SUMO proteins (small ubiquitin-like modifier) modify target lysine residues by a sequence of enzymatic processes (activation, conjugation, and ligation) similar to ubiquitination. About 1~5% of total recombinant

NtHttQ103 was either ubiquitinated or SUMOylated [77]. Recombinant expression of

NtHtt in Hela cells has shown that polyQ-expansion (Q103 and Q97) increased both ubiquitination and SUMOylation of NtHtt, in comparing with normal polyQ (Q25).

Additionally, the triple lysine mutant (K6,9,15R) that abolishes SUMOylation results in reduced NtHttQ103 protein accumulation. Therefore, SUMOylation may play a role in aberrant polyQ-expanded Htt accumulation. Indeed, SUMO-fused NtHttQ97-K6,9,15R

(SUMO-NtHttQ97-K6,9,15R) that mimics permanent SUMOylation has shown elevated accumulation in rat striatal cell line, as compared with NtHttQ97 and

NtHttQ97-K6,9,15R.

SUMOylation also influences the subcellular distribution of polyQ-expanded Htt aggregates. Cells expressing either NtHttQ97 or NtHttQ97-K6,9,15R exhibit a remarkable number of inclusions in the cytoplasm. In contrast, cytoplasmic inclusions of

SUMO-NtHttQ97-K6,9,15R are less abundant and more dispersed [77].

As to the cell viability, it is found that SUMO haplodeficiency (SUMO +/-) background ameliorates pathogenesis of compound eye degeneration in a HD fly model.

In contrast, ubiquitin haplodeficiency (Ubiquitin +/-) background modestly deteriorate this pathogenesis. In the complementary experiment, expressing NtHtt-Q97-K6,9,15R in the fly model substantially reduced this pathogenesis. Conclusively, preventing

SUMOylation by increasing ubiquitination or by mutating the key lysine residues is able to suppress polyQ-expanded Htt toxicity.

Investigating SUMOylation of polyQ-expanded Htt also raises the issue of whether aggregates are toxic. Since SUMOylation of polyQ-expanded Htt decreases cytoplasmic inclusions while increasing toxicity, protein aggregates could be protective and at least a fraction of soluble misfolded oligomer is toxic.

1-3-3. Phosphorylation

More than 10 phosphorylation sites in Htt have been identified and their relation to polyQ expansion is complex. Generally speaking, most of phosphorylations of Htt are reduced in the context of polyQ expansion, except phosphorylation of S431 and S432

(Table 1-3) [60, 78]. These phosphorylations have diverse effects on Htt regulation. For example, phosphorylation modulates Htt titer and anterograde transport of

BDNF-containing vesicles in neurons [79-82]. The following paragraphs describe the details of these Htt phosphorylation.

The best characterized phosphorylated residues are located in the Htt N17

(N-terminal 17 residues) region, in vicinity of ubiquitination/SUMOylation sites (K6, K9, and K15). For example, phosphorylation on serine residues 13 and 16 (S13, S16) (Fig

1-2B) modulates Htt distribution and cell toxicity.

Phosphorylation on these residues is carried out by IKK [79]. Overexpressing IKK leads to NtHttQ25 protein reduction. A phospho-mimicking mutant (NtHttQ25 S13,16E) which mimics permanent phosphorylation also results in decreased protein accumulation in steady state. In contrast, a phospho-dead mutant (NtHttQ25 S13,16A) which mimics permanent dephosphorylation leads to NtHttQ25 accumulation [79].

This phosphorylation also influences another modifications located at the Htt N17 region. Htt S13A mutant has shown the reduced ubiquitination and SUMOylation at the neighboring lysine residues, whereas ubiquitination and SUMOylation was retained in

S13D mutant [79]. In the same study, the phospho-mimicking mutant significantly increased NtHtt nuclear localization and reduced toxicity in primary neurons [79]. In another study investigating HD model mice showed that phospho-mimicking (S13,16D) but not phospho-dead (S13,16A) mutant abolished pathogenesis [83].

Threonine 3 residue is the other phosphorylation site in N17 region (Fig 1-2B) [84].

This phosphorylation is reduced in polyQ-expanded Htt. Intriguingly, both T3D

(phospho-mimicking) and T3A (phospho-dead) reduced cell death in HD fly model.

Additionally, T3D increased the number of cells containing aggregates. Therefore, these

T3D-associated aggregates may be protective and T3A underwent an alternative protective pathway. In contrast, unmodified threonine residue (T3) may be toxic itself through other mechanism [84]. In summary, PTMs in N17 region in concert with polyQ modulate HD pathogenesis.

The second group of phosphorylation sites is located between HEAT cluster 1 and

HEAT cluster 2 (Fig 1-2B), in proximity to caspase/calpain cleavage sites (Fig 1-2A, inset). In this group, S421 is intensively studied. Akt-mediated S421 phosphorylation and

S421D mutant rescued viability of striatal cells [85]. Additionally, Akt-mediated S421 phosphorylation and S421D mutant also restored the deficient BDNF transport due to polyQ expansion [81]. Inhibiting phosphatases (PP1 and PP2A) restored S421 phosphorylation and prevented striatal neurons from NMDA-induced cell death [86].

Interestingly, S421 phosphorylation was not affected in HD model mice expressing the

Htt with the caspase-6-resistant mutation (uncleavable). Thus, it suggests that S421 phosphorylation interconnects with Htt cleavage mediated by caspase-6 [86].

Phosphorylation at S434 also reduces Htt cleavage by caspase [87]. Cdk5 phosphorylates S434 and its activity is reduced in HD model mice. S434 phosphorylation also decreases aggregate formation. It has been shown that polyQ expansion reduces

S434 phosphorylation by disrupting the interaction of Cdk5 and p35, which is the activator of Cdk5 [87].

Unlike many other sites, phosphorylation at S431 and S432 is increased upon polyQ expansion [78]. Intriguingly, phospho-mimicking and phospho-dead mutation on these two sites showed opposite effects. For example, expressing S431D and S432A mutants maintained polyQ-expanded Htt toxicity whereas S431A and S432D rescued cell viability in HEK293 cells, suggesting different effects and mechanisms of phosphorylation on these sites. Additionally, S431D and S432A mutants increased polyQ-expanded Htt accumulation including both monomer and materials staying in stacking gel. Therefore, phosphorylation status on S431 and S432 is likely involved in Htt turnover.

S1181 and S1201, located between HEAT cluster 2 and HEAT cluster 3 (C-terminal to caspase/calpain cleavage sites, Fig 1-2A), are also the substrate of Cdk5 [88]. Reduced activity of Cdk5 decreases phosphorylation of S1181/S1201. In the context of normal polyQ (Q17), expressing phospho-dead mutant (S1181A, S1201A, or S1181,S1201A) in primary neuron culture leads to cell death. In contrast, phospho-mimicking mutant

(S1181D, S1201D, or S1181,S1201D) reduced the cellular toxicity from polyQ expansion. Phosphorylation of S1181/S1201 is elevated upon DNA damage (e.g. CPT treatment). Expressing S1181,S1201A mutant deteriorates cell death due to DNA damage.

Interestingly, knockdown or inhibition of p53 remarkably reduces toxicity of cell expressing HttQ17-S1181,S1201A or HttQ73. Therefore, it suggests that phosphorylation of S1181/S1201 is relevant to antagonize p53-mediated apoptosis in response to DNA damage [88].

PTMs in the C-terminal region of Htt are less described. A comprehensive study mapping the phosphorylation sites along full-length Htt has identified S2076, S2653, and

S2657 (Fig 1-2A) [89]. However, the biological significance as well as the interaction of

18 polyQ and these phosphorylation sites remain unknown.

1-3-4. Acetylation

Besides ubiquitin-proteasome system, autophagic-lysosomal pathway also regulates protein turnover. It has been shown that polyQ expansion increases Htt acetylation at

K444 (Fig 1-2B) [90]. In this study, they identify that K444 is acetylated by CBP

(CREB-binding protein) and the deacetylation is catalyzed by HDAC1 (histone deacetylase 1). Lentiviral expression of K444R mutant increases polyQ-expanded Htt accumulation in primary neuron culture and murine brains. HDAC knockdown or CBP expression increases polyQ-expanded Htt degradation in neuronal cell line treated with cycloheximide. Additionally, acetylated polyQ-expanded Htt but not K444R mutant increases colocalization with LC3 protein, an autophagosome marker. Inhibiting lysosomal enzyme also increases polyQ-expanded Htt accumulation. Therefore, it suggests K444 acetylation is required for polyQ-expanded Htt degradation by autophagic-lysosomal pathway [90].

1-3-5. Myristoylation

Autophagocytosis is regulated by myristoylated Htt fragments which are generated by caspase cleavages at D552 and D586 (Fig 1-2A, inset) [91]. The newly exposed G533 becomes the N-terminus for N-myristoylation (14-carbon lipidation) (Fig 1-2A, inset).

This lipidation increases hydrophobicity, promoting protein association with membranes.

Myristoylation at G533 is reduced by polyQ expansion. Myristoylated Htt553-585 fragments colocalize with Rab32, which is required for autophagy [91, 92].

Myristoylation of the Htt553-585 fragment and autophagic vesicle formation induced by the myristoylated Htt553-585 fragment are abrogated by the G553A mutation. Thus, myristoylated Htt553-585 is involved in regulation of autophagocytosis.

1-3-6. S-palmitoylation

Protein distribution can be regulated by aliphatic PTMs. In addition to myristoylation, it has been reported recently that Htt is modified by S-palmitoylation

(16-carbon lipidation; “S” stands for thio group) of cysteine residue 214 (C214) (Fig

1-2B) [93]. However, the stoichiometry of S-palmitoylated Htt relative to total Htt is not clear.

This modification is mediated by HIP14 (Htt-interacting protein 14, also called

ZDHHC17) [93], a palmitoyl acyltransferase that was first identified by yeast two hybrid screening [57, 94]. HIP14 is enriched in the brain and colocalizes with the Golgi complex marker protein, GM130 [94]. Overexpressing HIP14 in cortical neurons of YAC18 mice, which express human HttQ18 (polyQ=18), has shown colocalization of HIP14 and

HttQ18 in the peripheral area of Golgi complex [93].

PolyQ expansion in full-length Htt reduced its colocalization to the Golgi complex in neurons of YAC128 HD model mice, and also reduced Htt-HIP14 association, judged by coimmunoprecipitation [93, 94]. The same study shows that polyQ expansion reduces

~50% Htt S-palmitoylation in COS7 cells [93].

Therefore, polyQ can regulate Htt localization via S-palmitoylation. In the same study, overexpressing HIP14 increased HttQ128 S-palmitoylation, partially rescued

HttQ128 localization to Golgi complex, and reduced the number of neurons containing

HttQ128 aggregates. In contrast, the HttQ128 C214S mutant, which showed abolished

S-palmitoylation, exhibited an increased number of aggregate-containing neurons [93].

Additionally, NMDA-induced neuronal excitotoxicity was also modulated by Htt

S-palmitoylation. Hyperactivation of NMDA receptors by NMDA treatment results in excessive calcium ion influx. This excitotoxicity was elevated in cortical neurons with

HIP14 knockdown. Furthermore, the Htt C214S mutation accelerated NMDA-induced neuronal excitotoxicity. Cortical neurons expressing Htt1-548Q15 C214S fragment were more susceptible to NMDA-induced neuronal cell death than those expressing

Htt1-548Q15. Similar results were also observed for cortical neurons expressing

Htt1-548Q128 C214S fragment [93].

Intriguingly, mice lacking HIP14L (HIP14-like, also called ZDHHC13), a paralog of

HIP14, showed more severe HD-like pathology than HIP14-/- mice [95]. HIP14L is also enriched in brain and resides in the Golgi area. It also associates with Htt and results in

Htt S-palmitoylation [96, 97]. Therefore, S-palmitoylation may modulate Htt physiology and pathology in the context of different polyQ tracts. Whether HIP14 or HIP14L is sufficient to regulate Htt S-palmitoylation remains unclear.

The S-palmitoylated cysteine residues can be also S-nitrosylated. The details will be described in Chapter 2 and 3.

Figure 1-1.

Figure 1-1. Htt is a conserved protein. (A) Alignment of Htt sequences from multiple

22 species. Only the first ~150 a.a. are shown here. Sequences are from: Homo sapiens

(human), Pan troglodytes (Chimpanzee), Bos taurus (cattle), Mus musculus (mouse),

Cavia porcellus (guinea pig), Oryctolagus cuniculus (European Rabbit), Equus caballus

(domesticated horse), Monodelphis domestica (opossum), Gallus gallus (chicken),

Meleagris gallopavo (turkey), Anolis carolinensis (arboreal lizard), Xenopus tropicalis

(clawed frog), Danio rerio (zebrafish), Branchiostoma floridae (lancelet), and Ciona intestinalis (vase tunicate). (B) The pairwise identity score (%) of Htt sequences. The score is the number of identities of two sequences divided by the length of the alignment.

Multiple sequence alignment and the identity score were performed by Clustal 2.1.

Figure 1-2.

Figure 1-2. Full-length Htt, Htt N-terminal fragment and posttranslational modifications. (A) Wild-type (wt) full-length Htt. The vertical lines indicates all 70 Cys residues. The horizontal open boxes indicate HEAT repeat motif clusters (Tartari, M. et al., Mol Biol Evol. 2008). Anti-Htt antibodies (MAB2166, MCA2050, and MAB2168) and binding sites are labeled. The inset enlarges proteolysis region (the horizontal grey bar) that generate N-terminal and Htt553-585 fragments. G553 is myristoylated after cleavage at D552. (B) Htt N548 (1-548 a.a.) fragment and its posttranslational modifications. N548 approximates the size of proteolytic products of the full-length Htt.

The inset is an enlargement of this region. Cysteine residue numbers are indicated.

Arrows indicates the residues (C137 and C517) that do not show S-nitrosylation and

S-acylation in this study.

Table 1-1. Htt-interacting proteins

interaction upon polyQ Symbol annotated function expansion E2-25K/HAP1/HYPG ubiquitin-proteasome system increase

MAGA3/HYPD ubiquitin-proteasome system unclear

PSMD8/HYPF ubiquitin-proteasome system unclear

FBP11/HYPA mRNA processing increase

PR40B/HYPC mRNA processing unclear

Symplekin/HYPI mRNA processing unclear

α-adaptin/HYPJ vesicle trafficking decrease

Optineurin/HYPL vesicle trafficking unclear

SETD2/HYPB histone methylation increase

ZDHHC17/HIP14/HYPH palmitoyl acyltransferase decrease

Ywhae 14-3-3 signaling unclear

Kcnab1 voltage-gated K+ channel unclear Ca+/calmodulin-dependent cyclic Pde1b unclear nucleotide phosphodiesterase

Table 1-2. Age- and cerebellum-specific interactomes in HD mouse model

symbol annotated function

Ndufv1 mitochondrial respiratory chain

Sdhb mitochondrial respiratory chain

Uqcrc1 mitochondrial respiratory chain

Mfn2 mitochondrial fusion

Ndrg2 cell signaling

Rps6ka1 cell signaling

Ahcy adenosylhomocysteinase, involved in transmethylation regulation

Nos1 neuronal nitric oxide synthase

Gbas may be involved in vesicular transport

Sash1 may be involved in cell signaling

Table 1-3. Htt posttranslational modifications (PTMs)

change upon polyQ PTM residue expansion proteolysis G402 increase proteolysis T469 increase proteolysis D513 increase proteolysis S536 increase proteolysis D552 increase proteolysis D586 increase ubiquitination K6 increase ubiquitination K9 increase ubiquitination K15 increase SUMOylation K6 increase SUMOylation K9 increase SUMOylation K15 increase phosphorylation T3 decrease phosphorylation S13 decrease phosphorylation S16 decrease phosphorylation S421 decrease phosphorylation S431 increase phosphorylation S432 increase phosphorylation S434 decrease phosphorylation S1181 decrease phosphorylation S1201 decrease phosphorylation S2076 unclear phosphorylation S2653 unclear acetylation K444 increase Myristoylation G553 decrease S-palmitoylation C214 decrease

Chapter 2

Introduction of cysteine modifications

Summary

The oxidoreduction status of thio-groups of protein cysteine residues is dependent of various posttranslational modifications (PTMs). These modifications also influence protein hydrophobicity, protein conformation, and protein-protein interaction.

Consequently, PTMs of cysteine residues can regulate protein distribution and physiological function in cells [98-103]. This Chapter first introduces general properties of cysteine residue modifications and then focuses on the regulations of S-nitrosylation and S-acylation.

2-1. Overview of cysteine modifications

In addition to disulfide bonds formation, protein cysteine residues are able to be

S-nitrosylated, S-acylated, S-sulfenylated, S-glutathionylated, S-sulfhydrated,

S-glycosylated (“S” means sulfhydryl/thio group), or oxidized to sulfinic acid or sulfonic acid [98-103]. Many of these modifications are enzymatically reversible including disulfide bond formation, S-nitrosylation, S-acylation, S-sulfenylation,

S-glutathionylation, and S-sulfhydration [98]. Cysteine residue oxidation to sulfinic acid or sulfonic is generally considered irreversible, but emerging reports indicate reversible sulfinic acid formation mediated by sulphiredoxin (Srx) [98, 104-107]. Additionally,

S-glycosylation was recently identified in bacteria [99, 101]. The recent discovery of the bacterial glycosyltransferase that possesses both O- and S-glycosylation activities may imply a conserved mechanism in O-linked and S-linked glycosylation [101]. The

28 reversibility of cysteine modifications can regulate protein conformation and function. In the following sections, the focus will be on S-nitrosylation and S-acylation since they are most relevant to our study. As is further discussed below, there has been a single report of

S-nitrosylation of Htt and a single report of Htt acylation [93, 108].

2-2. S-nitrosylation

S-nitrosylation (SNO) is a reversible cysteine residue modification that influences a diverse set of physiologically important activities, protein-protein interactions, proteostasis, protein distribution, cell signaling, and enzyme activities [109].

S-nitrosylation is a reversible cysteine residue modification [110, 111]. Cultured cells treated with nitric oxide (NO) donors such as S-nitrosocysteine (CysNO) increases global

S-nitrosylation. Proteomic analysis suggests that the majority (>60%) of SNO modification have a half-life less than 30 minutes after cells have been treated with

CysNO [111].

SNO typically requires NO production mediated by nitric oxide synthase (NOS)

[112]. Corresponding transferases that are only incompletely characterized. The mammalian genome encodes three NOS isoforms: neuronal NOS (nNOS/NOS1), endothelial NOS (eNOS/NOS3), and inducible NOS (iNOS/NOS2) [113]. nNOS and eNOS are constitutively expressed in neurons and the vascular endothelium, respectively

[113], as well as in many other cell types. iNOS de novo expression is induced in most cell types upon inflammation [113]. Thus, virtually all cells are able to produce NO.

The abundance of NO production is critical to physiology and pathogenesis in vivo.

An early study indicated reduced mRNA expression of nNOS gene in the striatum of HD

29 patients, as compared with normal controls [114]. Increasing NOS may protect neurons from neurodegeneration. However, regulation of NOS is a double-edged sword for neuron viability. Nitric oxide production can be either protective or deleterious to neurons

[115-118].

- In physiologic conditions, local NO scavenges superoxide (O2 ) by forming reactive

- - peroxynitrite (ONOO ), which consequently rearranges to stable nitrate (NO3 ) in the absence of other reactive molecules. Nitric oxide also interacts with metal ions and forms metal-nitrosyl species, which prevent ROS (reactive oxygen species) production mediated by reaction of metal ion and peroxide [115, 118].

In pathogenic conditions, excess superoxide leads to peroxinitrite accumulation, resulting into oxidative stress [116, 118]. Therefore, the stoichiometry of NO is an important factor for modulating physiology and pathology.

Nitric oxide production catalyzed by NOS requires the precursor, L-arginine. HD model mice fed a diet lacking L-arginine manifested accelerated motor neuron disorder, judged by the roto-rod test, as compared with HD mice fed with 1.2% L-arginine (typical mouse chow). At week 17, maintaining time on the rod of HD fed without L-arginine is reduced to less than 80%, compared to control mice. In contrast, even at week 19, HD mice with normal diet still keep ~90% maintaining time, compared to controls (0% arginine at week 19: <60% maintaining time) [119]. Administration of high dose of the

NOS inhibitor L-NAME (L-NG-Nitroarginine methyl ester, inhibiting nNOS, eNOS, and iNOS) accelerates pathologic progression in HD model mice, suggesting that NOS activity modulates this process [120]. Furthermore, HD-related symptoms are accelerated in HD model mice lacking NOS (nNOS-/-) [121]. Altogether, disturbance of NOS/NO

30 system affect HD pathogenesis.

After produced by NOS, NO needs transfer to the targets. NO is able to form nitrogen oxide molecules with higher oxidization, such as N2O3, which react with the thio group of nearby cysteine residues of target proteins or glutathione (GSH) [112]. Because

NO and its derivatives are not stable, this reaction is limited to the area that is in proximity to the location of NOS [112]. The S-nitrosylated protein or

S-nitrosoglutathione (GSNO) subsequently transfers this moiety to nearby proteins [112].

Therefore, the specificity of S-nitrosylation depends on:

(1) The chemical reactivity of the cysteine that is influenced by the flanking sequence

(The consensus sequence is described in the following paragraph).

(2) Localization of NOS that limits the production and diffusion of NO.

(3) The S-nitrosylated proteins which obtain NO moiety from the local NO-derived molecules (i.e. N2O3 or GSNO) serve as transferases to transfer the NO moiety to the designated proteins.

The consensus sequence for SNO addition remains elusive [102, 122]; however, proteomic studies suggest that hydrophobicity and the charge of adjacent residues, as well as secondary structure, affect S-nitrosylation [123-135]. Recently, a striking study of iNOS-dependent S-nitrosylated proteome identified a consensus SNO modification motif

([I/L]-X-C-X2-[D/E]), suggesting the existence of proteins that facilitate specific

S-nitrosylation sites. [136]. Since previous studies have been unable to narrow down the consensus sequences, there may be several SNO transferases.

NOS localization also regulates SNO specificity. Sessa and colleagues first demonstrated that eNOS localization is relevant to regulatory functions [137].

Recombinant expression of wild-type endothelial NOS (eNOS) leads to the accumulation of eNOS and increased protein S-nitrosylation in the peripheral region of Golgi complex.

Thus, it delays protein secretion. In contrast, a mutant form of eNOS-NLS (nuclear localization signal) fusion protein that is confined to the nucleus elevates S-nitrosylation in the nucleus but doesn’t affect protein secretion [137].

nNOS and iNOS have different subcellular localizations. The sarcoplasmic reticulum is the major subcellular localization of nNOS in cardiac myocytes [138].

Another study has reported iNOS localization to peroxisomes in hepatocytes [139].

Whether subcellular NOS localization influences Htt S-nitrosylation is unknown.

SNO transferase activity has not been well-characterized because of the difficulty in recognizing the target sequences. A study of the iNOS-dependent S-nitrosylated proteome has identified a consensus SNO site ([I/L]-X-C-X2-[D/E]) [136]. These authors used

S-nitrosylated GAPDH as a model and identified proteins coprecipitated with GAPDH or iNOS by mass spectrometry (MS). Two proteins shorter than 120 a.a. were identified:

S100A8 and S100A9. Each has only one cysteine residue. They found that S-nitrosylated

Cys3 of S100A9 is required for iNOS-GAPDH association. On the other hand, Cys42 of

S100A8 is required for specific S-nitrosylation to Cys247 of GAPDH. S100A8 C42S mutation leads to S-nitrosylation to both Cys152 and Cys247 of GAPDH. The authors therefore suggest the existence of an iNOS/S100A8/S100A9 complex that interacts with target proteins and transfers NO to specific sites [136].

The status of S-nitrosylation is also regulated by enzymes that eliminate the NO moiety (denitrosylation). Currently three systems of denitrosylation have been found, including (1) GSNO reductase (GSNOR), (2) thioredoxin reductase (TrxR), and (3)

S-nitroso-coenzyme A reductases (SNO-CoA reductase) [105, 140-142]. These enzymes reduce the formation of S-nitrosylated form of glutathione (GSH), thioredoxin (Trx), and

CoA. Subsequently, reduced forms of GSH, Trx, and CoA are increased and remove the

NO moiety from S-nitrosylated proteins (Fig 2-1). These systems have specific targets.

GAPDH S-nitrosylation/denitrosylation is mediated by the SNO-CoA/SNO-CoA reductase system rather than GSNO/GSNOR [142]. However, the underlying mechanism of the specificity is unclear. Whether other elements are involved in the specific recognition remains elusive.

Alteration of protein S-nitrosylation is characteristic of several neurodegenerative diseases, including Huntington’s disease (HD), Alzheimer’s disease (AD), and

Parkinson’s disease (PD) [143]. Parkin S-nitrosylation inactivates the E3 ligase activity of

Parkin, impairing ubiquitination of its targets [144-146].

Recently Lipton’s group has reported that abnormal mitochondrial fission due to elevated SNO of Drp1 (dynamin-related protein 1) is observed in cell culture models and patients with AD and HD [108, 147]. Drp1 S-nitrosylation is required for the interaction of Drp1 with Htt [108].

Drp1 is a conserved protein required for mitochondrial fission (fragmentation)

[148-150]. Expansion of polyQ-expanded Htt increases the Drp1-Htt interaction, promotes mitochondrial fragmentation, and consequently leads to dysfunction [47, 48,

50]. Moreover, expression of polyQ-expanded Htt in cells increases NO production

(~2-fold) and leads to increased S-nitrosylation of Drp1 [108]. Coimmunoprecipitation experiment found that the polyQ-expanded Htt-Drp1 interaction requires SNO. Treatment with a NOS inhibitor or ascorbic acid (SNO reducer) abolishes this interaction.

On the other hand, overexpression of the Drp1 C644A mutant, which cannot be

S-nitrosylated, reversed mitochondrial fragmentation. Thus, mitochondrial fragmentation induced by polyQ-expanded Htt-Drp1 interaction is mediated by S-nitrosylation on Drp1.

Interestingly, the authors of the same study also detected an increase (~5-fold, based on the only one gel shown in the figure) of SNO modification of recombinant polyQ-expanded Htt by comparison to wild-type Htt in HEK293-nNOS cells, which overexpress neuronal NOS (nNOS). The authors suggest that the nitrosonium moiety may be transferred from polyQ-expanded Htt to Drp1. However, the authors didn’t have any experiment that blocks Htt-Drp1 interaction to examine whether this transnitrosylation depends on this interaction. Additionally, whether SNO modification of Htt is present in cells expressing endogenous NOS is still unknown.

2-3. S-acylation

S-acylation is a mechanism of cysteine residue lipidation by palmitic acid, stearic acid or oleic acid [102, 103]. A previous study applied GC-MS (gas chromatography-mass spectrometry) to measure the quantity of different acyl modifications of cysteine residues in platelet proteome [103]. They found that 74% were palmitate, 22% stearate, and 4% oleate. This study provides a first glance of lipid composition required for S-acylation in living cells, but the precise composition in different cell types needs further investigation. These fatty acids are not the direct substrates for the S-acylation. Instead, it requires acyl-CoA formation (fatty acid esterified to Coenzyme A) and the acyl-CoA subsequently serves as the substrate for

S-acylation [102, 151]. Since the CoA pool in cells is relevant to SNO-CoA/SNO-CoA

34 reductase, one of the systems regulating S-nitrosylation [142], it implies that the regulatory mechanisms of S-acylation and S-nitrosylation may be interconnected.

Protein S-acylation in eukaryotes is mediated by a group of palmitoyl acyltransferases (PATs). This group of PATs belongs to ZDHHC family. The activity of these enzymes is embedded into a conserved cysteine-rich zinc finger domain (~50 a.a.) in which the conserved DHHC (Asp-His-His-Cys) is present [152]. Most of these enzymes are transmembrane proteins located in ER and Golgi complex where they catalyze protein S-acylation and consequently regulate protein localization to membrane

[102, 152].

Consensus sequences for protein S-acylation mediated by ZDHHC families are not well characterized [102, 122]. Recently, it has been found that the akyrin repeat (AR) domains of HIP14 (ZDHHC17) and HIP14L (ZDHHC13) recognize ψβXXQP consensus

(ψ indicates aliphatic residue: Val, Ile, Ala, or Pro; β indicates C-β branched residue: Val,

Ile, or Thr; X: any residue) [153]. Nevertheless, the recognition mechanisms for the other

ZDHHC families lacking AR domain remain unknown.

Enzymatic deacylation is also an important mechanism that regulates S-acylation homeostasis. Presently, two groups of S-thioesteraases, which belong to a subgroup of serine hydrolases, are identified as the enzymes deacylating S-acylated proteins: (1) protein palmitoyl thioesterase 1 (PPT1) and (2) acyl protein thioesterases 1 (APTI/2) [102,

151]. PPT1 is a lysosomal enzyme catalyzing deacylation of S-acylated protein targeted to lysosome, whereas APTI and APT2 are cytoplasmic enzymes. A recent study screening serine hydrolases that promote N-Ras depalmitoylation has identified ABHD17 as a depalmitoylase [154]. It suggests an extended panel of protein families that regulate

35 protein deacylation.

S-acylation is required for protein localization. For example, Htt S-palmitoylation enhances Htt localization to the peripheral region of Golgi complex [93]. S-acylation is also in concert with N-myristoylation or prenylation to regulate membrane trafficking of proteins [102, 151]. Both S-acylation and N-myristoylation are required for eNOS localization. Loss of either S-acylation or N-myristoylation disrupts Golgi localization of eNOS. This mislocalization also reduces NO production [102, 155]. Thus, S-acylation regulates protein trafficking and protein localization that may influence S-nitrosylation.

2-4. Interaction between S-nitrosylation and S-acylation

It has been reported that protein activity and proteostasis are modulated by the interplay between S-nitrosylation and many PTMs, including phosphorylation, acetylation, redox modifications, ubiquitination, SUMOylation, and S-palmitoylation

[109]. Due to the relevance of our study in cysteine modifications, the following description will focus on the interaction between S-nitrosylation and S-acylation.

In several cases, S-nitrosylation and S-acylation appear to be regulated in reciprocal fashion and/or to occur on the same cysteine residues. For example, S-palmitoylation of proteins such as GAP-43 and SNAP-25 is inhibited when cells are exposed to nitric oxide donors [109]. The distinct roles of these two modifications is implied by the study in

PSD-95 (postsynaptic density protein 95). PSD95 is a scaffolding component of the postsynaptic density and is required for synaptic plasticity as well as spatial learning [156,

157]. Mutually regulated S-nitrosylation and S-palmitoylation on cysteine residues 3 and

5 (Cys3 and Cys5) of PSD-95 regulate its cell membrane targeting [158, 159].

Endogenous S-nitrosylated PSD-95 is undetectable in brains of nNOS knockout mice, suggesting the necessity of nNOS in regulating SNO of PSD95. In mouse cerebellar neurons, endogenous S-nitrosylated PSD-95 accounts for only 0.5~1% and

S-palmitoylated PSD-95 for only 3~5% of total PSD-95. Cys3 and Cys5 residues are required for both SNO and S-palmitoylation of PSD95. Double mutation (C3,5S) blocks both SNO and S-palmitoylation. Interestingly, S-palmitoylation of PSD95 is reduced in cells with activated nNOS and increased in cells treated with NOS inhibitor. Localization and clustering of PSD-95 at the cell membrane is regulated by S-palmitoylation [158].

This localization is reduced by nNOS activation and increased by nNOS inhibition.

Therefore, the authors propose a model in which the localization of PSD-95 is regulated through the competition of S-nitrosylation and S-palmitoylation on Cys3 and Cys5.

2-5. Cysteine modifications of the HEAT repeat motif

HEAT repeat motif was named after four founding proteins: Htt, Elongation factor 3, protein phosphatase 2A, and the yeast kinase TOR1 [25]. The secondary structure of

HEAT repeat motif is a solenoid-like α-helix [25, 27]. This motif is involved in protein-protein interaction. For example, HEAT repeat motif in CRM1/XPO1 (exportin 1) forms a binding groove targeting NES (nuclear export signal). Molecular modifications on this HEAT repeat motif interfere with the NES binding and consequently abolish nuclear export of cargo proteins [26, 28-30].

The cysteine residue 528 (Cys528) of CRM1/XPO1 is imbedded in the NES binding groove of HEAT repeat motif. It is known that covalent modification of this cysteine residue by the alkylation of leptomycin B (LMB) inhibits nuclear export [28, 160]. This

37 cysteine residue is conserved in species and the replacement of this cysteine residue by serine or threonine prevent the inhibition from LMB [28]. The further study has found that S-nitrosylation of Cys528 also displays the dysfunctional nuclear export [29]. The function of CRM1/XPO1 C528S mutant is not affected by S-nitrosylation. Interestingly, cells expressing C528W mutant showed nuclear accumulation of NES-containing proteins which was also observed in LMB- or GSNO-treated cells [29].

Therefore, S-nitrosylation of HEAT repeat motif can modify the local structure/environment, interfere protein-protein interaction, and consequently change the protein function.

Figure 2-1.

Figure 2-1. Reduced form of glutathione (GSH), thioredoxin (Trx), or coenzyme A

(CoA) denitrosylates SNO modifications via transnitrosylation. SNO-Trx is a transient product and converts to oxidized Trx rapidly.

Chapter 3

Polyglutamine expansion increases protein S-nitrosylation

Summary

It has been reported that expression of polyQ-expanded Htt elevate nitric oxide (NO) production in neuronal cells. This may increase Drp1 S-nitrosylation [108]. The authors also found S-nitrosylated Htt in HEK293 cells expressing recombinant nitric oxide synthase (NOS). Additionally, the study observed polyQ-dependent Htt S-nitrosylation

(SNO). However, whether Htt is S-nitrosylated at the physiological level of NOS remains unclear. Furthermore, whether polyglutamine tract (polyQ) expansion affects posttranslational modifications (PTMs) of multiple proteins are unknown. Here we use

Htt as a model protein to study the effect of polyQ expansion on Htt S-nitrosylation in different cell lines. We observe a parallel polyQ-dependent effect for Ataxin-1, the protein associated with spinocerebellar ataxia type 1 (SCA1).

3-1. Discovery of Huntingtin S-nitrosylation

To learn whether cysteine residues of Htt are S-nitrosylated or S-acylated, we expressed both full-length Htt and various Htt fragment constructs in cultured cell lines

(Fig 3-1). The N-terminal fragment (N548, residues 1-548 a.a.) is comparable in size to major in vivo cleavage products derived from full-length Htt [93, 161].

These experiments employed a “resin-assisted capture” (RAC) protocol that is based on selective retrieval of proteins from cell lysates using Thiopropyl Sepharose beads that covalently bind to the sulfhydryl groups which were exposed upon reduction of

S-nitrosylated cysteine residues (by ascorbic acid) or S-acylated residues (by hydroxylamine). The bound proteins were then eluted with sample buffer containing

β-mercaptoethanol and analyzed by Western blotting (Fig S1 and Appendix A6) [111,

162].

3-2. PolyQ expansion increases protein S-nitrosylation

We first analyzed S-nitrosylation and S-acylation of full-length Htt and the Htt

N-terminal fragment in COS7 and HEK293T cells without overproducing nitric oxide (i.e. at the physiological levels of nitric oxide). In summary, we found polyQ expansion causes a remarkable increase in S-nitrosylation (>5 fold) in both COS7 and HEK293T cells. Whereas a mild increase in S-acylation (<3 fold) is observed only in COS7 cells but not in HEK293T cells. We examined the generality of this effect by analyzing

S-nitrosylation and S-acylation of Ataxin-1 expressed in HEK293T cells. As for Htt,

S-nitrosylated Ataxin-1 increases with polyQ expansion. We also found that high molecular weight (HMW) species of Htt and Ataxin-1 are enriched in the S-nitrosylated and S-acylated forms. Therefore, these cysteine modifications may regulate protein conformation and/or affect protein-protein interactions.

3-2-1. PolyQ expansion increases Htt S-nitrosylation

We first evaluated S-nitrosylation and S-acylation using COS7 line (derived from green monkey) because this cell line was used to study S-palmitoylation of Htt [93]. After one day transfection of COS7 cells with either plasmid encoding N548 Q15 or N548

Q128, the proteins are readily detectable in the respective cell lysates (Fig 3-2A first two

41 lanes, input). S-nitrosylated and S-acylated Htt are also readily detectable, as judged by

Western blotting (Fig 3-2A, lane 3-6). The extent of both modifications depends on polyQ expansion. Thus, in experiments using two Htt N548 constructs (Q15, Q128) we observed that polyQ expansion is strongly linked to N548 S-nitrosylation (Fig 3-2A, lane

3 vs 4) and increased Htt SNO levels by ~15 fold (Fig 3-2B). A more modest increase of

S-acylation (~2.5 fold) also parallels polyglutamine expansion (Fig 3-2A, lane 5 vs 6, and

3-2B).

When similar experiments were performed with HEK293T (human) cells, we again detected both modifications and noted that polyQ expansion stimulates S-nitrosylation of

Htt much more strongly than S-acylation (Fig 3-2C). When normalized to total Htt, polyQ expansion increases SNO-Htt ~ 9.8 fold while the impact on S-acylation is not significant (Fig 3-2D).

A striking increase of S-nitrosylation and a lesser increase of S-acylation are also observed when full-length Htt +/- polyglutamine expansion is expressed in HEK293T cells (Fig 3-2E and 3-2F). Thus, in two different host cells, polyglutamine expansion

(Q128) consistently causes a major increase in the extent of S-nitrosylation and a lesser degree of S-acylation of Htt (Q128) by comparison to controls (Q15).

3-2-2. PolyQ expansion increases Ataxin-1 S-nitrosylation

To learn whether the relation between polyQ expansion and increased

S-nitrosylation is specific to Htt, we conducted similar studies of Ataxin-1, which causes spinocerebellar ataxia type 1 (SCA1) when its N-terminal polyQ tract is expanded (Fig

3-3A and 3-3B). For this purpose, we expressed a wild-type control, FLAG-tagged

Ataxin-1Q30 or pathogenic Ataxin-1Q85 in HEK293T cells. We again found that polyQ expansion increases S-nitrosylation (Fig 3-3C and 3-3D). Increased S-nitrosylation due to polyQ expansion therefore is characteristic of at least two proteins implicated in disease.

3-2-3. PolyQ-dependent high molecular weight species are highly cysteine-modified

The monomeric size of FLAG-Ataxin-1Q30 and FLAG-Ataxin-1Q85, based on protein sequence, are ~88 and ~95 kDa, respectively. Nevertheless, the MW estimated by gel mobility are ~115 (Q30) and ~130 (Q85) kDa. This deviation due to the polyQ tract is consistent with previous studies (Fig 3-3C) [163].

Surprisingly, a high molecular weight (HMW) species ~300 kDa of Ataxin-1Q85 was also seen in the samples that were RAC-enriched either for SNO or for S-acylation.

These species were barely observed in the input (Fig 3-4A). Importantly, such higher molecular bands were not seen in the similarly enriched proteins recovered from cells that express the control, FLAG-Ataxin-1Q30.

PolyQ-dependent HMW species were also observed in extracts of cells expressing

Htt N548Q128 when processed by SNO-RAC or acyl-RAC (Fig 3-4B). The calculated molecular weight of these species is ~290 kDa by comparison to the monomeric band detected by Western blotting (~140 kDa). By comparison to the input panel (Fig 3-4B), these HMW species were also enriched in the samples recovered by SNO-RAC or acyl-RAC. Nevertheless, the relative titer of HMW forms of N548Q128 is variable and these forms are much less abundant (by comparison to monomer) than for Ataxin-1Q85.

In all experiments, samples were prepared for SDS-PAGE by addition of

β-mercaptoethanol (β-ME) and boiling; however, the HMW material was also detected when reduction by β-ME was at 37ºC (Fig 3-5). Thus, these HMW species were not an artefact of boiling prior to SDS-PAGE.

3-3. S-nitrosylation and S-acylation occur at multiple sites of Htt

The primary structure of huntingtin includes several distinct domains (Fig 1-2). At the N-terminus upstream of the polyQ tract is a serine-rich phosphorylated short domain

(1-17 a.a.) that lacks cysteine residues. This is followed by the polyglutamine-containing segment and a short polyproline tract. After the polyproline region, much of the sequence of Htt can be modeled as being composed of clusters of HEAT motifs, each of which consists of a helix-turn-helix [27]. No specific functions have been attributed to the domains of Htt. The in vivo cleavage at any of several sites, as indicated in Fig 1-2A, is characteristic of pathogenesis [60]. We were wondering if there are a few specific cysteine residues that are modified. Later the results show that multiple sites are modified

(see below).

3-3-1. Htt N548 fragment S-nitrosylation and S-acylation

To locate the cysteine residues that are modified, COS7 and HEK293T cells were transfected to express the N548 N-terminal fragment (Q15 or Q128) of Htt and processed.

COS7 cells were of particular interest since they had been used to study S-palmitoylation of Htt [93]. HEK293T cells provide a higher yield of the recombinant proteins and therefore facilitate detection of modified cysteine residues.

To identify the modified cysteine residues, it is necessary to avoid changes of cysteine sulfhydryl status upon cell lysis. For this purpose, the cell lysis buffer included

N-ethylmaleimide (NEM) (Fig S2A). After elimination of excess NEM and reduction with ascorbate to selectively expose the cysteine sulfhydryl groups that had participated in S-nitrosylation, the samples were blocked with iodoacetamide and then immunoprecipitated (Fig S2B). In parallel, to detect sites that were S-acylated, samples were reduced with hydroxylamine before iodoacetamide-labeling and immunoprecipitation (Fig S2C). Samples were then separated by SDS-PAGE. The bands of purified Htt proteins were sliced for in-gel digestion. Tryptic peptides were then separated and analyzed by LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry). Detailed method is described in Appendix A7.

As summarized in Fig 3-6A and Table 3-1, seven of the nine cysteine residues in the

N548 fragment were identified and most were detected in both cell types. Moreover, all sites that could be S-nitrosylated were also found to be S-acylated. The representative spectra are shown in Fig S3.

Residues C204 and C214 were both in the same tryptic peptide, but SNO and

S-acylation on these residues can be individually identified on the basis of analysis of corresponding Cys→Ser point mutants (C204S and C214S) that we expressed in the same host cells. Two other sites (C105, C109) were also in a single tryptic peptide. MS detected both single and double modifications of this peptide (Table 3-1).

3-3-2. Full-length Htt S-nitrosylation and S-acylation

By expressing full-length Htt in HEK293T cells, we identified S-nitrosylation and

S-acylation of additional sites beyond the N-terminal 548 amino acids. Owing to the large size of Htt, the sequence coverage of the protein was incomplete (44%). Six additional

SNO sites and eight additional S-acylation sites beyond N548 were identified by mass spectrometry, as summarized in Fig 3-6B. Consistently, SNO-RAC and acyl-RAC experiments also detect S-nitrosylation and S-acylation of Htt C-terminal fragments

(585-3144 a.a.) expressed in cells (Fig 3-1F and Fig 3-7).

3-3-3. Identification of a major site of S-nitrosylation and S-acylation

Given that there are multiple SNO and S-acylation sites, some residues could be more frequently modified than others. Since S-palmitoylation of Htt on C214 has been reported in COS7 cells [93], we began by evaluating SNO and S-acylation of N548 wild-type versus the N548-C214S mutant using SNO-RAC and acyl-RAC.

In the context of normal polyQ (Htt N548Q15), Htt S-nitrosylation and S-acylation were reduced but not eliminated by this mutation (Fig 3-8A). SNO was reduced ~50% and S-acylation was reduced ~70% due to this mutation in N548Q15 (Fig 3-8B and

3-8C). By contrast, the equivalent mutation had no significant effect on N548Q128.

Therefore, the specificity of SNO and S-acylation in Htt appear to be modulated by the length of polyglutamine tract. Regulation of these modifications could contribute to the normal biology of Htt and to pathogenesis.

3-3-4. Htt Phosphorylation in response to nitric oxide donor treatment

Interestingly, in the samples prepared for S-nitrosylation detection, we also detected phosphorylation of Htt (Table 3-2). These tandem MS data identified phosphorylated

46 serine near C433 that can be S-nitrosylated and S-acylated. The phosphorylated sites are

S419 and/or S421, as well as S431, S432, S434, and/or S438. Because sample preparation was optimized to detect S-nitrosylation but not phosphorylation, the exact phosphorylated sites have not been studied further. Additionally, when SNO modifications are increased by eCysNO treatment (a chemical nitric oxide donor), phosphorylation increases. These results imply the interaction between S-nitrosylation and phosphorylation of Htt.

3-4. Polyglutamine-induced S-nitrosylation is not a global effect

Since polyQ expansion of Htt increases its S-nitrosylation and S-acylation, we asked whether expression of polyQ-expanded Htt changes SNO and S-acylation of other proteins in the same cells.

3-4-1. PolyQ-expanded Htt does not change global S-nitrosylation and S-acylation

We purified the complete S-nitrosylated and S-acylated proteomes of HEK293T cells using SNO-RAC and acyl-RAC. Coomassie blue staining showed that cells expressing polyQ-expanded Htt did not obviously change the global pattern of

S-nitrosylation or S-acylation (Fig 3-9).

3-4-2. PolyQ-expanded Htt does not affect gross S-nitrosylation and S-acylation of normal Htt

We asked whether the presence of polyQ-expanded Htt would increase SNO or

S-acylation of wild-type Htt. For this purpose, we co-expressed Htt exon1-Q97 along

47 with N548Q15 in HEK293T cells. No remarkable increase of S-nitrosylation or

S-acylation of N548Q15 was found in repeated experiments (Fig 3-10A to 3-10C).

Equivalent results were obtained in experiments with cells that co-express N548Q128 and full-length HttQ15 (Fig 3-10D to 3-10F).

The lack of stimulation might reflect the inability of these pairs of recombinant proteins to bind each other. Alternatively, there may be a limit to the amount of SNO or

S-acylation that can be accepted by HttQ15.

Figure 3-1.

Figure 3-1. Htt constructs. (A) Wild-type (wt) full-length Htt. The vertical lines indicates all 70 Cys residues. The horizontal open boxes indicate HEAT repeat motif clusters (Tartari, M. et al., Mol Biol Evol. 2008). The horizontal grey bar represents the region of proteolytic cleavage sites by caspases. Anti-Htt antibodies (MAB2166,

MCA2050, and MAB2168) and binding sites are labeled. (B) Full-length (FL) Htt constructs for transient expression. (C) Inducible FL Htt constructs expressed in PC12 cell. (D) Htt N-terminal 1-548 a.a. fragments that approximate proteolytic products of full-length Htt. (E) Constructs expressing Htt exon1 coding region (1-90 a.a.). (F)

C-terminal construct (585-3144 a.a.).

Figure 3-2.

Figure 3-2. PolyQ expansion increases S-nitrosylation of Htt. Recombinant proteins were expressed in cells for one day. SNO-RAC and acyl-RAC were used to recover

S-nitrosylated and S-acylated proteins, respectively. The negative control shows that non-specific binding is negligible. Western blotting was used to detect Htt. (A) SNO and

S-acylation of Htt N548 fragments expressed in COS7 cells. The longer exposure of the film shows the weak SNO signal from N548 with a normal polyQ tract. (B)

Quantification of S-nitrosylated and S-acylated N548 expressed in COS7 cells (n=3). (C)

SNO and S-acylation of Htt N548 fragments expressed in HEK293T cells. (D)

Quantitation of S-nitrosylated and S-acylated N548 expressed in HEK293T cells (n=6 for

S-nitrosylation and n=5 for S-acylation). (E) SNO and S-acylation of full-length Htt expressed in HEK293T cells. (F) Longer exposure for detecting S-nitrosylated Htt. SNO:

S-nitrosylation. S-acyl: S-acylation. ImageJ was used to determine band intensity in all figures. p-values from t-test are indicated if p<0.05. Error bars represent SEM in all figures.

Figure 3-3.

Figure 3-3. PolyQ expansion increases S-nitrosylation of Ataxin-1. (A) Diagram of full-length Ataxin-1. The horizontal open boxes indicate the AXH domain,

Ataxin-1/HBP1 (HMG box-containing protein 1 transcription factor). AXH of Ataxin-1 is a protein-protein interacting domain (Orr, H.T., Prog Neurobiol. 2012). The vertical lines indicates all 6 Cys residues. Cysteine residue numbers are indicated. Polyglutamine is interrupted by His residues. Ataxin-1 with uninterrupted polyQ≥ 39 is pathogenic. (B)

FLAG-tagged full-length Ataxin-1 proteins. Q30 and Q85 were used in this study. (C)

SNO and S-acylation of Ataxin-1 proteins expressed in HEK293T cells. (D) Quantitation of S-nitrosylated and S-acylated Ataxin-1 expressed in HEK293T cells (n=3).

Recombinant proteins were expressed in cells for one day. SNO-RAC and acyl-RAC

52 were used to recover S-nitrosylated and S-acylated proteins, respectively. The negative control shows that non-specific binding is negligible. Western blotting was used to detect

Ataxin-1 (FLAG-tagged). SNO: S-nitrosylation. S-acyl: S-acylation.

Figure 3-4.

Figure 3-4. PolyQ expansion in Ataxin-1 and Htt N548 proteins increases

S-nitrosylated and S-acylated high molecular weight (HMW) species. SNO-RAC and acyl-RAC were used to recover S-nitrosylated and S-acylated proteins followed by

Western blotting. (A) Top: S-nitrosylated and S-acylated monomer and high molecular weight (HMW) species of Ataxin-1 expressed in HEK293T cells. Bottom:

HMW/monomer quantity ratios for Ataxin-1Q85 (n=5). All experiments show that HMW material is enriched in S-nitrosylated and S-acylated proteins. (B) S-nitrosylated and

S-acylated monomer and HMW species of Htt N548 expressed in HEK293T cells.

Figure 3-5.

Figure 3-5. The high molecular weight (HMW) species of Ataxin-1Q85 do not disappear at 37ºC incubation with sample buffer. SNO-RAC and acyl-RAC were performed (3 hr bead binding) to purify S-nitrosylated and S-acylated Ataxin-1Q85. The supernatant of RAC was reserved to run the Western. In parallel, input control was incubated with or without reducer (ascorbic acid or NH2OH) for 3 hr. FLAG-tagged

Ataxin-1Q85 was transiently expressed in HET293T cells for 1 day. ASC: ascorbic acid.

SNO: S-nitrosylated proteins. S-acyl: S-acylated proteins. Negative control: no reagents to reduce S-nitrosylated/S-acylated proteins for pull-down.

Figure 3-6.

Figure 3-6. Summary of the SNO and S-acylation sites identified by LC-MS/MS. (A)

Htt N548 fragments. Vertical lines indicate all Cys residues in the N548 fragment. Stars label modified Cys. Modifications on C137 and C517 were not detected in three independent experiments. C137 and C517 are indicated by arrows. (B) Full-length Htt.

MS-detected modifications of Cys are labeled in full-length Htt.

Figure 3-7.

Figure 3-7. The fragment beyond the N-terminal region is S-nitrosylated and

S-acylated. The Htt C-terminal fragment is S-nitrosylated and S-acylated. Full-length

HttQ15 (FL) and the Htt C-terminal fragment (C-t, 585-3144 a.a.) were expressed in

COS7 cells for one day. Full-length HttQ15 serves as a positive control for

S-nitrosylation and S-acylation. SNO: S-nitrosylation. S-acyl: S-acylation. SNO-RAC and acyl-RAC were used to recover S-nitrosylated and S-acylated proteins, respectively.

The negative control (no ascorbic acid or hydroxylamine reduction) shows non-specific binding is negligible. Western blotting was used to detect Htt. MAB2050 detects

C-terminal regions.

Figure 3-8.

Figure 3-8. The C214S mutation reduces Htt S-nitrosylation and S-acylation in the context of the normal polyQ tract. (A) S-nitrosylation (SNO) and S-acylation (S-acyl) of Htt N548Q15, N548Q15-C214S, N548Q128, and N548Q128-C214S. Recombinant proteins were expressed in COS7 cells for one day. The stars indicate significant reduction due to the C214S mutation. C: C214; S: C214S. (B) Quantification of Htt N548

S-nitrosylation ratio to the total N548. The inset enlarges the scale for N548Q15 and

N548Q15-C214S. (C) Quantification of Htt N548 S-acylation ratio to the total N548.

Figure 3-9.

Figure 3-9. Expression of polyQ-expanded Htt does not significantly increase

S-nitrosylation and S-acylation of global proteins. Full-length Htt proteins or N548 fragments were expressed in HEK293T cells. Extracted proteins were used for

SNO-RAC and acyl-RAC. Purified S-nitrosylated and S-acylated proteomes were detected by Coomassie blue staining. N15: cell expressing N548Q15; N128: N548Q128;

NT: no transfection; F15: full-length HttQ15; F128: full-length HttQ128. The band indicated by the star has an expected gel mobility of N548Q128.

Figure 3-10.

Figure 3-10. Expression of polyQ-expanded Htt does not significantly increase

S-nitrosylation and S-acylation of wild-type Htt. Wild-type (normal polyQ<40) Htt protein (full-length or N548) was co-expressed with the polyQ-expanded Htt fragment

(N548 or exon1-coding region). For the control samples, wild-type Htt was co-expressed with an empty vector control (v), wild-type N548 or wild-type exon1. SNO-RAC and acyl-RAC followed by Western blotting were used to detect S-nitrosylation and

S-acylation of wild-type Htt co-expressed with other constructs. In this experiments,

MAB2166 recognizes full-length Htt and N548 but not exon1. MAB2168 recognizes the

C-terminal region of full-length Htt but not N548/exon1. N548 and exon1 are

EGFP-tagged. (A, B, and C) Co-expression of Htt exon1Q97 or N548Q128 does not increase S-nitrosylation and S-acylation of N548Q15 (bands indicated by stars). (A) Input loading control. (B) S-nitrosylation of N548Q15 co-expressed with other constructs. (C)

S-acylation of N548Q15 co-expressed with other constructs. (D, E, and F) Co-expression of Htt exon1Q97 or N548Q128 does not increase S-nitrosylation and S-acylation of full-length HttQ15 (bands indicated by stars). (D) Input loading control. (E)

S-nitrosylation of HttQ15 co-expressed with other constructs. (F) S-acylation of HttQ15 co-expressed with other constructs. SNO: S-nitrosylation. S-acyl: S-acylation.

Table 3-1. Summary of SNO and S-acylation sites in Htt N548Q15 and N548Q128*

IAA modified peptide (%)a Mean fold Site modified Exp 1b Exp 2b Exp 3b Identified peptide sequence change by IAA (N548) (N548-C214S) (N548-C204S) (Q128/Q15)c Q15 Q128 Q15 Q128 Q15 Q128

MVADECLNK C152 (SNO) 1.7 4.1 1.1 1.8 0.26 2.1 4.0

MVADECLNK C152 (S-acyl) 1.3 4.3 1.1 1.7 0.34 1.4 3.0

TAAGSAVSICQHSR C280 (SNO) 3.8 2.8 3.1 5.5 0.91 3.3 1.6

TAAGSAVSICQHSR C280 (S-acyl) 2.4 3 2.9 4.1 0.95 2.2 1.7

SGSIVELIAGGGSSCSPVLSR C433 (SNO) 3.7 2.6 1.4 2 1.0 0.96 1.0

SGSIVELIAGGGSSCSPVLSR C433 (S-acyl) 3.3 2.2 1.3 1.4 1.4 1.4 0.9

CRPYLVNLLPSLTRd C204 (SNO) N/A N/A 0e 2.1 N/A N/A Infinitee

CRPYLVNLLPSLTRd C204 (S-acyl) N/A N/A 0.92 1.8 N/A N/A 2.0

SRPYLVNLLPCLTRf C214 (SNO) N/A N/A N/A N/A 1.2 6.1 5.1

SRPYLVNLLPCLTRf C214 (S-acyl) N/A N/A N/A N/A 3.8e 28.1e 7.4e

C105/C109 VNHCLTICENIVAQSVRg N/A N/A 0.32 0.97 N/A N/A 3.0 (SNO)

C105/C109 VNHCLTICENIVAQSVRg N/A N/A 0.3 0.78 N/A N/A 2.6 (S-acyl)

*MS analysis is performed by Dr. Liwen Wang (Center for Proteomics and

Bioinformatics, CWRU). aCysteine S-nitrosylation (SNO) or S-acylation (S-acylation) was reduced and then modified by IAA. Percentage was calculated as IAA-modified/unmodified peptide. bCOS7 cells was used for exp1 and exp2. HEK293T was used for exp3. cMean of IAA-modified Q128 (%)/IAA-modified (%) Q15.

63 dC214S mutation and modifications on C204 were identified. eLow MS signal. Quantitation may not be accurate. fC204 mutation and modifications on C214 were identified. Low MS signal, identified by

MS precursor ion and LC retention time compared to unmodified species. gC105 and C109 are very close. Modifications on both residues were combined to calculate modification percentage.

Table 3-2. Summary of increased SNO and phosphorylation of Htt N548Q15 by the nitric oxide donor eCysNO*

eCysNO IAA modified identified peptide sequence phosphorylation percent (%) treatmenta (%)b

SGSIVELIAGGGSSCSPVLSRc untreated 1.2 (C433) 7.5 7.3 (S431/432/434/438)

(S419/421);

In vivo 1.4 (C433) 30.9 8.9 (S431/432/434/438) SGSIVELIAGGGSSCSPVLSRc (S419/421);

In vitro 41.1 (C433) 9.9 30.1 (S431/432/434/438) SGSIVELIAGGGSSCSPVLSRc (S419/S421);

MVADECLNK Untreated 0.32 (C152) N/A

MVADECLNK In vivo 1.9 (C152) N/A

MVADECLNK In vitro 6.9 (C152) N/A

TAAGSAVSICQHSR untreated 0.69 (C280) N/A

TAAGSAVSICQHSR In vivo 1.5 (C280) N/A

TAAGSAVSICQHSR In vitro 13.2 (C280) N/A

*MS analysis is performed by Dr. Liwen Wang (Center for Proteomics and

Bioinformatics, CWRU). aeCysNO treatment increases S-nitrosylation. in vivo: Cells were treated with eCysNO before harvesting. in vitro: Extracted proteins were treated with eCysNO. bIAA modified cysteine sulfhydryl group reduced from S-nitrosylated cysteine by ascorbate. cTandem MS results in this study distinguished two groups of phosphorylated fragments in the peptide: (S419/421) and (S431/432/434/438).

Chapter 4

Interaction of Huntingtin and nitric oxide synthases (NOS)

Summary

Nitric oxide synthase (NOS) is required for S-nitrosylation. We intend to investigate the interaction between Htt, polyQ, and NOS. We find that increased expression of NOS elevates perinuclear inclusions of normal Htt in cell culture. Additionally, we do not find remarkable increase of Htt-NOS interaction upon polyQ expansion.

4-1. NOS expression increases Htt inclusions

The Htt N548 fragment forms cytoplasmic inclusions in cultured cells in a polyQ-dependent manner [93]. It has been reported that polyQ expansion increases Htt aggregates [164]. We also find that the frequency of inclusions (GFP-tagged N-terminal

Htt) is greater in cells expressing N-terminal Htt with polyQ expansion (Q128) than with normal polyQ (Q15) (Fig 4-1A). Because polyQ expansion notably increases SNO modification of Htt, nitric oxide synthase (NOS) level could modulate the formation of inclusions. Indeed, we found that co-expression of NOS increased Htt N548 inclusion formation (Fig 4-1A). Typically, the inclusions are located adjacent to the nucleus (Fig

4-1B).

In three experiments, cells co-expressing NOS, eNOS, or iNOS had increased levels of inclusions of N548Q15 and N548Q128 (cells with inclusions/total

EGFP-positive cells), as compared to empty vector controls (Fig 4-1C and 4-1D). The modest p-values for these differences upon nNOS coexpression may be due to the small

66 sample size (n=3). Statistic “effect size” has been found to be a useful indicator of the treatment effect on the observed outcome [165]. It is not influenced by the sample size.

Based on Cohen's effect size standard (expressed as Cohen's d; d value), d> 0.80 indicates a large effect that cannot be neglected. The effect size calculated in this study was d= 1.02 for N548Q15 (nNOS coexpression versus vector coexpression control) and d= 1.85 for N548Q128 (nNOS coexpression versus vector coexpression control).

Therefore the effect of the nNOS expression cannot be neglected.

Although their size and shape are variable, the inclusions in different preparation are similar to each other (Fig 4-2). As illustrated, the frequency of inclusion formation

(N548Q15 and N548Q128) was increased significantly (p<0.05) upon expression of eNOS or iNOS, respectively (Fig 4-1C and 4-1D). Specifically, eNOS increased

N548Q15 inclusions ~2 fold whereas iNOS increased N548Q128 inclusions ~1.5 fold

(Fig 4-1C and 4-1D). We also detected ~2 fold increase in S-nitrosylated N548Q15 upon co-expression of eNOS or iNOS (Fig 4-3A to 4-3C), suggesting that SNO is involved in

Htt inclusion formation.

4-2. PolyQ-expansion does not markedly increase NOS-Htt interaction

It is also possible that the nitrosylation and acetylation machineries act only on abnormally aggregated Htt. Therefore, we examined the physical interaction between Htt and NOS. Nevertheless, experiments co-expressing nitric oxide synthase (NOS) and the

N548 fragment did not show remarkable co-immunoprecipitation of N548 and NOS due to polyQ expansion (Fig 4-3D and 4-3E).

Figure 4-1.

Figure 4-1. Co-expression of nitric oxide synthase (NOS) promotes Htt N548 inclusion formation. EGFP-tagged Htt N548 fragments were expressed in HEK293T cells for two days. Empty vector co-expression serves as the negative control by comparison to nNOS, eNOS, or iNOS. (A) Representative fluorescence microscopy

68 images of co-expression of NOS and N548 showing the fluorescent inclusions that are present. All images were acquired under the same parameters. (B) Htt N548Q15 and

N548Q128 inclusions are adjacent to the nucleus. A single focal plane (2 μm thickness) is shown. The DAPI signal is shown in red and EGFP-tagged N548 signal in green. (C) The percentage of cells with N548Q15 inclusions in the presence of NOS expression. Top: average of three experiments; bottom: individual experiment (n=3). (D) The percentage of cells with N548Q128 inclusions in the presence of NOS expression (n=3). Top: average of three experiments; bottom: individual experiments. For quantification, we divided the number of cells with inclusions by the total number of cells expressing EGFP signal. The error bar represents the SEM.

Figure 4-2.

Figure 4-2. Inclusions of N548-EGFP in cells expressing nitric oxide synthase.

Although their size and shape are variable, the inclusions in different preparation are similar to each other. EGFP-tagged Htt N548 fragments were expressed in HEK293T cells for two days. Empty vector co-expression serves as the control for co-expression of nNOS, eNOS, or iNOS. DAPI signal is shown in blue and EGFP-tagged N548 signal in green. All images were acquired under the same parameters. Two fields are illustrated for each condition. Magnification in these pictures is larger than Figure 4-1 so the morphology of inclusions is clearer.

Figure 4-3.

Figure 4-3. NOS co-expansion increases S-nitrosylation of Htt N548Q15 and

NOS-N548 interaction is not significantly affected by polyQ expansion. (A, B, and C)

NOS overexpression increases Htt N548Q15 S-nitrosylation. Recombinant proteins were expressed in HEK293T cells for one day. Htt N548Q15 was co-expressed with empty vector control (v), nNOS (n), eNOS (e), or iNOS (i). SNO-RAC and acyl-RAC were used to recover S-nitrosylated and S-acylated proteins, respectively. Western blotting was used to detect Htt, nNOS, eNOS, and iNOS. (A) Input loading controls for Htt N548Q15 71 co-expressed with empty vector (v), nNOS (n), eNOS (e), or iNOS (i). SNO:

S-nitrosylation. (B) S-nitrosylation of N548Q15 co-expressed with empty vector control

(v), nNOS (n), eNOS (e), or iNOS (i). (C) The quantification of S-nitrosylated N548Q15.

SNO N548Q15 content is normalized to input N548Q15. Empty vector co-expression is set to one fold. ImageJ was used to determine band intensity. (D and E) PolyQ expansion in Htt N548 fragment does not significantly affect physical association of nitric oxide synthase (NOS) and N548 fragments. Htt N548 fragments were co-expressed with nNOS, eNOS, or iNOS in HEK239T cells for one day. Immunoprecipitation (IP) and Western blotting (WB) were used to detect NOS-Htt interaction. (D) N548 fragments recovered by IP with anti-EGFP. (E) Co-precipitated NOS with N548 fragments. Co-precipitation of nNOS or eNOS was detected whereas coprecipitated iNOS was not detectable. PolyQ expansion in N548 did not significantly change the N548-nNOS or N548-eNOS interaction.

Chapter 5

Discussion: Polyglutamine expansion and protein S-nitrosylation

Summary

We have identified many cysteine residues that are both S-nitrosylated and

S-acylated. The S-nitrosylation of Htt and Ataxin-1 is elevated due to polyglutamine tract expansion. We simulated the 3-dimentional structure of Htt HEAT motif cluster 1 and found that several modified cysteine residues are spatially clustered. Further study focusing on these cysteine residues may unveil the importance of S-nitrosylation in the early stages of Hungtinton’s disease pathogenesis.

5-1. PolyQ modulates specificity of S-nitrosylation and S-acylation

We found that S-nitrosylation and S-acylation of Htt are reduced if C214S is introduced into Htt N548 fragment with a normal polyQ tract (Q15). In contrast to the normal polyQ tract, a Htt fragment with a polyQ expansion (Q128) does not show significant reduction of S-nitrosylation and S-acylation due to C214S (Fig 3-8). These results suggest that C214 is the major site of S-nitrosylation and S-acylation in the wild-type Htt fragment whereas–since polyQ expansion increases S-nitrosylation and

S-acylation on the other cysteine residues–C214S does not have a comparable effect on

S-nitrosylation and S-acylation after polyQ expansion.

5-2. S-acylation and S-palmitoylation

An early study has found that polyQ expansion reduces Htt S-palmitoylation [93].

However, we did not detect reduction of S-acylation due to the polyQ expansion. This difference could result from the difference of detection methods. First, we measured

S-acylation, not S-palmitoylation. Second, this early study detected S-palmitoylation by treating cells with isotope-labeled palmitic acid, whereas we detected endogenous

S-acylation. Third, in contrast to the SDS-containing cell lysis buffer that we used, the early study didn’t include a strong detergent.

5-3. Inspecting a S-nitrosylated HEAT repeat motif of Htt

Htt is composed of several HEAT repeat motifs (Htt, Elongation factor 3, protein phosphatase 2A, and the yeast kinase TOR1), which is an α-helix dominant solenoid-like structures [25, 27]. Since it is reported that modifications of cysteine residue embedded in HEAT repeat motif of CRM1/XPO1 (exportin 1) abolish its function of protein-protein interaction [28, 29], we were wondering if there is any structural clue that could highlight the function of Htt.

We therefore performed computational simulation using the MMM server, based on the prediction of Tartari et al. [166] (Fig 5-1A). The template is the first 8 HEAT repeats of the PR65/A structure (1B3U) since Andrade et al. classified Htt HEAT motifs into the group represented by PR65/A [27]. Several clusters of HEAT motifs are found in the Htt sequence [60, 167] (Fig 1-2A).

The N-terminal 548 a.a (N548) fragment on which we have concentrated includes the first cluster (Fig 1-2A) and the first seven of nine cysteine residues, all of which are conserved and are located in helices. C137, by contrast, is unmodified and is situated in an intervening loop. As shown in Fig 5-1B, C105, C109, and C152 may be adjacent to

74 each other and therefore collectively influence the structure of the α-helices in which they reside. C204 and C214, on the other hand, are located in neighboring helices and may have their sulfhydryl groups oriented in such a fashion that interactions are unlikely.

C280 is further downstream and far-separated from other modified cysteine residues.

5-4. Significance of S-nitrosylation of Htt

Huntingtin is highly conserved among vertebrates and chordates [168] and most of its many cysteine residues are conserved among vertebrates. In human Htt, there are seventy cysteine residues. Htt undergoes S-nitrosylation and S-acylation, and

S-nitrosylation is remarkably increased by polyQ expansion. This relation seems to be of general significance for polyQ-containing proteins since parallel increases are seen for

Ataxin-1. Further examples may well be found in additional proteins with polyQ tracts.

Judging from the present observations on both the N548 fragment and the C-terminal region (585-3144 a.a.) of Htt, many of the cysteine residues of the full-length protein may actually be modified. These posttranslational modifications would be expected to affect local features of Htt domains and might cause deleterious gain of function. Moreover, their widespread distribution could have broader impact on global properties of Htt.

A fundamental question that emerges from these studies is the mechanism by which polyQ expansion increases cysteine modifications. One might envisage a change in levels of nitric oxide (NO) or of transferases and in fact there has been a report of elevated NO in cells expressing polyQ-expanded Htt [108]. We have observed that recombinant nNOS and eNOS can be co-immunoprecipitated with Htt N548 fragments expressed in

HEK293T cells (Fig 4-3E). Nevertheless, we did not detect a remarkable increase in the

75 degree of NOS association due to polyQ expansion.

Considering that cysteine residues are broadly distributed along the length of the protein (Fig 1-2A), the observation that polyQ expansion has a major effect on the overall level of modification may best be attributed to global conformational features of Htt that alter reactivity of these residues as well as their interactomes, which may include NOSs and nitrosylases [136, 169, 170]. PolyQ expansion may also increase NOS activity in the cytoplasm.

We have in fact noticed one indication of higher-level reorganization of Ataxin-1 and Htt upon polyQ expansion. This is the presence of high molecular weight species that are S-nitrosylated and S-acylated. None of these species is eliminated by reduction with

β-ME, suggesting novel chemistry and perhaps linkage to other proteins. A recent report has found that disulfide formation, which may be promoted by S-nitrosylation [171] can mediate Htt oligomerization [172]. The HMW species could correspond to a distinct Htt conformation that is resistant to β-ME.

Our mass spectrometry data identified multiple cysteine residues that are

S-nitrosylated. When we align the amino acid sequence of 21-mers centered on these

S-nitrosylated residues, we find that seven of nine are flanked by both negatively charged

(D/E) and positively charged (K/R/H) residues (Fig 5-2), fitting a canonical acid-base motif model [124, 127]. The only exception is C280 (which is also modified), perhaps because of its interaction with other proteins. Our experiments show that C137 and C517 are not S-nitrosylated. Neither of these cysteines is flanked by positively charged residue in the 21-mers. C137 does however lie in a context (I/L-X-C-X2-D/E) that is predicted to be nitrosylated by the iNOS/S100A8/S100A9 complex [136]. We do not know whether

76 this complex is formed in COS7 and HEK293T cells. Alternatively, Htt may be regulated by other NOS complexes that do not include S100A8/S100A9. The striking presence of multiple sites in Htt suggests an atypical role for its S-nitrosylation, considering that many S-nitrosylated proteins have no more than a few modified site [105].

There is no a priori reason to expect selected residues to be preferentially modified.

However, we observe that removal of a single site (C214) has a significant effect on the level of S-nitrosylation and S-acylation of wild-type Htt (but only a modest effect on polyQ expanded Htt). C214 is also a site of Htt S-palmitoylation by HIP14 [93], a palmitoyl acyltransferase enriched in the brain where it colocalizes with the Golgi [57,

94].

Several further observations stand out:

 S-nitrosylation and S-acylation often occur at the same sites.

 Some of the modifications localize to putative helical portions of HEAT

repeats.

 Although the stoichiometry of SNO and S-acyl modification of Htt is low,

our microscopic observations indicate a widespread impact of increased NOS

levels on Htt distribution.

Since the chemical properties of NO and acyl moieties are very different in size and hydrophobicity, it is likely that a shift from one to the other will cause changes of spatial relations with nearby residues. These potential interactions could be either in cis, between separate copies of Htt, or between Htt and neighboring proteins. In several other proteins,

S-nitrosylation and S-acylation do occur on the same cysteine residues and may be regulated in reciprocal fashion. Well-established examples concern GAP-43, SNAP-25

77 and PSD-95. PSD-95 is of central importance for regulation of NMDA receptors and neuronal survival [109, 158, 159].

Many proteins undergo S-nitrosylation or S-acylation but the extent of modification is often unknown. Some of these modifications could regulate specific functions and reactivities, while others could have more global effects on protein structure. In this view, considering the length of huntingtin and the broad distribution of its cysteine residues, its entire structure might undergo repeated fluctuations due to these (and other) modifications. It is also possible that these modifications reflect the age of Htt molecule.

In summary, we document a general effect of polyglutamine expansion on protein

S-nitrosylation and S-acylation. These changes could modulate protein conformation and progression of disease. We also observe the Htt inclusions increase in response to NOS overexpression and that they show an intimate relation to nuclei. The increase of

N548Q128 inclusions is less than for N548Q15. Considering that S-nitrosylation is elevated in N548Q128, this suggests that expression of NOS doesn’t remarkably affect

N548Q128 inclusion formation. Since multiple lines of evidence have suggested that large inclusions or aggregate formation can protect cells from toxic oligomer formation

[17-19], S-nitrosylation of Htt may be involved in this protective mechanism.

Additionally, we have found endogenous Htt S-nitrosylation in multiple mouse tissues (Fig 5-3A and 5-3B). PolyQ-dependent S-nitrosylation is also observed in the

PC12 pheochromocytoma cell line (Fig 5-3C and 5-3D), which has been used as a neuronal cell model [173]. Therefore we hypothesize that polyQ-dependent modifications impact the dynamics of local, and perhaps global protein conformation. On a broader scale, such effects may provide a rationalization for a general function of cysteine

78 residues of proteins in the relatively reducing cytosolic environment. We therefore suggest that Htt S-nitrosylation is involved in early dysfunction in Huntington’s disease, such as proteostasis, protein-protein interactions, calcium homeostasis, mitochondrial fission, gene expression, and vesicle transport [167] (Fig 5-4),

5-5. Future directions

In this study, we use Huntingtin (Htt) as the model to study the effect of polyglutamine tract (polyQ) expansion on S-nitrosylation (SNO). Understanding the biological significance of Htt SNO is imperative. Since we show that polyQ expansion increases Htt SNO and that elevated expression of nitric oxide synthase (NOS) increases

Htt inclusions, it will be helpful to identify which cysteine residues are required for SNO and Htt inclusion formation in response to NOS expression. Additionally, since SNO of a

HEAT repeat motif in CRM1/XPO1 (exportin 1) reduces protein-protein interactions, comparison of the binding partners of S-nitrosylated Htt and unmodified Htt should reveal the importance of Htt SNO. Since early cellular dysfunction eventually leads to neuronal cell death, it is relevant to elucidate early events (Fig 5-4) in which Htt

S-nitrosylation is involved. A C. elegans model expressing N-terminal Htt (171 a.a.) has been used for screening neuronal protective drugs [174]. Further studies of this practical model could unveil the importance of S-nitrosylation in early HD pathogenesis.

Figure 5-1.

Figure 5-1. Local environment of S-nitrosylated cysteine residues of Htt. (A)

Computer-simulated Htt HEAT repeat cluster 1 (79-397 a.a.). MS-identified

S-nitrosylation and S-acylation sites are indicated in red. In three independent experiments, we found no evidence of modification of C137. The sequence between polyQ and Gly79 is the 38-residue long polyproline tract

(PPPPPPPPPPPQLPQPPPQAQPLLPQPQPPPPPPPPPP). (B) Enlarged side view of

79-165 a.a. region from (A). Residues beyond Leu165 are masked. Structure simulation is performed by Dr. Tsan Sam Xiao (Department of Pathology, CWRU).

Figure 5-2.

Figure 5-2. Flanking sequences of S-nitrosylated cysteine residues of Htt. The

21-mers centered on cysteine residues are presented. The iNOS complex contains iNOS,

S100A8, and S100A9 proteins. CRM1 (Exportin 1) C517 is the SNO and leptomycin alkylation site.

Figure 5-3.

Figure 5-3. Full-length Htt is S-nitrosylated in mouse tissues and polyQ expansion increases S-nitrosylation of full-length Htt expressed in neuron-like PC12 cells. (A)

Endogenous wild-type Htt proteins in B6 mouse tissues are S-nitrosylated. Samples without ascorbic acid treatment are the negative control of SNO-RAC. (B) Input loading control for endogenous Htt in mouse tissues. Some degradation of full-length Htt was observed even the protease inhibitor cocktail was in the lysis buffer. (C) PolyQ expansion increases full-length Htt S-nitrosylation in PC12 pheochromocytoma cell line. Inducible recombinant HttQ23 or HttQ73 construct was induced by adding ponasterone A (5 μM) for two days. Nitric oxide donor eCysNO (S-nitrosocysteine ethyl ester) increases

S-nitrosylation. Living cells treated with eCysNO (100 mM) for 10 min before harvesting were used to the positive controls for SNO. Samples without ascorbic acid treatment are the negative controls. (D) Input loading control for recombinant HttQ23 or HttQ73

82 expressed in PC12 cells. SNO: S-nitrosylation. S-acyl: S-acylation. SNO-RAC and acyl-RAC were used to recover S-nitrosy lated and S-acylated proteins, respectively. The negative control (no ascorbic acid or hydroxylamine reduction) shows non-specific binding is negligible. The SNO-RAC experiment for mouse tissues is performed by Dr. Fabio Vasconcelos Fonseca (Institute for Transformative Molecular Medicine, CWRU).

Figure 5-4.

Figure 5-4. Cellular dysfunction results from Htt oligomers. Full-length Htt with polyQ expansion undergoes proteolysis that produces a N-terminal fragment which forms oligomers. Htt oligomers interfere with multiple cellular functions including proteostasis

(e.g. proteasome inhibition and abnormal protein-protein interactions), vesicle transport, gene expression (e.g. inhibition of BDNF expression), mitochondrial fission, and calcium homeostasis.

Chapter 6

Zygote formation in S. cerevisiae

Summary

In this chapter, I will describe a quite different project: Remodeling of the yeast transcriptome during zygote formation. In forming zygotes, haploid yeast cells undergo cell cycle arrest, polarized growth, and nuclear fusion. Section 5-1 describes the background of yeast mating types, zygote formation, and cellular mechanisms relevant to zygote formation. Section 5-2 starts with our transcriptome analysis in which we compare various yeast cell types: haploid cells, pheromone-stimulated haploids, zygotes, and diploids. The transcriptome analysis in this study is validated by comparing with previous reports: (1) many pheromone-responsive genes are identified in both previous studies and in this study; (2) most haploid- and diploid-specific genes reported in earlier studies are also identified in this study.

Finally, I highlight zygote-specific transcripts that are identified for the first time in this lab. These genes are involved in (1) mating loci regulation, (2) chromosome/spindle proteins organization, and (3) the mitochondrial respiratory chain. Interestingly, most genes that are relevant to chromosome/spindle organization are upregulated whereas genes related to the mitochondrial respiratory chain are downregulated. Upregulation of chromosome/spindle proteins and downregulation of ATP production therefore may be important to zygote formation.

6-1. Cell-cell fusion and budding yeast zygote formation

Animal cell fusion and polyploid formation are involved in physiologic and

85 pathogenic processes. For example, many bone-marrow cells migrate to liver, fuse with local cells, and then form polyploid bone-marrow-derived hepatocytes. Transplantation experiments in mice have shown that specific genomic markers of bone-marrow donors can be found in the polyploid hepatocytes of recipients [175].

Additionally, oncogenic viruses trigger cell-cell fusion and produce tetraploid cells

[176]. If these cells escape from checkpoint-dependent cell cycle arrest, tetraploid cells undergo multipolar division, which is mediated by multiple centrosomes, leading to formation of aneuploid cells [177].

Zygote formation is a canonical cell-cell fusion process which initiates development for many organisms. A recent experimental study indicates that nuclear fusion is required for early zebrafish embryogenesis [178]. Mitosis of early stages in zebrafish embryos produces cells containing karyomeres, which are intermediate nuclear structures that enclose one or several chromosomes in membrane-bound micronuclei that finally fuse together to form a single nucleus before the next mitosis. Defects in the nuclear protein

Bmb (Brambleberry) result in the failure of karyomere fusion. Interestingly, Bmb is homologous to yeast Kar5 [178], a protein required for nuclear fusion during yeast zygote formation. Therefore, underlying mechanisms of cell-cell fusion and karyogamy are evolutionarily conserved.

6-1-1. Two mating types of budding yeast and the pheromone response

Investigating zygote formation in budding yeast (Saccharomyces cerevisiae) has expanded knowledge of cell-cell fusion. Inspecting yeast zygote formation provides opportunities to study cell membrane fusion and karyogamy, including nuclear

86 congression and nuclear fusion [179, 180].

Budding yeast zygote formation requires two mating types of haploid cells, MATa and MATα. MATa cells release a mating factor (factor “a”) and respond to mating factor “α”, and vice versa (Fig 6-1). Once stimulated by mating factor (pheromone), the haploid cell cycle arrests in G1 phase and is committed to polarized growth. The resulting projection tip is oriented toward neighboring mating partner [181].

6-1-2. Cell membrane fusion and nuclear envelope fusion

When the projection tips of two mating cells contact each other, cell wall reorganization (local degradation and reformation) and cytoskeleton reorganization of the projection tip lead to formation of a narrow connection between the mating partners

[182-185]. Subsequently, nuclear congression and nuclear envelope fusion result in mononuclear zygotes (Fig 6-1B).

In budding yeast, the spindle pole body (SPB), which is the microtubule organizing center (MTOC) (Fig 6-2), anchors and orients microtubule cables that connect SPBs during nuclear congression (Fig 6-1B, II) [186]. Congression depends on a motor protein,

Kar3 (a kinesin) that is required to bring the two nuclei to the midzone of the zygote

[187]. When two nuclei contact to each other, the outer nuclear membrane fuses, followed by inner membrane fusion and nuclear envelope dilation [188]. Finally, fusion of adjacent SPBs unifies the microtubule organizing center (MTOC) [189]. Once karyogamy has finished, the diploid zygote reenters into the mitotic cell cycle, replicates

DNA, and produces diploid progeny. Thus, the SPB has an important role in karyogamy.

6-1-3. The yeast spindle pole body

The budding yeast spindle pole body (SPB) structure is composed of: outer plaque, central plaque, inner plaque, half bridge, and satellite (Fig. 6-2). It is the most intensively investigated microtubule organizing center in eukaryote cells [170, 180, 186, 190]. This multilayered organelle is embedded in the nuclear envelope (Fig 6-2 and Table 6-1) [170,

186, 191]. Central plaque peripheral proteins are required for SPB localization to nuclear envelope [192-194]. The outer and inner plaques orient the γ-tubulin complex, which serves as a microtubule (MT) nucleation platform [195-197]. The outer plaque modulates cytoplasmic MTs (cMTs/astral MTs), whereas the inner plaque organizes nuclear MTs

(nMTs). Despite the similarity of protein composition and shared functions in microtubule nucleation, the outer plaque is connected to the γ-tubulin complex by Spc72, but the inner plaque is connected by Spc110 [198, 199]. This difference allows distinct regulation of cytoplasmic and nuclear MT.

A structure including membrane proteins, called the “half-bridge”, associates with one side of the central plaque (Fig 6-2) [186]. Additionally, a satellite structure localized at the cytoplasmic face of the half-bridge appears in early stages of the cell cycle. This structure is the precursor of the new SPB [186]. Cytoplasmic microtubules are nucleated at the cytoplasmic face of the half-bridge before completion of SPB duplication and separation of the two SPBs. During the remaining phases of the cell cycle, the cytoplasmic microtubule nucleation center is transferred to the outer plaque of the SPB

[186, 200]. In contrast, during nuclear congression, microtubule cables initiate from the half-bridge but not from the outer plaque [186, 200]. By the end of nuclear congression,

SPB fusion initiates with contact between satellite-bearing half-bridges [186]. Therefore,

88 the half-bridge can regulate SPB duplication, SPB fusion, and cytoplasmic microtubule organization.

6-1-4. Karyogamy deficiency

A first genetic clue connecting karyogamy deficiency and the SPB involves the Kar1 protein that localizes to the cytoplasmic face of the half-bridge of the SPB (Fig 6-2). The kar1-1 mutant is deficient in nuclear congression due to failed microtubule organization

[201, 202]. Rose and colleagues found additional genes whose products (proteins) are required for karyogamy and classified karyogamy mutants into two groups. Most do not carry mutations in SPB genes [180].

The first group of genes (KAR1, KAR3, KAR4, and KAR9) regulates nuclear congression. Kar3 is a kinesin motor protein required for nuclear migration [187]. Kar4 is a transcription factor that activates expression of KAR3 and PRM2 genes, which are required for nuclear fusion [203, 204]. Kar9 protein localized at the projection tip of pheromone stimulated cells, and the kar9 mutant shows defective microtubule orientation toward the projection tip [205]. Kar9 also directs old/new SPBs segregation in the vegetative cell cycle by interacting with both plus ends of astral microtubules from the old SPB and actin cables that terminates at the budding site. Consequently, the old SPB moves into daughter cell [206].

The second group of genes (KAR2, KAR5, KAR7, and KAR8) is involved in nuclear fusion. Intriguingly, this group of genes is also involved in ER-targeted polypeptide translocation and chaperone function [207-211]. Dr. Tartakoff has demonstrated that nuclear fusion is abolished in the presence of DTT, a chemical reducing reagent, implying

89 that impaired disulfide-bond formation in ER leads to nuclear fusion defect, presumably by sequestering Kar2 [188]. Additionally, a temperature-sensitive mutant of SEC18, which can form Sec18/Sec17 (α-SNAP) complexes to disassemble SNARE complex, also results in defective nuclear fusion at the restrictive temperature [188].

These results imply that SNARE proteins in ER could be the direct fusogens that cause nuclear fusion in zygotes. Indeed, a recent publication has shown that a subgroup of SNAREs, encoded by BOS1, SEC20, UFE1, and USE1, which mediate trafficking between the ER and Golgi complex, are essential for nuclear fusion [212]. In summary,

ER chaperone- and SNARE-mediated vesicle fusion are essential for nuclear fusion.

Although most of the classical karyogamy mutants (kar) described above do not show the importance of SPB’s for karyogamy, SPB components are of central importance for nuclear congression, as are ER chaperones.

Spc72 re-localization from the outer plaque of the SPB to the half bridge is required for nuclear congression. Protein-protein interaction of Spc72 and Kar1 is essential for this re-localization [202, 213]. Another SPB half-bridge protein, Mps3, interacts with Jem1, which is an ER lumen DnaJ-like chaperone protein. Defects in Mps3-Jem1 interactions lead to weak nuclear fusion deficiency [214, 215]. Additionally, disrupting the

Mps3-Mps2 interaction by deleting protein-protein interacting domains impairs nuclear congression and nuclear fusion [216]. Deficiency of the γ-tubulin complex (Spc97, Spc98, and Tub4) also impairs karyogamy [191].

Thus, SPB components are of central importance for karyogamy.

6-1-5. Ploidy and chromosome tethering

Regulation of MTOC duplication is required for genome stability. Indeed, ploidy increase and supernumerary MTOCs have been reported to cause chromosome instability and aneuploidy in higher eukaryotes [177]. Nevertheless, physiological MTOC fusion after cell-cell fusion and MTOC duplication are poorly understood. By contrast, SPB inheritance is well understood in haploid cells. For example, pulse-chase experiments indicate that the preexisting SPB (the old one) migrates into the daughter cell while the newly formed SPB stays in the mother cell [217]. SPB inheritance has not been investigated in zygotes.

It is unclear how the MTOC handles genomes and ploidy increase; however, SPBs are larger in haploid cells than that in haploid cells [190, 218], SPB size could parallel chromosome number. By screening libraries of yeast gene deletion and temperature-sensitive strains, Pellman and colleagues identified 39 lesions, which are viable in haploid and diploid cells but are lethal in tetraploid cells [219]. Among these genes, 18 are involved in spindle, kinetochore, and SPB components including Cmd1,

Cnm67, Spc97, Spc110, and Tub4 (Fig 6-2). They also demonstrated that cell volume parallels ploidy although pre-anaphase spindle length does not. Furthermore, rates of chromosome loss and homologous recombination are drastically increased in tetraploid cells in comparison to diploid cells. Altogether, they suggest that geometric constraints of

SPB-microtubule relations are relevant to genomic stability.

The studies mentioned above focus on polyploid cells, but not on early stages of cell-cell fusion. Organelle dynamics during zygote formation is the interest of our lab. As described below, we have performed genome-wide analysis to compare the transcriptome of haploid cells, pheromone-stimulated haploids, zygotes, and diploid cells. We expected

91 that SPB components would be of particular interest since the roles of the SPB change during zygote formation.

6-2. Transcriptome profiles of yeast zygotes

Investigating zygote formation in budding yeast (Saccharomyces cerevisiae) has expanded knowledge of nuclear congression and nuclear fusion [179, 180]. The following sections explain how we purified zygotes and studied their transcriptome.

6-2-1. Budding yeast zygote purification and transcriptome analysis

Purifying zygote cells is a prerequisite for studying their transcriptome. We previously reported an improved zygote purification protocol in physiological conditions

(Fig S4 and Appendix A11) [220]. In brief, each mating type expressed either a mCherry- or EGFP-tagged fluorescent protein. Mixing the two mating types allowed the formation of zygotes which express both mCherry- and EGFP-tagged proteins, making it possible to purify zygotes by flow cytometry. We then analyzed transcriptomes from replicated samples of haploid cells, pheromone-stimulated haploids, zygotes, and from diploid cells. Transcripts that did not show ≥1.5 fold change or that did not exhibit a statistically significant change within any binary comparison of cell types were eliminated from our data set. The results allow us to identify zygote-specific genes and make it possible to assess changes in gene expression profiles in response to pheromone stimulation and increased ploidy.

6-2-2. Genetic determinants of budding yeast cell types

We first tested whether our dataset can identify MATa-, MATα-, and diploid-specific genes identified in previous studies [221].

Cell type-specific gene expression in yeast haploid MATa, MATα, or diploid cells is controlled by distinct regulators of transcription. Their downstream genes can be classified into three groups: (1) MATa-specific genes (a-specific), (2) MATα-specific genes (α-specific), and (3) Haploid-specific genes that are expressed in both MATa and

MATα. These genes are repressed by a1/α2 complex in diploid cells. (Fig 6-3) [221].

Expressing or silencing of these three groups of genes by specific transcriptional regulators establishes cell-type specificity.

MATa cells express Mcm1 and a1 transcription factors. Mcm1 alone promotes a-specific gene expression. MATα cells express Mcm1, α1, and α2. The Mcm1/α1 complex activates α-specific genes, whereas the Mcm1/α2 complex inhibits a-specific genes.

The diploid genome encodes both MATa and MATα transcriptional regulators.

Therefore, diploid cells can express Mcm1, a1, and α2 (but not α1, see explanation below). In contrast to haploid cells, diploid specificity is established by inhibiting haploid-specific genes rather than activating another group of genes. In diploid cells,

Mcm1/α2 inhibits a-specific genes. Additionally, a1/α2 complex inactivates another group of haploid-specific genes that are expressed in both MATa and MATα (Fig 6-3).

Because α1 expression is inhibited by a1/α2, α-specific genes are not activated in the absence of α1.

Galgoczy et al. applied chromosome immunoprecipitation (ChIP against transcription regulators), whole cell microarray data (cell-type comparison), and binding

93 motif analysis (cross-species consensus sequences in fungi) to identify the three groups of genes described above: (1) a-specific gene, (2) α-specific gene, and (3) other haploid-specific genes that are expressed in both MATa and MATα [221]. By comparing their results with our data for actively growing cells, we identified 7 of 8 genes in group

(1), 4 of 5 genes in group (2), and 16 of 19 genes in group (3) (Fig 6-4 and Fig 6-5). Thus, our microarray data detects cell-type specific genes. This encourages us to identify zygote-specific genes.

6-2-3. The pheromone response transcriptome

To further explore transcriptome changes, we identified genes whose expression was affected when yeast haploid cells were exposed to pheromone (mating factor). Mating factors target specific cell types. For example, haploid MATa cells respond to α-factor that is secreted by haploid MATα cells. We therefore compared our database with the research by Roberts et al. [222].

Our pheromone response data were derived from mating mixtures of MATa and

MATα cells followed by flow cytometric purification, whereas Roberts et al. added synthetic α-factor to MATa cells. We selected genes from their dataset obtained with 50 nM α-factor treatment which contain at least one datum yielding p< 0.05 at any indicated time point after α-factor treatment (0, 15, 30, 45, 60, 90, or 120 min). In our study, we collected pheromone-stimulated haploid MATa cells at 1.75 hr (105 min) or 2.5 hr (150 min) after mixing the two mating types of haploid cells.

We eliminated genes which do not possess any p<0.05 datum with a fold change greater than 1.5 (up- or down-regulation; ≥ +1.5 or ≤ -1.5), as compared with non-treated

94 controls. The rank order was then established based on the magnitude of significant change.

To compare our results with those of Roberts et al., we listed the top 100, 200, 300, etc. responsive genes in both datasets. Many pheromone-responsive genes are shared in both datasets (Fig 6-6A). For example, if 100 out of 5000 genes are pheromone-responsive, the chance to pick up this kind of gene in a random pool is 2%, i.e. 2 genes. Instead, we detected 11 genes out of 100 top pheromone-responsive genes in both datasets. The enrichment of genes in our list is therefore biologically significant.

Since Roberts et al. treated haploid cells with external mating factor in liquid culture while we established a microenvironment on the surface of agar, which seems much closer to physiological conditions, and therefore some variation between the lists is expected.

Many genes appear in both lists. For example, when comparing the top 100 pheromone-responsive genes (Table 6-2), 11 genes (AGA1, FIG2, PRM1, PRM6, FIG1,

FUS2, PRM2, PRM3, SPG4, YRO2, and MRH1) are shared in both lists (Table 6-2, dark-grey column). Nine genes (AGA1, FIG2, PRM1, PRM6, FIG1, FUS2, PRM2, PRM3, and SPG4) were upregulated and two (YRO2 and MRH1) were downregulated in both lists (Fig 6-6B and 6-6C). The annotated functions of these 11 genes are described in the following paragraphs.

Many of these genes are obviously relevant to zygote formation. AGA1 [223-225],

FIG2 [226, 227], PRM1 [184, 228], and PRM6 [184, 228] are critical for cell adhesion or membrane fusion. FIG1 mediates cell fusion and Ca2+ influx [226, 229]. FUS2 [185, 230,

231], PRM2 [184, 204, 228], and PRM3 [184, 228, 232, 233] are involved in nuclear

95 fusion. Intriguingly, SPG4 is known as an essential gene for stationary phase survival at high temperature [234]. It has been also reported that the expression of SPG4 under different growth conditions correlates with genes involved in intra-Golgi transport and v-SNARE [235]. Interestingly, cells lacking SPG4 resist rapamycin, the inhibitor of TOR kinase [236].

YRO2 and MRH1 were downregulated by pheromone. The significance of these two proteins during zygote formation is unclear. A recent study shows that their deletion impairs acetic acid tolerance in yeast [237]. Additionally, phosphorylated forms of these proteins were found in purified mitochondria [238, 239]. Curiously, Mrh1 was also found primarily on the plasma membrane, and also at the nuclear envelope [240]. The Mrh1 protein sequence shows 67% and 34% identity to Yro2 and to yeast Hsp30, respectively

[240]. Yeast Hsp30 is a novel heat shock-induced protein that localizes to the plasma membrane. It does not belong to the family of cytoplasmic heat shock proteins [241].

Yro2, Mrh1, and Hsp30 sequences are homologous to the seven-transmembrane microbial rhodopsins [242]. Specifically, the Yro2 protein sequence has 18% identity and

55% similarity to bacteriorhodopsin [242]. Since Hsp30 has been reported to be a regulatory component of plasma membrane H+-ATPase (Pma1) function [243], our data suggest that Yro2- and Mrh1-regulate membrane potential and that this is required for mating.

The 89 additional genes among our top 100 pheromone-responsive genes (Table 6-2, white column) are involved in diverse functions including amino acid metabolism, DNA repair, etc. We also identified a downregulated gene PRY3 of unknown function, which is suppressed by transcription factor Ste12 after pheromone stimulation [244]. Roberts et al.

96 however did not detect downregulation of PRY3. On the other hand, of the remaining 89 genes in Roberts et al. top 100 pheromone-responsive list (Table 6-2, light-grey column),

Roberts et al. identified 15 pheromone-responsive genes involved in mating and zygote formation (FUS1, FUS3, KAR3, KAR4, KAR5, PRM4, PRM5, PRM8, PRM10, AFR1,

ASG7, HYM1, NQM1, PCL2, and SCW10; Table 6-3). Our dataset identifies ten of these but misses five of these genes (FUS3, PRM5, PRM8, HYM1, and SCW10); however, these ten are not in our top 100 pheromone-responsive gene list. (Their rank in this study,

FUS1: #233, KAR3: #822, KAR4: #182, KAR5: #150, PRM4: #159, PRM10: #137, AFR1:

#724, ASG7: #1372, NQM1: #672, and PCL2: #218.)

Thus, results from our study and Roberts et al. suggest that:

(1) Pheromone-responsive genes are exaggeratedly elevated by external α-factor

treatment.

(2) More physiological pheromone also activates many of the same

pheromone-responsive genes identified in previous studies using synthetic

pheromone (AFR1, ASG7, FUS1, KAR3, KAR4, KAR5, NQM1, PCL2, PRM4,

and PRM10).

(3) The upregulation and downregulation of other genes in our list will need further

study.

6-2-4. Zygote-specific transcriptome

Several features of zygotes may require proteins that are of lesser importance for haploid and diploid cells. For example, zygote formation could require equipment that facilitates nuclear congression and fusion, SPB unification, cohabitation and stabilization

97 of the two parental genomes, and any equipment needed for fusion between heterotypic mitochondria or management of mitochondrial genomes. Zygotes may also be equipped with proteins that ensure that they will no longer express mating type-specific genes.

For our special interest in cellular functions of zygotes, a missing puzzle of the yeast cell life cycle, we focused on genes which are specifically upregulated or downregulated in this stage, but are steadily expressed in haploid cells, mating factor-stimulated haploids, and conventional diploid cells. A total of 81 genes satisfy these criteria (Fig 6-7 and

Table 6-4). We classified them into four groups according to their statistical significance and the threshold of expression fold change in ZE and ZL stages. Zygotes in ZE stage are early zygotes (1.75 hr after mating) whereas zygotes in ZL stage are later zygotes (2.5 hr after mating); however, both are still producing initial buds.

Type I genes (Table 6-5): Transcript expression in both ZE and ZL was ≥ 1.5 times fold change (FC) greater than that in mating factor-stimulated haploid cells.

Type II genes (Table 6-5) were expressed in ZE (≥ 1.5 FC with statistical significance), and expression level in ZL did not differ from mating factor-stimulated haploids (≤ 1.5 FC, no statistical significance).

In contrast, type III genes (Table 6-5) were expressed only in ZL (≥ 1.5 FC with statistical significance).

The remaining genes are classified as type IV (Table 6-5). Genes in Type IV are significantly upregulated or downregulated (≥ 1.5 FC with statistical significance) in only one of the zygote stages (ZE or ZL). Thus, expression in the other zygote stage (ZL or ZE) was not statistically significant or the expression magnitude was small (≤ 1.5 FC), i.e. either ≥ 1.5 FC without statistical significance or ≤ 1.5 FC with statistical significance.

In order to identify the common functions of these genes, we applied Gene ontology analysis (GoMiner [245]) on these 81 zygote-specific genes. The result suggests that cell cycle regulation and chromosome segregation are important in zygotes (Fig 6-7C).

Because this result is rather non-specific, we also inspected the list for candidate functions.

Among the genes whose transcript levels change in zygotes, those that change at both time points are statistically most secure (Type I). Judging from visual inspection of the data, this group of 14 genes includes a subset that is plausibly related to the zygote state. The most conspicuous is the increase of FKH1, Fkh1 is a transcriptional regulator required for silencing HMR locus and activating HML locus (HML and HMR loci are hidden MAT left and hidden MAT right, respectfully. Each of these loci carries a silenced set of mating type-specific alleles.) [246, 247]. Fkh1 also suppresses CLB2 transcription, which is required for the G2/M phase transition [246]. Thus, Fkh1 may have regulatory roles in zygotes.

It is curious that ASH1 is downregulated since Ash1 inhibits transcription of the

HO endonuclease that initiates mating-type conversion [248]. Nevertheless, conventional laboratory strains of yeast have been engineered so that conversion does not occur. These unusual circumstances could contribute to the observed reduction of ASH1 transcripts.

Bud9 is one of the cell polarity factors that is required for bipolar budding; however, mutations of BUD9 have also been linked to aneuploidy [249, 250]. The reduction of

BUD9 transcripts in zygotes therefore could be related to yet-unidentified features of genome maintenance that are unique to circumstances in which the two parental genomes have encountered each other. Additionally, two transcripts related to microtubule-based

99 transport are affected. NDL1 (downregulated) and VIK1 (upregulated) encode proteins associating with motor proteins [251, 252]. Their involvement could reflect a need to ensure that the efficiency of nuclear fusion is maximized. The only Type I transcript obviously related to mitochondria is MIC14 that is downregulated. Although its molecular functions are not known, Mic14 is thought to localize to the intermembrane space of mitochondria [253]. In addition to these six transcripts, the significance of several others can be rationalized only by invoking indirect arguments. These are listed in

Table 6-6.

If one includes the transcripts whose titer has changed at one of the two time points

(ZE/1.75 hr or ZL/2.5 hr), other possible targets are evident. As summarized in Table 6-7, these include components of the SPB and kinetochores, as well as proteins that contribute to the organization of chromatin. For example, Smc3 is a component of cohesin complex, which is required for sister chromatid cohesin, and Smc6 is required for DNA repair as well as resolution of the supercoiling during DNA replication [254]. Furthermore, there are a number of transcripts encoding proteins that are needed for respiration, all of which are downregulated. The possible involvement of the SPB and kinetochores, as well as cohesin subunits, could be attributed to the mechanics of genome unification that is one of the hallmarks of zygote formation. Nevertheless, it is difficult to understand just why these specific proteins may be affected. Inspecting the SPB (Fig 6-2), kinetochore (Fig

6-8), and the canonical cohesin ring that encircles chromosomes (Fig 6-8), does not explain why the proteins in question are critical for zygotes.

The unprecedented downregulation of transcripts encoding mitochondrial proteins is also puzzling. As shown in Fig 6-9, many the affected transcripts encode proteins that are

100 involved in respiratory complexes, as well as accessory proteins. For example, ubiquinol-cytochrome c oxidoreduction, ATP synthesis, and iron/copper transfer in mitochondria. Thus downregulation of these genes implies that decreased activity of the respiratory chain and reduced ATP production in mitochondria are important to the zygotes.

6-3. Discussion

In combination with transcriptome research of yeast meiosis/sporulation [255] and the mitotic cell cycle [256], we are now able to add the critical missing stage, zygotes.

The zygote is an important missing element for understanding how cells regulate ploidy change. Our dataset could allow other laboratories to study this process. Additionally, our dataset also detects pheromone-responsive genes in a defined experimental process that is close to physiological conditions.

By tracking gene expression profiles during the entire yeast life cycle, we can comprehensively explore involvement of genes known to function in cell-cell contact, organelle fusion/fission, macromolecule transport, cell proliferation, cell differentiation/development, etc.

Since zygote cells were collected after mixing haploid MATa and MATα cells for

1.75 or 2.5 hr, they are partially synchronized with regard to cell cycle progression. Some upregulation or downregulation of zygotes-enriched transcripts thus could emphasize one phase of the cell cycle. We therefore inspected the peak expression of these 81 genes in budding yeast haploid cells by searching an online database Cyclebase 3.0

(http://www.cyclebase.org) [257]. We find that 23 of 36 zygote-specific genes that show

101 increases have peak expression at G1 and/or S phase. On the other hand, 28 of 45 that decreased show peak expression at G2 and/or M phase (Table 6-8). Therefore, some genes identified in this study may reflect cell cycle progression whereas others may be more relevant to zygote physiology per se. Especially genes whose expression increases in zygotes but do not show peak expression in the G1/S phase and those that decrease in zygotes but do not show peak expression in G2/M phase may be of zygotic relevance

(Table 6-8).

6-4. Future directions

In addition to identifying genes that are regulated specifically in zygotes, our findings provide the opportunity to scrupulously look into the gene markers specifically regulated at each stage in the yeast life cycle. With further study of these genes, we can identify those of greatest importance for the cell cycle of zygotes. We need to track the expression and turnover of the cyclins in zygotes and the length of the G1, S, G2, and M phases. We will then be able to determine the exact window of up- or down-regulation of the genes. Identification of checkpoints that operate in zygotes could also support future studies to determine the importance of these transcriptional changes at different stages of the zygote cell cycle. Further investigation of candidate transcripts enumerated in Tables

6-6 and Tables 6-7 will need to confirm the modulation of their titer and then explore the consequences of their deletion or downregulation with regard to zygote formation. Those listed in Tables 6-6, being of greatest statistical significance, should be the first priority.

102

Figure 6-1.

Figure 6-1. Life cycle of budding yeast (S. cerevisiae). (A) Life cycle of yeast. a:

MATa. α: MATα. Z: zygote. D: diploid. (B) The detailed processes of yeast zygote formation and initial budding illustrated in (A). SPB: spindle pole body. MT: microtubule.

Zygote formation requires two mating types of haploid cells (MATa and MATα). MATa and MATα secrete mating factor (pheromone) “a” and mating factor “α”, respectively.

After stimulation by pheromone secreted from the opposite mating type, haploid cell is arrested in G1 phase of cell cycle and engaged in polarization growth. Conjugation of

103 two mating types forms a dumbbell-like zygote cell. Then zygote starts to bud diploid cells. Diploid buds daughter cells (mitosis) in the proper environment but undergoes sporulation (meiosis) in stress such as starvation. Spores germinate in proper environment to form haploid cells. Haploid cells can independently live and proliferate (mitosis) before sensing the mating factors released from the opposite mating type.

104

Figure 6-2.

Figure 6-2. Structure and protein components of budding yeast spindle pole body.

For detail: Jaspersen, S.L. and Winey, M., Annu Rev Cell Dev Biol. 2004. Red frames indicate proteins which are encoded by zygote-specific genes identified in this study. cMTs: cytoplasmic microtubules. nMTs: nuclear microtubules. Zygote-specific genes identified in this study are highlighted by rectangles.

105

Figure 6-3.

Figure 6-3. Yeast cell-type specificity regulation. For detail: Galgoczy, D.J. et al.,

PNAS. 2004. MATa cells express Mcm1 and a1 transcription factors. Mcm1 alone promotes a-specific gene expression. MATα cells express Mcm1, α1, and α2. The

Mcm1/α1 complex activates α-specific genes whereas the Mcm1/α2 complex inhibits a-specific genes. The diploid genome encodes both MATa and MATα transcriptional regulators. Therefore, diploid cells can express Mcm1, a1, and α2 (but not α1, see explanation below). In contrast to haploid cells, diploid specificity is established by inhibiting haploid-specific genes rather than activating another group of genes. In diploid cells, Mcm1/α2 inhibits a-specific genes. Additionally, a1/α2 complex inactivates another group of haploid-specific genes that are expressed in both MATa and MATα. Because α1 expression is inhibited by a1/α2, α-specific genes are not activated in the absence of α1.

106

Figure 6-4.

Figure 6-4. Upregulated haploid MATa- and MATα-specific genes that are identified in both previous studies and in this study. Gene lists are from Galgoczy, D.J. et al.,

PNAS. 2004. Heat map: The haploid-specific genes identified in this study. Red: upregulated. Green: downregulated. FC (for the scale bar): fold change. Upper group: haploid MATa-specific genes (a-specific). Lower group: MATα-specific genes

(α-specific).

107

Figure 6-5.

Figure 6-5. Downregulated genes in diploid cells that are identified in both previous studies and in this study. Gene list is from Galgoczy, D.J. et al., PNAS. 2004. Heat map:

The downregulated genes in diploid cells identified in this study. Red: upregulated. Green: downregulated. FC (for the scale bar): fold change.

108

Figure 6-6.

Figure 6-6. Pheromone-responsive genes identified in both studies and their expression profiles. (A) The number of overlapping genes in both this study and Roberts et al., according to the size of rank (e.g. top 100, top 200, etc.). Among top 100

109 pheromone-responsive genes, 11 genes are overlapping in both lists. The expression profiles of these 11 genes are described in (B) and (C). (B) Time course of expression of physiological pheromone-responsive genes in this study. Each bar represents the logarithm value (with base 2) of the ratio of stimulated to non-stimulated signal. (C)

Time course of expression of synthetic pheromone-responsive genes in the list of Roberts et al. Each bar represents the logarithm value (with base 10) of the ratio of treated to non-treated signal. Lack of values in certain time points (labeled with stars) is due to the absence of statistical significance.

110

Figure 6-7.

111

Figure 6-7. Zygote-specific genes and clustered gene functions. (A) Zygote-specific gene expression profiles identified in this study: upregulated (red bars) or downregulated

(green bars) in zygotes (early or later) without showing significant changes in other stages. Four types (I-IV) are classified based on expression levels (1.5 fold change; FC) and statistical significance. Type I transcripts are changed in both early and later zygotes with significant change of ≥ 1.5 FC. Type II transcripts are changed in early but not later zygotes (significant with ≥ 1.5 FC) whereas Type III transcripts are changed in later but not early zygotes (significant with ≥ 1.5 FC). Type IV has four subgroups: (i) significant change of ≥ 1.5 FC in early zygotes and significant change of < 1.5 FC in later zygotes,

(ii) significant change of ≥ 1.5 FC in early zygotes and nonsignificant change of ≥ 1.5 FC in later zygotes, (iii) significant change of < 1.5 FC in early zygotes and significant change of ≥ 1.5 FC in later zygotes, and (iv) nonsignificant change of ≥ 1.5 FC in early zygotes and significant change of ≥ 1.5 FC in later zygote. S*: statistically significant. H: haploid cell. H*: haploid cell stimulated by pheromone. Z: zygote cell. D: diploid cell. (B)

Expression profiles of 81 zygote-specific genes, represented by a heat map. FC (for scale bar): fold change. Early: 1.75 hr incubation. Later: 2.5 hr incubation. Annotations of these genes are listed in Table 6-4. (C) Clustered gene functions by GoMiner (gene ontology analysis).

112

Figure 6-8.

Figure 6-8. Identified genes whose products are involved in the chromosome organization and segregation. Dashed arrows indicate the localizations and/or regulated targets of the gene products. KMN complex, CCAN complex, and the centromeric

DNA/histone form the major components of the kinetochore. SPB: spindle pole body.

Smc6, which is homologous to Smc3, is not shown here since it forms another ring structure that is required for DNA repair and the resolution of supercoiling during DNA replication.

113

Figure 6-9.

Figure 6-9. Identified genes whose products are involved in the respiratory chain of mitochondria. Dashed arrows indicate the localizations and/or regulated targets of the gene products. Complex I: NADH dehydrogenase. Complex II: succinate dehydrogenase.

Complex III: ubiquinol-cytochrome c oxidoreductase. Complex IV: cytochrome c oxidase.

Complex V: ATP synthase. Q: coenzyme Q/ubiquinone. cyt c: cytochrome c. H+: proton. e-: electron. Copper and iron are required for the electron transfer in the respiratory chain.

Aim39 is not shown here because its localization is unclear. Null mutation of the AIM39 leads to increased mitochondrial genome loss.

114

Table 6-1. Spindle pole body genes

SPB SPB localization Gene deficiency effects on zygote formation Genes TUB4 γ-tubulin complex Unknown SPC97 γ-tubulin complex Unknown SPC98 γ-tubulin complex Unknown SPC72 outer plaque, half-bridge Deficient spindle orientation and karyogamy [202, 213, 258] outer plaque, satellite, inner layer CNM67 Nuclear migration deficiency in zygote mitosis [200, 202] 1 NUD1 outer plaque, satellite Unknown Defective Cmd1-Spc110 interaction leads to karyogamy deficiency CMD1 central plaque [259] central plaque, satellite, inner SPC42 Unknown layer 2 Defective Cmd1-Spc110 interaction leads to karyogamy deficiency SPC110 central plaque, inner plaque [259] NBP1 central plaque, SPB periphery Unknown SPC29 central plaque, satellite Unknown BBP1 SPB periphery Unknown Mutant forms monopolar spindles, leads to deficiency of nuclear MPS2 SPB periphery congression and nuclear fusion [216, 260] NDC1 SPB periphery Unknown CDC31 half-bridge Unknown KAR1 half-bridge Defective for nuclear fusion [201] Mutant forms monopolar spindles, leads to deficiency of nuclear MPS3 half-bridge congression and nuclear fusion [216, 261]

115

Table 6-2. Pheromone-responsive genes (top 100)

rank identified gene rank identified genes overlapping (this study)a (this study)a (Roberts et al.)b (Roberts et al.) b genesc 6 PRM6 9 PRM6 PRM6 10 FIG1 14 FIG1 FIG1 12 PRM3 23 PRM3 PRM3 13 PRM1 20 PRM1 PRM1 14 PRM2 10 PRM2 PRM2 16 FIG2 21 FIG2 FIG2 17 SPG4 33 SPG4 SPG4 25 FUS2 27 FUS2 FUS2 27 AGA1 26 AGA1 AGA1 57 YRO2 53 YRO2 YRO2 97 MRH1 64 MRH1 MRH1 1 MF(ALPHA)1 1 YET2 2 MF(ALPHA)2 2 STU2 3 MET17 3 SRS2 4 MATALPHA1/HMLALPHA1 4 SPO1 5 ECM13 5 SPH1 7 AAD4 6 SPC25 8 BOP2 7 SPC24 9 FRM2 8 SNU56 11 FLR1 11 NUD1 15 MMP1 12 MCH2 18 MET2 13 KAR5 19 MHT1 15 EMP46 20 AAD16/AAD4 16 DIT1 21 MET32 17 DCS1 22 STE3 18 CLN1 23 HXT11/HXT12 19 MNN1 24 IMD1/IMD2 22 AFR1 26 NCE103 24 ASG7 28 HAC1 25 SPT21 29 STP4 28 CIK1 30 TMA10 29 API2 31 YRF1-1 to YRF1-8 30 CLB1 32 YPK2 31 FUS1 33 SAG1 32 ACM1 34 DIP5 34 SWI5 35 FRE1 35 BUD4 36 SPT5 36 SIP4 37 FRE3 37 ECM18 38 MET14 38 HYM1 39 ECM4 39 RTT107 40 VHT1 40 IME4 41 COX19 41 ICY1 42 YAT2 42 NSE4 43 PUS2 43 AIM44 44 AGP1 44 PCL2

116

45 ATR1 45 PUT1 46 CIT3 46 MCD1 47 RGM1 47 TMN2 48 SFG1 48 PRM4 49 RPR2 49 NQM1 50 PRY3 50 ACE2 51 RIP1 51 FUS3 52 PHM8 52 KAR4 53 SDH2 54 YHP1 54 ALD6 55 HTA1 55 BUD5 56 CLB2 56 HMX1 57 TDP1 58 MUP1 58 PRM10 59 CYT1 59 HTB2 60 FMP23 60 HTB1 61 SOK2 61 INO2 62 RAX1 62 ALG14 63 OPT1 63 SRL1 64 HEM3 65 HHF2 65 PDR8 66 GPG1 66 GSH1 67 RNR3 67 CWP1 68 HHF1 68 GIP1 69 RNR1 69 GTT2 70 HTA2 70 ARN2 71 SPO16 71 DUR3 72 TOS4 72 BDF2 73 PMU1 73 DSE1 74 KAR3 74 NKP2 75 EGT2 75 NCA3 76 YPS3 76 TPO1 77 PRM8 77 PUT4 78 AMS1 78 PIC2 79 GSC2 79 WHI3 80 PMS1 80 SAM3 81 SPO77 81 COS5/COS7 82 MSG5 82 PTP3 83 AIM34 83 MIR1 84 PRB1 84 ZRT1 85 PRM5 85 SOL4 86 HHT1 86 GRX8 87 PGM1 87 SRO9 88 PHO12 88 LRO1 89 HEK2 89 IMP2' 90 YOX1 90 FRE4 91 ELO1 91 GDH2 92 CYC3 92 PIG1 93 HST3 93 COX5A 94 PUG1 94 WSC4 95 SCW10

117

95 DHH1 96 PGU1 96 YKE4 97 ESC8 98 SOL1 98 PLB2 99 ADY2 99 CRG1 100 POT1 100 NCE102

aTop 100 pheromone-responsive genes identified in this study (white column) bTop 100 pheromone-responsive genes identified in Roberts et al. (light-grey column) cThe dark-grey column shows overlapping 11 genes identified in both this study and

Roberts et al.

118

Table 6-3. A subset of pheromone-responsive genes

gene annotated function (SGD)a Membrane protein localized to the shmoo tip; required for cell fusion; expression regulated by mating FUS1 pheromone; proposed to coordinate signaling, fusion, and polarization events required for fusion; potential Cdc28p substrate. Mitogen-activated serine/threonine protein kinase involved in mating; phosphoactivated by Ste7p; substrates include Ste12p, Far1p, Bni1p, Sst2p; inhibits invasive growth during mating by FUS3 phosphorylating Tec1p, promoting its; inhibits recruitment of Ste5p, Cdc42p-mediated asymmetry and mating morphogenesis. Minus-end-directed microtubule motor; functions in mitosis and meiosis, localizes to the spindle pole KAR3 body and localization is dependent on functional Cik1p, required for nuclear fusion during mating; potential Cdc28p substrate. Transcription factor required for response to pheromones; also required during meiosis; exists in two KAR4 forms, a slower-migrating form more abundant during vegetative growth and a faster-migrating form induced by pheromone. Protein required for nuclear membrane fusion during karyogamy; localizes to the membrane with a KAR5 soluble portion in the endoplasmic reticulum lumen, may form a complex with Jem1p and Kar2p; similar to zebrafish Brambleberry protein; expression of the gene is regulated by pheromone. Pheromone-regulated protein proposed to be involved in mating; predicted to have 1 transmembrane PRM4 segment; transcriptionally regulated by Ste12p during mating and by Cat8p during the diauxic shift Pheromone-regulated protein, predicted to have 1 transmembrane segment; induced during cell PRM5 integrity signaling; PRM5 has a paralog, YNL058C, that arose from the whole genome duplication. Pheromone-regulated protein; contains with 2 predicted transmembrane segments and an FF sequence, PRM8 a motif involved in COPII binding; forms a complex with Prp9p in the ER; member of DUP240 gene family; PRM8 has a paralog, PRM9, that arose from a segmental duplication. Pheromone-regulated protein; proposed to be involved in mating; predicted to have 5 transmembrane PRM10 segments; induced by treatment with 8-methoxypsoralen and UVA irradiation. Protein required for pheromone-induced projection (shmoo) formation; regulates septin architecture AFR1 during mating; has an RVXF motif that mediates targeting of Glc7p to mating projections; interacts with Cdc12p; AFR1 has a paralog, YER158C, that arose from the whole genome duplication. Protein that regulates signaling from G protein beta subunit Ste4p; contributes to relocalization of Ste4p ASG7 within the cell; specific to a-cells and induced by alpha-factor. Component of the RAM signaling network; is involved in regulation of Ace2p activity and cellular HYM1 morphogenesis, interacts with Kic1p and Sog2p, localizes to sites of polarized growth during budding and during the mating response. Transaldolase of unknown function; transcription is repressed by Mot1p and induced by alpha-factor NQM1 and during diauxic shift; NQM1 has a paralog, TAL1, that arose from the whole genome duplication Cyclin, interacts with cyclin-dependent kinase Pho85p; member of the Pcl1,2-like subfamily, involved in the regulation of polarized growth and morphogenesis and progression through the cell cycle; PCL2 localizes to sites of polarized cell growth; PCL2 has a paralog, PCL9, that arose from the whole genome duplication. Cell wall protein with similarity to glucanases; may play a role in conjugation during mating based on SCW10 mutant phenotype and its regulation by Ste12p; SCW10 has a paralog, SCW4, that arose from the whole genome duplication.

aAnnotated function from SGD: http://www.yeastgenome.org/

119

Table 6-4. Zygote-specific genes

gene profilea annotated function (SGD)b Putative protein of unknown function; null mutant displays elevated frequency of AIM39 TYPE IV mitochondrial genome loss. Transcription factor involved in regulation of arginine-responsive genes; acts with Arg81p ARG80 TYPE IV and Arg82p. Chorismate mutase, catalyzes the conversion of chorismate to prephenate to initiate the ARO7 TYPE IV tyrosine/phenylalanine-specific branch of aromatic amino acid biosynthesis. Component of the Rpd3L histone deacetylase complex; zinc-finger inhibitor of HO transcription; mRNA is localized and translated in the distal tip of anaphase cells, resulting ASH1 TYPE I in accumulation of Ash1p in daughter cell nuclei and inhibition of HO expression; potential Cdc28p substrate. Alcohol acetyltransferase, may play a role in steroid detoxification; forms volatile esters ATF2 TYPE III during fermentation, which is important for brewing and winemaking. Scaffold protein responsible for phagophore assembly site organization; regulatory subunit of an autophagy-specific complex that includes Atg1p and Atg13p; stimulates Atg1p ATG17 TYPE IV kinase activity; human ortholog RB1CC1/FIP200 interacts with p53, which inhibits autophagy in human cells. Component of autophagosomes and Cvt vesicles; unique ubiquitin-like protein whose conjugation target is the lipid phosphatidylethanolamine (PE); Atg8p-PE is anchored to ATG8 TYPE IV membranes, is involved in phagophore expansion, and may mediate membrane fusion during autophagosome formation; deconjugation of Atg8p-PE is required for efficient autophagosome biogenesis. NAD-dependent (R,R)-butanediol dehydrogenase, catalyzes oxidation of BDH1 TYPE I (R,R)-2,3-butanediol to (3R)-acetoin, oxidation of meso-butanediol to (3S)-acetoin, and reduction of acetoin; enhances use of 2,3-butanediol as an aerobic carbon source BDS1 TYPE I Bacterially-derived sulfatase required for use of alkyl- and aryl-sulfates as sulfur sources Protein involved in bud-site selection; mutant has increased aneuploidy tolerance; diploid mutants display a unipolar budding pattern instead of the wild-type bipolar pattern, and bud BUD9 TYPE I at the distal pole; BUD9 has a paralog, BUD8, that arose from the whole genome duplication Essential kinetochore protein; component of the CBF3 multisubunit complex that binds to the CDEIII region of the centromere; Cbf2p also binds to the CDEII region possibly CBF2 TYPE IV forming a different multimeric complex, ubiquitinated in vivo; relative distribution to the spindle pole body decreases upon DNA replication stress Component of the spindle pole body outer plaque; required for spindle orientation and CNM67 TYPE III mitotic nuclear migration; CNM67 has a paralog, ADY3, that arose from the whole genome duplication Copper metallochaperone that transfers copper to Sco1p and Cox11p for eventual delivery COX17 TYPE IV to cytochrome c oxidase; contains twin cysteine-x9-cysteine motifs Subunit of the Cop9 signalosome, which is required for deneddylation, or removal of the CSI1 TYPE IV ubiquitin-like protein Rub1p from Cdc53p (cullin); involved in adaptation to pheromone signaling; functional equivalent of canonical Csn6 subunit of the COP9 signalosome Replication fork associated factor; required for stable replication fork pausing; component CSM3 TYPE IV of the DNA replication checkpoint pathway; required for accurate chromosome segregation during meiosis; forms nuclear foci upon DNA replication stress Basic leucine zipper (bZIP) transcription factor, in ATF/CREB family; mediates transcriptional activation of NCE103 (encoding carbonic anhydrase) in response to low CST6 TYPE IV CO2 levels such as in the ambient air; proposed to be a regulator of oleate responsive genes; involved in utilization of non-optimal carbon sources and chromosome stability; CST6 has a paralog, ACA1, that arose from the whole genome duplication Sorting factor, central regulator of spatial protein quality control; physically and functionally interacts with chaperones to promote sorting and deposition of misfolded CUR1 TYPE IV proteins into cytosolic compartments; involved in destabilization of [URE3] prions; CUR1 has a paralog, BTN2, that arose from the whole genome duplication Putative protein of unknown function; non-essential gene identified in a screen for mutants DAS2 TYPE IV with increased levels of rDNA transcription; weak similarity with uridine kinases and with phosphoribokinases 120

Ser/Thr kinase involved in transcription and stress response; functions as part of a network of genes in exit from mitosis; localization is cell cycle regulated; activated by Cdc15p DBF2 TYPE IV during the exit from mitosis; also plays a role in regulating the stability of SWI5 and CLB2 mRNAs Putative ATP-dependent RNA helicase of the DEAD-box protein family; mutants show DBP1 TYPE III reduced stability of the 40S ribosomal subunit scanning through 5' untranslated regions of mRNAs; protein abundance increases in response to DNA replication stress Phosphoesterase involved in downregulation of the unfolded protein response, at least in DCR2 TYPE IV part via dephosphorylation of Ire1p; dosage-dependent positive regulator of the G1/S phase transition through control of the timing of START Integral membrane component of endoplasmic reticulum-derived COPII-coated vesicles, EMP46 TYPE IV which function in ER to Golgi transport Putative purine-cytosine permease, very similar to Fcy2p but cannot substitute for its FCY21 TYPE IV function Forkhead family transcription factor; rate-limiting replication origin activator; evolutionarily conserved lifespan regulator; binds multiple chromosomal elements with distinct specificities, cell cycle dynamics; regulates transcription elongation, chromatin FKH1 TYPE I silencing at mating loci, expression of G2/M phase genes; facilitates clustering, activation of early-firing replication origins; binds HML recombination enhancer, regulates donor preference during mating-type switching. Mitochondrial matrix protein, required for assembly or stability at high temperature of the FMC1 TYPE IV F1 sector of mitochondrial F1F0 ATP synthase; null mutant temperature sensitive growth on glycerol is suppressed by multicopy expression of Odc1p Tail-anchored ER membrane protein of unknown function; interacts with homolog Frt1p; promotes growth in conditions of high Na+, alkaline pH, or cell wall stress, possibly via a FRT2 TYPE I role in posttranslational translocation; potential Cdc28p substrate; FRT2 has a paralog, FRT1, that arose from the whole genome duplication Subunit of GID complex; involved in proteasome-dependent catabolite inactivation of gluconeogenic enzymes FBPase, PEPCK, and c-MDH; forms dimer with Rmd5p that is FYV10 TYPE IV then recruited to GID Complex by Gid8p; contains a degenerate RING finger motif needed for GID complex ubiquitin ligase activity in vivo, as well as CTLH and CRA domains; plays role in anti-apoptosis; required for survival upon exposure to K1 killer toxin Regulatory subunit of protein phosphatase 1 (Glc7p); involved in glycogen metabolism and chromosome segregation; proposed to regulate Glc7p activity via conformational GLC8 TYPE IV alteration; ortholog of the mammalian protein phosphatase inhibitor 2; protein abundance increases in response to DNA replication stress Proposed gamma subunit of the heterotrimeric G protein that interacts with the receptor GPG1 TYPE III Gpr1p; involved in regulation of pseudohyphal growth; requires Gpb1p or Gpb2p to interact with Gpa2p; overproduction causes prion curing GTPase-activating protein (GAP) for the yeast Rab family member, Ypt6p; involved in GYP6 TYPE IV vesicle mediated protein transport Transcriptional activator involved in adaptation to weak acid stress; activates transcription of TPO2, YRO2, and other genes encoding membrane stress proteins; HAA1 has a paralog, HAA1 TYPE I CUP2, that arose from the whole genome duplication; relocalizes from cytoplasm to nucleus upon DNA replication stress HIP1 TYPE IV High-affinity histidine permease, also involved in the transport of manganese ions Aurora kinase subunit of the conserved chromosomal passenger complex (CPC; Ipl1p-Sli15p-Bir1p-Nbl1p), involved in regulating kinetochore-microtubule attachments; IPL1 TYPE IV helps maintain condensed chromosomes during anaphase and early telophase; required for SPB cohesion and prevention of multipolar spindle formation; localizes to nuclear foci that become diffuse upon DNA replication stress Mitochondrial membrane localized inositol phosphosphingolipid phospholipase C, ISC1 TYPE IV hydrolyzes complex sphingolipids to produce ceramide; activated by phosphatidylserine, cardiolipin, and phosphatidylglycerol; mediates Na+ and Li+ halotolerance Member of the Puf family of RNA-binding proteins, interacts with mRNAs encoding JSN1 TYPE IV membrane-associated proteins; involved in localizing the Arp2/3 complex to mitochondria; overexpression causes increased sensitivity to benomyl Serine/threonine protein kinase involved in regulation of exocytosis; localizes to the KIN1 TYPE IV cytoplasmic face of the plasma membrane; closely related to Kin2p

121

Nuclear protein involved in asymmetric localization of ASH1 mRNA; binds LOC1 TYPE II double-stranded RNA in vitro; constituent of 66S pre-ribosomal particles Component of the EGO complex, which is involved in the regulation of microautophagy, MEH1 TYPE IV and of the GSE complex, which is required for proper sorting of amino acid permease Gap1p; loss results in a defect in vacuolar acidification Mitochondrial intermembrane space protein of unknown function; required for normal MIC14 TYPE I oxygen consumption; contains twin cysteine-x9-cysteine motifs; protein abundance increases in response to DNA replication stress Putative metal transporter involved in mitochondrial iron accumulation; closely related to MMT2 TYPE IV Mmt1p Component of the SSU processome and 90S preribosome, required for pre-18S rRNA MPP10 TYPE IV processing, interacts with and controls the stability of Imp3p and Imp4p, essential for viability; similar to human Mpp10p Protein that forms heterodimers with Msh3p and Msh6p that bind to DNA mismatches to initiate the mismatch repair process; contains a Walker ATP-binding motif required for MSH2 TYPE IV repair activity and involved in interstrand cross-link repair; Msh2p-Msh6p binds to and hydrolyzes ATP Nuclear encoded protein needed for efficient splicing of mitochondrial COX1 aI5beta MSS18 TYPE IV intron; mss18 mutations block cleavage of 5' exon - intron junction; phenotype of intronless strain suggests additional functions Putative GTPase peripheral to the mitochondrial inner membrane, essential for respiratory MTG1 TYPE IV competence, likely functions in assembly of the large ribosomal subunit, has homologs in plants and animals One of two type I myosins; localizes to actin cortical patches; deletion of MYO3 has little MYO3 TYPE IV effect on growth, but myo3 myo5 double deletion causes severe defects in growth and actin cytoskeleton organization Homolog of nuclear distribution factor NudE, NUDEL; interacts with Pac1p and regulates NDL1 TYPE I dynein targeting to microtubule plus ends Essential component of the MIND kinetochore complex (Mtw1p Including NNF1 TYPE IV Nnf1p-Nsl1p-Dsn1p) which joins kinetochore subunits contacting DNA to those contacting microtubules; required for accurate chromosome segregation Component of the spindle pole body outer plaque; acts through the mitotic exit network to NUD1 TYPE IV specify asymmetric spindle pole body inheritance Protein with a possible role in phospholipid biosynthesis; null mutant displays an OPI10 TYPE IV inositol-excreting phenotype that is suppressed by exogenous choline; protein abundance increases in response to DNA replication stress Essential protein of unknown function; exhibits variable expression during colony PAM1 TYPE I morphogenesis; overexpression permits survival without protein phosphatase 2A, inhibits growth, and induces a filamentous phenotype Member of the seripauperin multigene family encoded mainly in subtelomeric regions; PAU5 TYPE IV induced during alcoholic fermentation; induced by low temperature and also by anaerobic conditions; negatively regulated by oxygen and repressed by heme Putative peroxisomal membrane protein required for import of peroxisomal proteins, PEX22 TYPE IV functionally complements a Pichia pastoris pex22 mutation Phosphoglucomutase, minor isoform; catalyzes the conversion from glucose-1-phosphate PGM1 TYPE IV to glucose-6-phosphate, which is a key step in hexose metabolism PAS domain-containing serine/threonine protein kinase; coordinately regulates protein synthesis and carbohydrate metabolism and storage in response to a unknown metabolite PSK1 TYPE IV that reflects nutritional status; PSK1 has a paralog, PSK2, that arose from the whole genome duplication Subunit of the ubiquinol-cytochrome c oxidoreductase complex which includes Cobp, QCR10 TYPE IV Rip1p, Cyt1p, Cor1p, Qcr2p, Qcr6p, Qcr7p, Qcr8p, Qcr9p, and Qcr10p and comprises part of the mitochondrial respiratory chain Subunit 7 of the ubiquinol cytochrome-c reductase complex, which is a component of the QCR7 TYPE IV mitochondrial inner membrane electron transport chain; oriented facing the mitochondrial matrix; N-terminus appears to play a role in complex assembly Checkpoint protein, involved in the activation of the DNA damage and meiotic pachytene RAD17 TYPE III checkpoints; with Mec3p and Ddc1p, forms a clamp that is loaded onto partial duplex DNA; homolog of human and S. pombe Rad1 and U. maydis Rec1 proteins

122

DNA helicase; proposed to promote replication fork regression during postreplication repair by template switching; RING finger containing ubiquitin ligase; stimulates the RAD5 TYPE IV synthesis of free and PCNA-bound polyubiquitin chains by Ubc13p-Mms2p; required for error-prone translesion synthesis; forms nuclear foci upon DNA replication stress Putative RNA exonuclease possibly involved in pre-rRNA processing and ribosome REX4 TYPE IV assembly Protein that binds to the Rap1p C-terminus and acts synergistically with Rif2p to help RIF1 TYPE II control telomere length and establish telomeric silencing; deletion results in telomere elongation Ribonuclease H2 subunit, required for RNase H2 activity; related to human AGS3 that RNH203 TYPE II causes Aicardi-Goutieres syndrome Protein component of the small (40S) ribosomal subunit; homologous to mammalian RPS9A TYPE IV ribosomal protein S9 and bacterial S4; RPS9A has a paralog, RPS9B, that arose from the whole genome duplication Protein involved in 7-aminocholesterol resistance; has seven potential membrane-spanning RTA1 TYPE I regions; expression is induced under both low-heme and low-oxygen conditions; member of the fungal lipid-translocating exporter (LTE) family of protein CTD phosphatase; dephosphorylates S5-P in the C-terminal domain of Rpo21p; has a RTR1 TYPE III cysteine-rich motif required for function and conserved in eukaryotes; shuttles between the nucleus and cytoplasm Histone acetyltransferase (HAT) catalytic subunit of the SAS complex SAS2 TYPE IV (Sas2p-Sas4p-Sas5p), which acetylates free histones and nucleosomes and regulates transcriptional silencing; member of the MYSTacetyltransferase family Subunit of the tRNA splicing endonuclease, which is composed of Sen2p, Sen15p, Sen34p, SEN34 TYPE IV and Sen54p; Sen34p contains the active site for tRNA 3' splice site cleavage and has similarity to Sen2p and to Archaeal tRNA splicing endonuclease Mitotic spindle protein; interacts with components of the Dam1 (DASH) complex, its SHE1 TYPE IV effector Sli15p, and microtubule-associated protein Bim1p; also localizes to nuclear microtubules and to the bud neck in a ring-shaped structure; inhibits dynein function Subunit of the multiprotein cohesin complex required for sister chromatid cohesion in SMC3 TYPE II mitotic cells; also required, with Rec8p, for cohesion and recombination during meiosis; phylogenetically conserved SMC chromosomal ATPase family member Component of the SMC5-SMC6 complex; this complex plays a key role in the removal of SMC6 TYPE IV X-shaped DNA structures that arise between sister chromatids during DNA replication and repair; homologous to S. pombe rad18 Inner plaque spindle pole body (SPB) component, links the central plaque component SPC29 TYPE II Spc42p to the inner plaque component Spc110p; required for SPB duplication Putative protein of unknown function; the authentic, non-tagged protein is detected in TBS1 TYPE IV highly purified mitochondria in high-throughput studies; TBS1 has a paralog, HAL9, that arose from the whole genome duplication Largest of six subunits of the RNA polymerase III transcription initiation factor complex TFC3 TYPE IV (TFIIIC); part of the TauB domain of TFIIIC that binds DNA at the BoxB promoter sites of tRNA and similar genes; cooperates with Tfc6p in DNA binding TFIIA large subunit; involved in transcriptional activation, acts as antirepressor or as TOA1 TYPE IV coactivator; homologous to largest and second largest subunits of human and Drosophila TFIIA Polyamine transport protein specific for spermine; localizes to the plasma membrane; TPO3 TYPE II member of the major facilitator superfamily Enzyme that mediates the conjugation of Rub1p, a ubiquitin-like protein, to other proteins; UBC12 TYPE IV related to E2 ubiquitin-conjugating enzymes Protein that interacts with Ulp1p, a Ubl (ubiquitin-like protein)-specific protease for Smt3p UIP4 TYPE I protein conjugates; detected in a phosphorylated state in the mitochondrial outer membrane; also detected in ER and nuclear envelope Putative subunit of U3-containing 90S preribosome complex; complex is involved in UTP30 TYPE IV production of 18S rRNA and assembly of small ribosomal subunit Cytoplasmic protein of unknown function; identified as a high-copy suppressor of the VHS2 TYPE IV synthetic lethality of a sis2 sit4 double mutant, suggesting a role in G1/S phase progression; similar to Mlf3p Protein that forms a complex with Kar3p at the spindle pole body, possible regulator of VIK1 TYPE I Kar3p function in microtubule-mediated processes; required for sister chromatid cohesion; 123

has similarity to Cik1p Protein kinase related to mammalian glycogen synthase kinases of the GSK-3 family; YGK3 TYPE I GSK-3 homologs (Mck1p, Rim11p, Mrk1p, Ygk3p) are involved in control of Msn2p-dependent transcription of stress responsive genes and in protein degradation Protein of unknown function; authentic, non-tagged protein is detected in highly purified ZRG8 TYPE IV mitochondria in high-throughput studies; GFP-fusion protein is localized to the cytoplasm; transcription induced under conditions of zinc deficiency

aType of expression profile (please see section 6-2-4). bAnnotated function from SGD: http://www.yeastgenome.org/

124

Table 6-5. Zygote-specific genes (ordered by type)

gene profilea gene profilea gene profilea ASH1 TYPE I ARG80 TYPE IV MSH2 TYPE IV BDH1 TYPE I ARO7 TYPE IV MSS18 TYPE IV BDS1 TYPE I ATG17 TYPE IV MTG1 TYPE IV BUD9 TYPE I ATG8 TYPE IV MYO3 TYPE IV FKH1 TYPE I CBF2 TYPE IV NNF1 TYPE IV FRT2 TYPE I COX17 TYPE IV NUD1 TYPE IV HAA1 TYPE I CSI1 TYPE IV OPI10 TYPE IV MIC14 TYPE I CSM3 TYPE IV PAU5 TYPE IV NDL1 TYPE I CST6 TYPE IV PEX22 TYPE IV PAM1 TYPE I CUR1 TYPE IV PGM1 TYPE IV RTA1 TYPE I DAS2 TYPE IV PSK1 TYPE IV UIP4 TYPE I DBF2 TYPE IV QCR10 TYPE IV VIK1 TYPE I DCR2 TYPE IV QCR7 TYPE IV YGK3 TYPE I EMP46 TYPE IV RAD5 TYPE IV LOC1 TYPE II FCY21 TYPE IV REX4 TYPE IV RIF1 TYPE II FMC1 TYPE IV RPS9A TYPE IV RNH203 TYPE II FYV10 TYPE IV SAS2 TYPE IV SMC3 TYPE II GLC8 TYPE IV SEN34 TYPE IV SPC29 TYPE II GYP6 TYPE IV SHE1 TYPE IV TPO3 TYPE II HIP1 TYPE IV SMC6 TYPE IV ATF2 TYPE III IPL1 TYPE IV TBS1 TYPE IV CNM67 TYPE III ISC1 TYPE IV TFC3 TYPE IV DBP1 TYPE III JSN1 TYPE IV TOA1 TYPE IV GPG1 TYPE III KIN1 TYPE IV UBC12 TYPE IV RAD17 TYPE III MEH1 TYPE IV UTP30 TYPE IV RTR1 TYPE III MMT2 TYPE IV VHS2 TYPE IV AIM39 TYPE IV MPP10 TYPE IV ZRG8 TYPE IV

aType of expression profile (please see section 6-2-4).

125

Table 6-6. Additional Type I zygote-specific genes

expression gene annotated function (SGD)a in zygote BDH1 decrease butanediol dehydrogenase BDS1 increase sulfatase FRT2 decrease ER membrane protein of unknown function HAA1 decrease transcriptional activator of TPO2 (polyamine transporter gene) and YRO2 (unknown function) PAM1 decrease overexpression induces filamentous growth RTA1 increase member of the fungal lipid-translocating exporters UIP4 decrease unknown function, may be involved in SUMOylation YGK3 increase related to mammalian glycogen synthase kinases of the GSK-3 family

aAnnotated function from SGD: http://www.yeastgenome.org/

126

Table 6-7. Type II-IV zygote-specific genes with manually-clustered functions

expression categorya gene annotated function (SGD)a in zygote SPB/spindle proteins CNM67 decrease component of the SPB outer plaque SPB/spindle proteins NUD1 increase component of the SPB outer plaque SPB/spindle proteins SHE1 increase mitotic spindle protein SPB/spindle proteins SPC29 increase component of the SPB inner plaque chromosome CBF2 increase kinetochore protein organization/segregation chromosome regulator of protein phosphatase 1 for chromosome GLC8 decrease organization/segregation segregation chromosome IPL1 increase Aurora kinase regulating chromosome segregation organization/segregation chromosome NNF1 increase kinetochore protein organization/segregation chromosome SMC3 increase cohesin component regulating chromosome segregation organization/segregation chromosome component of Smc5-Smc6 complex for removal of SMC6 increase organization/segregation X-shaped DNA structures during DNA replication and repair null mutant increases mitochondrial genome loss; unknown mitochondria AIM39 decrease function metallochaperone that promotes copper delivery to complex mitochondria COX17 decrease IV matrix protein required for assembly or high temperature mitochondria FMC1 decrease stability of complex V mitochondria JSN1 decrease localizing the Arp2/3 complex to mitochondria Putative metal transporter, involved in mitochondrial iron mitochondria MMT2 decrease accumulation mitochondria MSS18 decrease protein involved in splicing of mitochondrial COX1 intron putative GTPase may be involved in mitochondrial mitochondria MTG1 decrease translation, peripheral to the inner membrane mitochondria QCR10 decrease Subunit of the complex III mitochondria QCR7 decrease Subunit of the complex III transcription factor involved in regulation of transcription regulator ARG80 increase arginine-responsive genes transcription regulator CST6 decrease basic leucine zipper transcription factor transcription regulator RTR1 increase phosphatase regulating CTD of RNA polymerase II transcription regulator SAS2 increase histone acetyltransferase (HAT) catalytic subunit subunit of the RNA polymerase III transcription initiation transcription regulator TFC3 decrease factor complex transcription regulator TOA1 decrease TFIIA large subunit involved in transcriptional activation ribosome assembly MPP10 increase required for pre-18S rRNA processing ribosome assembly RPS9A increase protein component of the small (40S) ribosomal subunit ribosome assembly UTP30 increase putative subunit of U3-containing 90S preribosome complex

aBrief classifications and annotated functions were made based on SGD: http://www.yeastgenome.org/ 127

Table 6-8. Peak expression of zygote-specific genes in haploid cells

peak peak expression expression gene expression gene expression in zygote in zygote in haploida in haploida ARO7 increase G1 phase AIM39 decrease G1 phase BDS1 increase G1 phase BDH1 decrease G1 phase CSM3 increase G1 phase BUD9 decrease G1 phase DBP1 increase G1 phase CNM67 decrease G1 phase MSH2 increase G1 phase CUR1 decrease G1 phase RAD5 increase G1 phase EMP46 decrease G1 phase RIF1 increase G1 phase FRT2 decrease G1 phase RTA1 increase G1 phase GLC8 decrease G1 phase SEN34 increase G1 phase GPG1 decrease G1 phase SMC3 increase G1 phase PAM1 decrease G1 phase SMC6 increase G1 phase PAU5 decrease G1 phase DCR2 increase G1/S phase RAD17 decrease G1 phase FCY21 increase G1/S phase TFC3 decrease G1 phase IPL1 increase G1/S phase TOA1 decrease G1 phase RNH203 increase G1/S phase UBC12 decrease G1/S phase SAS2 increase G1/S phase ATG17 decrease S phase SPC29 increase G1/S phase PSK1 decrease S phase

CBF2 increase S phase JSN1 decrease G2 phase ISC1 increase S phase MEH1 decrease G2 phase NNF1 increase S phase QCR7 decrease G2 phase NUD1 increase S phase TBS1 decrease G2 phase PGM1 increase S phase TPO3 decrease G2 phase SHE1 increase S phase VHS2 decrease G2 phase ARG80 increase G2 phase ATG8 decrease G2/M phase DAS2 increase G2 phase CST6 decrease G2/M phase FKH1 increase G2 phase MYO3 decrease G2/M phase HIP1 increase G2 phase PEX22 decrease G2/M phase REX4 increase G2 phase ZRG8 decrease G2/M phase UTP30 increase G2 phase ASH1 decrease M phase VIK1 increase G2 phase COX17 decrease M phase ATF2 increase G2/M phase CSI1 decrease M phase LOC1 increase G2/M phase DBF2 decrease M phase MPP10 increase G2/M phase FMC1 decrease M phase YGK3 increase G2/M phase FYV10 decrease M phase RPS9A increase M phase GYP6 decrease M phase RTR1 increase M phase HAA1 decrease M phase

128

KIN1 decrease M phase MIC14 decrease M phase MMT2 decrease M phase MSS18 decrease M phase MTG1 decrease M phase NDL1 decrease M phase OPI10 decrease M phase QCR10 decrease M phase UIP4 decrease M phase

aPeak expression data from Cyclebase 3.0 (http://www.cyclebase.org). Genes in white column are increased in zygotes whereas genes in light grey column are decreased in zygotes.

129

Appendix “Materials and Methods”

A1. Reagents

Collagen (C5533), HEPES (H4034), EDTA (E5134), Diethylene triamine pentaacetic acid (DTPA; D6518), Neocuproine hydrochloride (72090), SDS (L6026),

S-Methyl methanethiosulfonate (MMTS; 64306), N-Ethylmaleimide (NEM; 04260),

N-Methylmaleimide (NMM; 389412), Iodoacetamide (I1149), Sodium L-ascorbate

(11140), Hydroxylamine hydrochloride (55459), Anti-FLAG antibody (F1978; 1:2000 for the western), Anti-actin antibody (A1978; 1:2000 for the Western), poly-L-lysine solution

(P4707), and histology mounting medium (with DAPI, F6057) were from Sigma. DMEM medium (11965092), F12K medium (21127022), Horse serum (26050088), FBS

(26140079), and HBSS buffer (14025076) were from Gibco. Protease inhibitor cocktail

(05892791001) and Protein G-agarose (11719416001) were from Roche. Anti-NOS1

(H-299, sc-8309; 1:1000 for the Western), anti-NOS2 (H-174, sc8310; 1:1000 for the

Western), and anti-NOS3 (C-20, sc654; 1:1000 for the Western) were from Santa Cruz

Biotechnology. Anti-Htt (N-terminal, clone 1HU4C8; MAB2166; 1:2500 for the Western) and Anti-Htt (C-terminal, clone HU-2E8; MAB2168; 1:2500 for the Western) were from

EMD Millipore. SDS-Out SDS Precipitation kit (20308) and Imperial protein stain

(24615) were from Thermo. BCA assay (23225) was from Pierce. Anti-Htt (C-terminal, clone HDB4E10; MCA2050; 1:2500 for the Western) was from AbD Serotec.

Immun-Star AP chemiluminescence (1705010) was from Bio-rad. Polyclonal anti-GFP

(for immunoprecipitation; ab290) was from Abcam. Monoclonal anti-GFP (for Western;

632381; 1:5000 for the Western) was from Clontech. Thiopropyl Sepharose 6B

130

(17042001) was from GE. MS grade trypsin (V5111) was from Promega. Lipofectamine

3000 (L3000015) was from Invitrogen. Ponasterone A (P-1083) was from A.G. Scientific.

NP40 (19628) was from USB.

A2. Mammalian cell culture and recombinant protein expression

Cells were grown at 37°C, with 5% CO2. HEK293T (gift from Dr. H-Y. Kao,

CWRU) and COS7 (gift from Dr. M. Weiss, CWRU) cells were grown in DMEM with

5% FBS. Recombinant proteins were expressed in these cell lines by using Lipofectamine

3000, following the instructions of the manufacturer. Cells in a well of 6 well plate was transfected with 2.5 μg. Cells were harvested 1-2 days after transfection. No obvious growth reduction or toxicity was observed in our cell models expressing Htt N548 +/- polyQ expansion over a week.

PC12 cells were grown in F12K medium with 2.5% FBS and 15% horse serum.

Tissue culture plates were coated with collagen before use. For this purpose, a stock of 1 mg/mL collagen in HBSS buffer/0.25% acetic acid was diluted to 0.2 mg/mL in HBSS buffer. Plates were coated for 20 min, the liquid was aspirated and the plates were then air-dried overnight. PC12 cells expressing inducible full-length Htt constructs (HttQ23 and HttQ73) were the kind gift from Dr. X. Qi (CWRU). Recombinant HttQ23 and

HttQ73 were induced by adding ponasterone A (5 μM) for two days.

After harvesting by scraping cells in the presence of PBS including the chelator,

DTPA (100 μM), cell pellets were washed twice with PBS/DTPA and snap frozen at

-80°C.

131

A3. Mouse tissues

Tissue samples were from a B6 mice (3 months). Tissue homogenates were prepared in the buffer containing protease inhibitor cocktail that is also used for

SNO-RAC/acyl-RAC (see below). All experimental procedures conducted upon live animals were first approved by the Institutional Animal Care and Use Committee of

CWRU School of Medicine and were conducted in accordance with the National

Institutes of Health Guide for the Care and Use of Laboratory Animals. CWRU is a

PHS-assured institution (Assurance # A-3145-01) and the institution is fully accredited by AAALAC.

A4. Plasmids used in this study

Htt constructs are shown in Fig 1. Full-length Htt and N-terminal 548 a.a. constructs (pCINeoHttFL.15Q.wt.HA, pCINeoHttFL.128Q.wt.HA, pCINeoHtt1955.15Q.wt.GFP, pCINeoHtt1955.128Q.wt.GFP, pCINeoHtt1955.15Q.C214S.GFP, and pCINeoHtt1955.128Q.C214S.GFP) and Htt

C-terminus (585-3144) construct were the kind gifts from Dr. M. Hayden (University of

British Columbia). The Htt exon1 constructs (pcDNA3.1 HttEx1-25Q-EGFP and pcDNA3.1 HttEx1-97Q-EGFP) were gifts from Dr. W. Yang (University of California,

Los Angeles). Ataxin-1 constructs (pcDNA1 Flag ATXN1[30Q] and pcDNA1 Flag

ATXN1[85Q]) were the gifts from Dr. Orr (University of Minnesota). Generation of nitric oxide synthase constructs (pCDNA3-nNOS, pCDNA3-eNOS, and pCDNA3-iNOS) was described in the previous study [262].

132

A5. Site-directed mutagenesis

To create the C204S mutant, the Htt N-terminal 548 a.a. constructs were used as template. Site-directed mutagenesis was done using a kit (Agilent Technologies, 200519).

The C204S forward primer was:

5’-cggcctcagaaaagcaggccttacctggtgaac-3’.

The C204S reverse primer was:

5’- gttcaccaggtaaggcctgcttttctgaggccg-3’.

A6. Detection of S-nitrosylation and S-acylation by resin-assisted capture

Resin-assisted capture (RAC) of S-nitrosylated proteins (SNO-RAC) or

S-acylated proteins (acyl-RAC) was described in previous studies [111, 162]. The procedures are also illustrated in Fig S1. Cells were lysed on ice in HENS buffer (HEPES

100mM, EDTA 5 mM, Neocuproine 0.1%, and SDS 1%; pH8.0) containing NP40 1%,

MMTS 0.1%, and a protease inhibitor cocktail. The extracts were then incubated in blocking buffer (HEPES 100mM, EDTA 5 mM, Neocuproine 0.1%, SDS 2.5%, and

MMTS 0.1%; pH8.0) at 50°C for 20 min. This step blocks free thio-groups so that later addition of thiopropyl sepharose 6B beads will interact exclusively with the thio-groups resulting from selective reduction of S-nitrosylated or S-acylated residues, using ascorbate or hydroxylamine, respectively. After blocking, the samples were precipitated with acetone at -80°C for 15 min.

Protein pellets were washed with 70% acetone (4°C) three times and then resuspended in the binding buffer (HEPES 100mM, EDTA 5 mM, Neocuproine 0.1%,

SDS 1%, and protease inhibitor cocktail; pH8.0). Protein was quantitated using a BCA

133 assay.

For selective capture, the binding mixture contained Thiopropyl Sepharose 6B beads and the appropriate specific reducing reagent. For SNO-RAC, S-nitrosylated Cys residues were reduced by adding ascorbic acid (50 mM) so that beads covalently bound to them. For acyl-RAC, hydroxylamine (200 mM) was used to reduce S-acylated residues.

A “buffer control” (no reducing reagent) was performed in parallel to detect any non-specific binding. Binding was performed on a shaker in the dark at room temperature for 3 hr. The beads were then washed four times with HENS buffer and two times with 10% HEN/1% SDS buffer.

After elimination of excess liquid, protein was eluted from the beads using SDS sample buffer with β-mercaptoethanol. After 20 min incubation at room temperature and

4 min at 90°C, the eluate was collected and the samples were run in 7.5% gel for

SDS-PAGE and analyzed by Western blotting. Quantification of images was with ImageJ.

A7. Detection of S-nitrosylation and S-acylation sites by LC-MS/MS

The procedures are also illustrated in Fig S2. Free thiol-groups of proteins in cell extracts were first blocked with NEM, followed by acetone precipitation and resuspension in HENS buffer containing a protease inhibitor cocktail. Samples were then divided into two equal parts. One part was incubated with ascorbate (50 mM) and IAA

(100 mM) for 1 hr to reduce S-nitrosylated residues and label them. The other part was incubated with hydroxylamine (200 mM) and IAA (100 mM) for 1 hr to reduce

S-acylated residues and label them.

When completing the labeling reaction, protein was again acetone-precipitated

134 and then resuspended in IP buffer (Tris-HCl 20 mM, NaCl 137 mM, EDTA 2 mM, NP-40

1%, glycerol 10%, and protease inhibitor cocktail, pH 8.0) plus SDS 1%. After resuspension, SDS was eliminated using a SDS precipitation kit (Thermo, 20308).

Samples were pre-cleared by incubating with a protein G-agarose slurry (50%, 20 μL) in the cold for 1 hr. The supernatant was collected and incubated with antibody (~30 μg) in the cold for 1 hr. EGFP-tagged Htt N548 fragments were immunoprecipitated by anti-GFP (ab290; 1:50 for immunoprecipitation) and full-length Htt proteins by anti-Htt

(C-terminal, MCA2050; 1:50 for immunoprecipitation). The bead slurry (50%, 50 μL) was then added for an additional 3 hr. After washing three times with PBS, protein was eluted by incubating with SDS sample buffer containing β-mercaptoethanol. After 20 min incubation at the room temperature and 4 min at 90°C, the eluate was recovered and proteins in samples were resolved by SDS-PAGE on gradient gels. For the high molecular weight (HMW) species, a group of samples were incubated at 37°C to determine these HMW species were not induced by heat.

After staining with Imperial Protein Stain (Thermo), protein bands were excised, destained in a mixture of 50% 100 mM ammonium biocarbonate and 50% acetonitrile and reduced with DTT, followed by excess NMM to reduce disulfides and to block the resulting free cysteine residues. After washing with 100 mM ammonium carbonate and

100% acetonitrile - alternately - three times, gel bands were spin-dried. A sequencing grade trypsin (Promega) solution (500 ng in 50 μL 100 mM ammonium bicarbonate) was incubated with the dried bands overnight at 37C.

Htt tryptic peptides were subjected to LC-MS/MS analysis using an Orbitrap Elite hybrid mass spectrometer (Thermo Scientific). Nano-reverse phase liquid

135 chromatography separations were performed on a UPLC (Waters, Milford, MA) directly connected to a nanospray emitter (10 μm, New Objectives). LC separation was conducted by using mobile phases A (0.1% formic acid in water) and B (100% acetonitrile) with a

90 min linear gradient starting with 1% B and increasing to 40% phase B. All data were acquired in positive ion modes. Full MS scans (m/z 300-2000) were followed by MS2 scans of the ten most abundant peptide ions at normalized collision energy of 35%. High mass accuracy FT/MS was performed to detect precursor ions (resolution, 60,000; mass accuracy, 5 ppm).

A8. MS data analysis

The representative spectra of MS results are in Fig S3. Bioinformatics software

MassMatrix (2.4.2, Feb 22, 2012) [263] was used to search tandem MS data against a database containing the Htt sequence and decoy sequences with reversed sequence of this protein. We targeted trypsin-digested peptides containing cysteine residues. The M/Z shift of targeted peptides due to cysteine modifications [iodoacetamide (IAA),

N-ethyl-maleimide (NEM), and N-methyl-maleimide (NMM)], were selected for detection by mass spectrometry. According to the sample preparation described above,

NEM-labeled cysteines were unmodified, IAA-labeled cysteine residues were

S-nitrosylated/S-acylated, and the remaining cysteine residues with other modifications such as disulfide bond were labeled by NMM after DTT reduction.

Precursor ions were searched with 10 ppm mass accuracy and product ions were searched with 1 Da mass accuracy. Peptide identifications with or without modifications were determined with PP scores greater than 5.0 and PPtage scores greater than 2.0. All

136 of the detected modified peptides mass spectra were manually checked. LC-MS/MS and

MS analysis were performed in the Center for Proteomics and Bioinformatics, Case

Western Reserve University.

A9. Fluorescent microscopy study of EGFP-tagged Htt N548 inclusions

HEK293T cells were grown in 12 well tissue culture plate with poly-lysine-coated cover slips. Htt N548-EGFP construct mixed with empty vector or NOS construct in 1:1 ratio (ng) was used to transfect HEK293T cells by Lipofectamine 3000 (L3000015). After two days expression, cells were fixed by 4% formaldehyde at room temperature in dark for 10 minutes. Then rinsed the samples with PBS for three times. The mounting medium

(with DAPI) was then applied to the sample incubation at the room temperature in dark for 5 minutes, followed by sealing with nail polish. The EGFP-positive cells were counted to calculate the percentage of cells with N548-EGFP inclusion(s). Fluorescent signals from DAPI and EGFP were acquired by LEICA DMI4000 B microscopy/LEICA

DFC345 FX camera. To obtain high resolution image, DeltaVision RT wide-field fluorescence imaging system (Applied Precision, Inc) and CCD digital camera

(Photometrics CoolSnap HQ) were used. To remove out-of-focus light, deconvolution software (Applied Precision, Inc) was applied.

A10. Htt HEAT repeat motif simulation

The MMM server (http://manaslu.aecom.yu.edu/MMM) [264] was used to create the molecular models for the A, B, C and D clusters of HEAT repeats (Tartari et al., 2008)

[166] using the crystal structure of the first eight HEAT repeats from protein phosphatase

137

2A PR65/A subunit (1B3U) as a template (Groves et al., 1999) [265]. The choice of the

PR65/A HEAT repeats was based on work by Andrade and colleagues [27], which suggested that the HEAT repeats of Htt belong to the AAA class represented by the

PR65/A structure.

A11. Yeast strain growth and zygote purification

Budding yeast (S. cerevisiae) cells of S288C background (BY4741 strain) were grown in complete synthetic medium (CSM) with 2% glucose at room temperature.

Haploid MATa cells (ATY4552) expressed polysome-targeting mCherry (pAJ1661,

URA3/CEN, encoding RPL25 fused to mCherry) and haploid MATα cells (ATY4442) expressed cytoplasmic EGFP (pEG220, URA3/Yip, encoding cytoplasmic EGFP). In this thesis, we refer to red naïve (RN) and green naïve (GN) for unstimulated ATY4552 and

ATY4442, respectively. Diploid cells were prepared by crossing RN and GN (mCherry+ diploid: D+), or MATa BY4741 with GN (mCherry- diploid: D-). The comparison between D+ and D- was used to subtract the effect of fluorescent protein expression.

Zygote formation and purification procedures are described in our previous report

(Fig S4) [220]. In brief, haploid cells of both mating types in exponential growth were mixed on CSM/glucose agar plates to incubate at room temperature. After 1.75 hr, suspended early zygotes (referred as ZE; red+/green+) and stimulated haploids (referred as RE; red+/green-) were recovered from the mixture by flow cytometry-based sorting.

(Emission for green fluorescence: 520 nm; red fluorescence: 615 nm)

Another sample was collected after 2.5 hr. These purified late zygotes and stimulated haploids were referred as ZL and RL, respectively. Flow cytometry was

138 performed in the Cytometry & Imaging Microscopy Core Facility of the Case

Comprehensive Cancer Center (P30 CA43703) [220]. Purified cells were re-incubated in

CSM/glucose medium for 30 min at room temperature and then samples were frozen prior to the RNA extraction.

A12. RNA purification and microarray performance

Total RNA was extracted from sorted cells: RN, GN, RE, RL, ZE, ZL, D+, and D-.

RN (red naïve; MATa; red+/green-) and GN (green naïve; MATα; red-/green+) are haploid cells expressing polysome-targeting mCherry and EGFP fluorescent proteins, respectively. Both RE and RL are mCherry-expressing (red+/green-) haploid MATa cells stimulated by mating factor that was released by EGFP-expressing (red-/green+) haploid

MATα cells. RE (early) was stimulated by mating factor for 1.75 hr whereas RL (late) was 2.5 hr. ZE (early) and ZL (late) are zygote cells collected after mixing two mating types for 1.75 and 2.5 hr, respectively. D+ and D- are diploid cells with EGFP expression

(EGFP+). D+ also expresses mCherry protein (red+) but D- does not (red-).

The total RNA purification was done by phenol/chloroform method. Briefly, cell pellet was resuspended in LET buffer (LiCl 100 mM/EDTA 20 mM/Tris 25 mM pH8.0) and then cells were homogenized by vortex with glass beads in the presence of phenol/chloroform at room temperature for 5 min. Extracted RNA in the aqueous layer was precipitated by absolute ethanol in -80°C freezer overnight. RNA pellet was washed by 70% ethanol and dried out before resuspension in DEPC water. RNA quantity and quality were spectrometrically determined by A260 and A260/A280 ratio (≥2.0), respectively.

139

Yeast Genome 2.0 Array (Affymetrix) was applied to detect global RNA quantity.

Sample hybridization to microarray and signal detection were performed in the Gene

Expression and Genotyping Facility, CWRU.

A13. Microarray data analysis

Microarray data set of independent replicate from 8 groups of yeast cell types (2 from GN, 2 RN, 2 RE, 2 RL, 4 ZE, 4 ZL, 2 D+, and 2 D-) were processed by Affymetrix

Expression Console (EC) software (version 1.1).

(EGFP+). D+ also expresses mCherry protein (red+) but D- does not (red-).

The exported multigroup microarray data were analyzed by BAMarray 3.0

(Bayesian ANOVA Analysis of Variation of Microarrays) to determine the differences between each pair of groups and avoid incorporating false positive results [266]. To build up a comprehensive gene list for transcriptome dynamics of the yeast life cycle, we set up two criteria. First, genes without significant difference in any binary comparison were eliminated. Second, only genes with at least a significant fold-change (FC) ≥ 1.5 between cell types were kept. Consequently, a list containing 2609 genes was processed by

140 complete linkage hierarchical clustering with the aid of Cluster version 3.0, as developed by Michael Eisen [267, 268]. The heat map was created for illustration by TreeView version 1.60 (http://rana.lbl.gov/EisenSoftware.htm).

To identify the mating factor-responsive genes, two screening criteria were applied and genes satisfying both criteria were kept: (1) Either the RE/RN (105 min response) or

RL/RN (150 min response) comparison shows a significant change (FC) determined by

BAMarray. (2) The ratio of either RE/RN or RL/RN is greater than 1.5 FC (either upregulated or downregulated). The strength of the mating factor response was then ranked in the order of fold-change value (either RE/RN or RL/RN).

Since we are especially interested in genes whose expression profiles did change in zygote but not in haploid, pheromone-stimulated haploid, and diploid cells, we identify these zygote-specific genes by the following data processing: (1) Genes that had any

BAM array-determining statistic change or fold-change value ≥1.5 in any binary comparison between RN, RE, RL, and D+ were eliminated. (2) Genes which satisfied both significant change and fold-change value ≥1.5 in either ZE/RE, ZE/RL, ZL/RE or

ZL/RL were kept.

A14. Gene Ontology (GO) analysis

The clustered biological process of changed genes in zygote or stimulated haploid cells were analyzed by the High-Throughput GoMiner [245]. (web-based software: http://discover.nci.nih.gov/gominer/GoCommandWebInterface.jsp)

141

Figure S1.

Figure S1. Detection of protein S-nitrosylation by SNO-RAC and protein

S-acylation by acyl-RAC. (A to B) S-nitrosylation detection by resin-assisted capture

142

(SNO-RAC). (A to C) S-acylation detection by acyl-RAC. Blocking in (A) is essential to distinguish unmodified and modified cysteines. In SNO-RAC and acyl-RAC, both NEM and MMTS can be used to block free thio groups. (B) S-nitrosylated proteins are reduced by ascorbic acid (ASC) to interact with beads covalently for precipitation. SDS-PAGE sample buffer containing β-ME is applied to elute S-nitrosylated proteins. Western is performed to detect specific protein that is S-nitrosylated. Parallel aliquot without ASC treatment serves as the negative control to evaluate non-specific binding. (C) S-acylated proteins are reduced by hydroxylamine (NH2OH). The remaining processes are identical to (B) except the detected protein is S-acylated. Parallel aliquot without NH2OH treatment serves as the negative control. The acyl group here is represented by palmitate but acylation is not limited to palmitoylation. Detailed method is described in Appendix

A6.

143

Figure S2.

Figure S2. Determination of S-nitrosylated and S-acylated cysteine residues by Mass

144 spectrometry (MS). (A to B) S-nitrosylated cysteine determination by MS. (A to C)

S-acylated cysteine determination by MS. Blocking in (A) is essential to distinguish unmodified and modified cysteines. (B) S-nitrosylated cysteine residues are reduced by ascorbic acid (ASC) to interact with IAA covalently. The target protein is enriched by immunoprecipitation. After SDS-PAGE separation, the target protein is recovered for in-gel digestion followed by LC-MS/MS analysis. The shift of M/Z due to IAA-labeling indicates the cysteine residues that are S-nitrosylated. (C) S-acylated s are reduced by hydroxylamine (NH2OH). The remaining processes are identical to (B) except the detected IAA-labeled cysteine residues are S-acylated. In order to distinguish the cysteine residues that are not modified (free thio group), the alkylation/blocking reagents should be irreversible (e.g. NEM/IAA) but not reversible (e.g. MMTS) because strong reducer

DTT is used for in-gel digestion. The acyl group here is represented by palmitate but acylation is not limited to palmitoylation. Detailed method is described in Appendix A7.

145

Figure S3.

Figure S3. MS/MS analysis. Representative iodoacetamide-labeled peptides indicate modification on C105, C109, C152, C280, C433, and C204. The C204-containing

146 peptide was from the C214S mutant. Peaks that matched expected peptides are labeled in red. The IAA-labeled C214 was detected in the tryptic peptide containing C204S mutation. However, this peptide did not provide good resolution for sequencing in tandem MS so that the picture is not shown here. MS analysis is performed by Dr. Liwen

Wang (Center for Proteomics and Bioinformatics, CWRU).

147

Figure S3. Continued.

148

Figure S3. Continued.

149

Figure S4.

Figure S4. Separation of yeast zygote cells and pheromone-stimulated haploid cells by flow cytometry. For detail: Zapanta Rinonos, S. et al., J Vis Exp. 2012. Haploid

MATa cells express red florescent proteins (mCherry-tagged) whereas MATα cells express green fluorescent proteins (EGFP-tagged). In contrast to haploid cells, zygote cells, which are formed after mixing MATa and MATα cells, express both red and green fluorescent proteins. The cell mixture of MATa and MATα cells is incubated for 1.75 or

2.5 hr before separation by flow cytometry. Detailed method is described in Appendix

A11.

150

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