The Function of the Qcr7 of the Mitochondrial Ubiquinol- c Oxidoreductase of Saccharomyces cerevisiae

by

Suzann Malaney

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Biochemistry University of Toronto

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Doctor of Philosophy, 1997 Suzann Malaney Department of Biochemistry, University of Toronto

ABSTRACT The respiratory chain enzyme ubiquinol-cytochrome c oxidoreductase (also termed bcl complex or complex III) of Saccharomyces cerevisiae contains 1 0 subunits and resides in the inner mitochondrial membrane (IMM). This multisubunit enzyme complex is involved in the transfer of electrons from ubiquinol to cytochrome c and in the establishment of an by translocation of protons across the IMM. A previous study has shown that inactivation of the yeast nuclear gene QCR7, which encodes subunit 7 (also referred to as Qcr7 protein or 14 kDa subunit) of the bci complex leads to an inactive enzyme. The bel complex of the mutant strain lacks holocytochrome b and has reduced levels of apocytochrome b, the Rieske iron-sulfur protein (ISP), and the 11 kDa subunit. Although this study showed that the Qcr7 protein is essential for respiration, the exact role of this subunit is not known. In the present study 1 have shown by circular dichroism that an amino-terminal peptide of subunit 7 is a-helical. I have also studied the effect of mutations in the amino-terminus of--this protein on mitochondrial targeting, assembly of the bel complex, and proton translocation. Mutant were analyzed by overexpression of mutated qcr7 genes in the strain YSM-qcr7A, which contains a disrupted chromosomal QCR7 gene, and by comparison to Qcr7 protein from a YSM-qcr7A strain in which the wild type QCR 7 gene is overexpressed. Respiration proficiency and the activity of the bci complex were monitored. Based on these preliminary data, strains expressing mutated proteins that lacked the N-terminal 7, 10, 14, and 20 residues (after Met-1) of subunit 7 referred to as Qcr7p-~7,-AI 0, -a14, and -a20, respectively, and strains expressing versions of Qcr7p-A7 that contained point mutations RI OK, Dl3V, and RiOI/GlZV were chosen for further study. Al1 the mutated versions of the Qcr7 protein, with the exception of Qcr7p-al0 and Qcr7p-a7(D13V), are present in the mitochondria (frorn cells grown at 300C) at reduced levels of approximately 55% when compared to Qcr7 protein from the strain overexpressing wild type QCR7. In contrast, Qcr7p-~lO is not detectable and Qcr7p-~7(013V)is present at wild type levels. This may implicate the Qcr7 protein amino-terminus in import or in conferring stability to the protein. The activity of complex III in mitochondria from strains with Qcr7p-a7 and Qcr7p-a7(Rl OK) was normal. In contrast, strains overexpressing qcr7 genes encoding Qcr7p-~lO, Qcr7p-A14, Qcr7p-~20,Qcr7p-~7(D13V), and Qcr7p- A~(R1 OVG12V) were respiration-deficient. Western blot analyses from this latter group of mutants indicate that the bcl complex in respiration-deficient cells displays a significant variation of

iii reduced levels of the 11 kDa subunit, as well as combined intermediate and mature ISP and cytochrome cl. Spectrophotometric analyses indicate that the amount of holocytochrome b was reduced in the strain containing Qcr?p-~7(RlOI/G12V) and lacking in the strain with Qcr7p-d7(D13V). ATP synthesis (an indirect measure of proton translocation) in mitochondria was comparable to the wild type in al1 respiration-proficient strains tested, including the strain with Qcr7p-A7. Based on the results of this work, I concluded that the amino- terminus of the Qcr7 protein is essential for the functional assembly of ubiquinol-cytochrome c oxidoreductase. In addition to the proposed function in assernbly, the N-terminal seven residues may facilitate import into rnitochondria. DEDICATION

This work is dedicated to my two little daughters Maxine and Michelle. May it be an inspiration to them throughout their lives. This work is also dedicated to my husband Robert for his love. ACKNOWLEDGEMENTS

I would like to thank my supervisor, Dr. Brian H. Robinson. While in his laboratory for five years I have learned a tremendous amount about science and life. He has always been supportive, encouraging, and full of ideas. I thank him for always having an open door no matter how busy he was. I also thank him for accepting me into his laboratory and giving me the opportunity to accomplish this work.

I would like to thank my CO-supervisors Dr. Charles Deber and Dr. David Williams for al1 their encouragement, support, and constructive criticism. I would also like to acknowledge contribution to the thesis by Dr. Jacqueline Segall. I am very grateful to Dr. Bernard L. Trumpower from Dartmouth Medical School from whom I have gained much knowledge over the years. I would also like to thank Dr. Peter Lewis, Dr. Reinhart Reithrneier, and Dr. Shelagh Ferguson-Miller (outside examiner from Michigan State University) for critically proofreading this manuscript and for many useful comments.

Somebody who deserves special mention is my husband Robert. A scientist himself (Astrophysicist), he has been an inspiration and a hero to me from the second year of my undergraduate degree. Without him this work would have never been accomplished. His dedication as a father and a husband has made it possible for me to complete my research. I would like to thank rny fellow graduate students and CO- workers, some of whom deserve special mention. Dr. Frank Merante and Dr. Sandeep Raha were never too busy to help out, talk about science, or play practical jokes. Thank you to Tomoko Myint for her endless friendship and encouragement. I would also like to thank Dr. Sandeep Raha, and Dr. Mingfu Ling for critically proofreading this dissertation and for many useful comrnents. Thank you to Maureen Waite, our administrative assistant, for easing my work on many occasions.

I would like to thank my family, especially my mother, for her never-ending support and for believing in me. Her encouragement has given me strength. 1 also thank my mother as well as my sister- in-law Linda for coming from abroad to babysit.

Thank you Maxine and Michelle for brightening up even those days on which none of my experiments worked. TABLE of CONTENTS

DEDIUTON...... v

ACKNOW LEDGEM ENTS...... vi

TABLE of CONTENTS...... viit a..

a.* a.* UST of FIGURES ...... XIII

LIST of TABLES...... ,...... m*

ABBREVWONS ...... xvii

CtIAFl€R 1: Introduction...... 1

Components of the mitochondrial respiratory chain...... 1 O 1. NADH dehydrogenases...... 1 0 II. Succinate-ubiquinone reductase...... 1 1 III. Ubiquinolcytochrorne c oxidoreductase ...... 1 2

vii i The Rieske ironsulfur protein...... 1 7 What about the supernumerary subunits of complex [Il?...... 19 The supernumerary subunits of complex III...... 20 Subunit 6 may regulate the activity of complex III...... 20 Subunit 7: the protein of the current study...... 21 Subunit 8: a ubiquinone-binding protein ? ...... 23 Subunit 9...... 24 Subunitl O...... 25 The core proteins...... 25 Functional assembly of complex III...... 26 N . Cytochrome oxidase...... 29 V . ATP synthase...... 31 Nucleo-rnitochondrial interactions...... , ...... 33 How do mitochondria and the nucleus communicate ? ...... 34 The expression of many complex III subunits is dependent on the need for oxidative metabolism...... 36 Do mitochondria exert regulation on nuclear gene products ? ...... 38 Protein import into mitochondria...... 39 Import into the mitochondrial matrix...... 40 The targeting sequence...... 43 Cytoçolic chaperones ...... 44 Receptors in the outer mitochondrial membrane...... 46 Pase

The translocation channel and the mitochondrial chaperones ...... 48 lmport into the intermembrane space (IMS) ...... 49 lmport into the inner mitochondrial membrane (IMM) ...... 51 lmport into the outer mitochondrial membrane (OMM) ...... 52 . . Objectives of the research...... 52

CHAPTER 2: Inactivation of the QCR7 gene ...... 54 2.1 . INTROOUCTiON...... S S 2.2. MATERIALS AND METHODS...... 57 Materials...... 57 2.2.1 . Disruption of the chromosomal copy of the QCR7 gene ...... 58 2.2.2. Confiming inactivation of the QCR7 gene ...... 60 . . Selection of diploids...... 60 Northem and Southem bbtting...... 60 Isolation of yeast mitochondria...... 61 Western analysis...... 62 2.2.3. Raising an antibody in chicken against the Qcr7 protein ...... 63 Isolation of anti-Qcr7 antibody from rabbit and chicken ...... -63 2.3. RESULTS...... 65 Disruption of the QQP7 gene...... 65 Confirmation of the qcr7 gene inactivation...... *...... *...... 69 2.4. DISCUSSION ...... 73 CHAPTER 3: Mutagenesis of the QCR7 gene...... 74 3.1 . INTRODUCTION...... -75 3.2. MATERIALS AND MmK)DS ...... 85 Materials...... 85 Mutagenesis by PCR ...... 85 Sitedirectecd mutagenesis using the M 1 3 phage...... 88 . Enzyme assays ...... 89 Growth studies...... 89 3.3. RESULTS...... 91 Characterization of mutants by growth...... 91 ... Enzyme activities ...... 95 3.4. DISCUSSION...... 99

CHAPTER 4: The role of the amino-terminus of the Qcr7 protein in rnitochondrial targeting. complex III asse mbly. and proton pumping...... 102 4.1 . INTRODUCTION...... 1 03 4.2. MATERUILS AND METHODS...... 1 04 Materials...... 104 Isolation of yeast rnitochondria and Western analyses...... 104 ATP synthesis assays...... 105 Circular Dichrosm spectra...... 105 Spectra of the cytochrornes...... 106 ... Co-immunoprecipitation...... 106 Buffers ...... A07 Procedures...... ,., ...... 107 . . Cross-hnking...... 108 ...... 4.3. RESULTS ...... 10 Circular Dichroism spectra of amino-terminal peptides...... 170 The amino-terminus of the Qcr7 protein may facilitate import into mitochondria...... 113 The amino-terminus of the Qcr7 protein is essential for assernbly of the bel complex ...... 120 ... Co-immunoprecipitations...... 129 Deletion of seven residues from the amino-terminus does not impair proton pumping ...... 131 The strain expressing Qcr7p-~7is temperature sensitive ...... 132 4.4. DISCUSSION ...... ,...... 13 5

CHAPTER 5: Discussion and Future Directions ...... W...... 1 39 DISCUSSION...... ,...... 140 Future Directions ...... 158 Confirmation of assembly of the Qcr7 protein N- terminus with cytochrome b and/or the 11 kDa subunit and identification of contact sites ...... 158 Testing for the involvement of the Qcrï protein N-terminus in mitochondrial import...... 161

xii LIST of FIGURES

Figure 1-1 High resolution scanning electron micrograph of a ...... 5

Figure 1-2 The mitochondrial respiratory chah of S. cerewae...... -9

Figure 1-3 mechanism...... 1 4

Figure 1-4 lmport of proteins into the mitochondrial rnatrix ...... 42

Figure 2-1 DNA construct for disrupting the QCR7 gene...... 59

Figure 2-2 Southern blot analysis of the parental strain W303-1 B and the disrupted strain, qcr7A:LEU2 ...... 68

Figure 2-3 Northern blot analysis of qcr7d:LEUZ ...... 71

Figure 2-4 Western blot analysis of qcr7A:LEUZ and qcr7A:LEUZ transformed with the wild type

Figure 3-2 Alignment of yeast Qcr7 protein and its 1 3.4 kDa homolog from beef heart mitochondrial complex III...... 82

Figure 3-3 Growth on SD agar plate with ethanol/glycerol and 0.1 % glucose, or SD media plate . - containing glucose...... 94

Figure 4-1 Circular Dichroism spectra ...... 1 12

Figure 4-2 Helical wheel projections...... 1 14

Figure 4-3 Western blot analyses of mitochondrial proteins from YSM-qcr7~strains overexpressing wild type and N-terminally truncated proteins Qcr7p-~7,Qcr7p-Al O, Qcr7p-~14. Qg7pQO ...... 1 16

Figure 4-4 Quantification of complex III subunits in various Qcr7 mutant strains by densitometry ...... 117

xiv Figure 4-5 Western blot analyses of mitochondrial proteins from YSM-qcr7A strains overexpressing wild type Qcr7, Qcr7p-~7(DI3V). Qcr7p-a7(Rl OK), and Qcr7p-~7(RI01/61 ZV), respectively...... 1 23

Figure 4-6 Difference spectra of the cytoc hromes...... 1 28

Figure 4-7 Western blot analysis perforrned with mitochondrial membranes prepared from YSM-qcr7a (grown at 37aC) overexpressing Qcr7p-a7 and the wild type Qcr7 protein...... 134 LIST of TABLES Page

Table 1-1 Complex III subunit composition...... 13

Table 3-7 Prirners used for mutagenesis...... 87

Table 3-2 Sumrnary . Characterization of mutants according to growth and enzyme activities...... 98

Table 4-1 ATP syn thesis ...... 1 33

xvi LIST of A8BREVIATIONS

ATP adenosine 5'-triphosphate BClP 5-bromo-4-chloro-3-indolyl phosphate-toluidine D7-r ditheiothreitol EDTA ethylenediamine tetraacetate FlSH fluorescence in situ hybridization h hour IMM inner mitochondrial membrane IMS intermembrane space ISP Rieske iron-sulfur protein of complex III kDa kilodaIton min minute mt mitochondrial NBT p-nitro blue tetrazoliurn chloride OMM outer mitoc hondrial membrane PAGE polyacrylamide gel electrophoresis PBS phosphate-buffered saline PMSF phenylrnethylsulfonyl fluoride RT room temperature SD media synthetic def icient media SDS sodium dodecyl sulfate

xvii CHAPTER 1

Introduction CHAPTER 1 Introduction

The focus of this study is subunit 7 (also referred to as Qcr7 protein or 14 kDa subunit) of ubiquinol-cytochrome c oxidoreductase (also termed complex III or bc, complex) of the mitochondrial respiratory chain in Saccharomyces cerevisiae. Complex III is involved in the establishment of the protonmotive force which is used to drive ATP synthesis. QCR7, the gene encoding subunit 7, is nuclear encoded and therefore has to be imported into mitochondria. Hence, for the functional assembly of complex III, a coordination of nuclear and mitochondrial genomes is required.

To give a brief account of the topics involved in the study of the Qcr7 protein, an overview is first given as to where, in the study of metabolism, mitochondria fit in. Following this overview is a general section on mitochondria and a detailed description of the enzyme complexes in the mitochondrial respiratory chain with an emphasis on the bc,cornplex and assembly thereof. After that, nucleo-mitochondrial interactions are discussed and a current update of mitochondrial protein import is given. The introduction closes with a brief account of the objectives of this study (a more detailed account is given at the beginning of Chapter 3).

If not mentioned otherwise, al1 the information given in this dissertation and al1 the terminology used are specific to Saccharomyces cerevisiae, also referred to simply as "yeast" throughout this study.

Ovewiew Living organisms are not at equilibrium. Rather, they need a continuous influx of free energy to maintain order in a universe bent on maximizing disorder (1). Free energy is derived from the intake of nutrients, and metabolism is the process that liberates the free energy so that it can be utilized to drive the various functions needed to sustain life. There are two categories of metabolic pathways: reactions involved in catabolism and those involved in anabolism. The driving force for the endergonic reactions involved in anabolisrn uses ATP and NADPH which are generated by the exergonic breakdown of a variety of substances during catabolism. These substances, carbohydrates, lipids, and proteins, are provided by the nutrient intake of the organism and are catabolized to their component monomeric units: glucose, gfycerol and fatty acids, and amino acids, respectiveiy. Subsequently, these metabolites are converted to the common mitochondrial intermediate acetyl- coenzyme A which is then further oxidized to COp by the enzymes of the citric acid cycle with the concurrent reduction of NAD' and FAD to NADH and FADH2. NADH and FADH2 enter the mitochondrial where they are reoxidized, the final acceptor of electrons being O*. The complete breakdown of a substance entering the living system therefore results in the final products HzO and CO2. The energy generated during this process is used to drive the synthesis of one of the bioenergetically most important molecules, ATP. Thus, living organisms are not at equilibrium, but rather maintain a steady state; despite the intake of large amounts of water and nutrients, the weight of an adult does not change significantly over his/her lifetime.

Mitochondria Mitochondria (Fig. 1-1) constitute the site of eukaryotic oxidative metabolism. The enzymes of the pyruvate dehydrogenase complex, the citric acid cycle, as well as those involved in fatty acid oxidation (in humans), electron transport and oxidative phosphorylation are al1 located in the mitochondrion. In addition to serving as the cell's "power plant", mitochondria, however, also play roles in the generation of precursors for anabolic pathways and in calcium homeostasis of the cell (2). Mitochondria, which can occupy up to 20% of the cytoplasm of a eukaryotic ceII (31, consist of four areas, each of which embodies a distinct set of proteins. The smooth outer mitochondrial membrane (OMM) contains the pore- forming protein porin which renders the membrane permeable to diffusion for a number of molecules. Receptor components involved in import of nuclear encoded mitochondrial proteins are also embedded in this membrane (4-W. The enzymes of the respiratory chain and oxidative phosphorylation are located in the intermembrane space (IMS) and the extensively invaginated inner rnitochondrial membrane (IMM). The number of invaginations (cristae) in the IMM and sites for oxidative phosphorylation Vary with the metabolic demand of the cell type. Thus, the surface area of the inner membrane is larger in a heart muscle cell with a high Fig. 1-1. High resolution scanning electron rnicrograph of a rnitochondrion (M) typically found in striated rat muscle (x8000).(17*) The cristae are densely packed into the matrix space and show both shelf or plate (short arrow) and tubular (long arrow) morphology. 1, inner mitochondrial membrane (IMM); O, outer mitochondrial membrane (OMM); T, T-tubule. rnetabolic demand than in a liver cell. The IMM contains more proteins than the OMM, about 70% by weight, and in beef heart half of al1 these integral membrane proteins are involved in oxidative phosphorylation. The remaining 30% of the membrane is lipid in nature, composed of approximately 15% cardiolipin, 40% phosphatidylcholine, and 35% phosphatidylethanolamine (1 2). Unlike the OMM, the IMM is freely permeable only to Op,COZ, and H20 (2). This limited permeability of the IMM permits the generation of ionic gradients, but it also calls for a number of transport mechanisms. The fourth and innermost region of the mitochondrion is the matrix, a gel-like compartment, that consists of approximately 50% water. The matrix contains the pyruvate dehydrogenase complex, the enzymes of the citric acid cycle and fatty acid oxidation, as well as substrates for the electron transport chain, nucleotide cofactors, inorganic ions, and the mitochondrial genetic and protein synthetic machineries (1 3).

Theories as to the origin of mitochondria now favor the endosymbiosis hypothesis, such that mitochondria contain their own genetic code, DNA replication and protein synthetic machineries. As can be seen from fixed sections under the electron microscope, mitochondrial (mt) DNA may exist in three alternative forrns: closed circular, linear, or aggregates of large and small circles. The size of the DNA molecule can Vary extensively from 16 kb in animals to

100 kb in plants (2). Saccharomyces cerevisiae has one of the largest mt DNA molecules seen in fungi, a circular molecule of 70- 75 kb which is 21 -25 pm long. Yeast mt DNA also contains introns and intergenic spacers. On the other hand, the circular, 16.5 kb mt DNA molecule of humans is highly efficiently organized and is therefore one of the smallest ones, being only 4.8-5.1 pm in length. Yeast mt DNA encodes 24 tRNAs, as weli as large and small rRNAs; although yeast mt DNA may encode more than one small and one large rRNA, only one of each is functional. In addition, yeast rnt DNA also encodes cytochrome oxidase subunits 1, 11, III, cytochrome b, ATPase subunits 6, 8, and 9, RNA maturases, an intron transposition endonuclease, an RNA component RNase-P-like enzyme and a ribosome-associated protein (3. '3). The introns of the cytochrome b gene are believed to encode maturases required for the splicing of its RNA transcript (14). Some strains of S. cerevisiae have a split 21S rRNA gene; the intron of this gene encodes a protein, however, with a function other than RNA splicing (2). All genes are transcribed from the same strand, except for the tRNA for threonine. Initiation of transcription is marked by the sequence S-atataagta- 3', whereby the last adenosine constitutes the first nucleotide of the translational initiation codon atg. In yeast, there are 20 different primary transcripts which al1 contain at least two coding sequences (1 5).

The simple and highly organized mitochondrial genome of humans encodes only two rRNAs, 22 tRNAs, and a small number of proteins of the respiratory chain and oxidative phosphorylation. The human mitochondrial genome does not contain any introns or intergenic spacers, nor does it encode any regulatory elernents. In al1 animal mitochondria, both strands are transcribed from a single promoter and the prirnary transcripts contain al1 the information encoded in each of the two strands. There are no 5' leaders in the transcripts; however the tRNA genes which are present between

most genes signify the start and end of a gene (15). From the small mitochondrial genome in animals it is evident that only a few of the hundreds of mitochondrial proteins are encoded by mitochondrial DNA. Hence, most of the respiratory chain enzymes as well as most of the components of the mitochondrial genetic system such as mitoc hondrial DNA polyrnerase, mitochondrial RNA polymerase, ribosomal proteins, and translation factors are encoded by nuclear

DNA (3).

The mitochondrial respiratory chain consists of five enzyme complexes which are ernbedded in the inner mitochondrial membrane (Fig. 1-2). Electron transport through complexes III (also referred to as ubiquinol-cytochrome c reductase or bc, complex) and IV (cytochrorne oxidase) in S. cerevisiae and complexes I (NADH- ubiquinone reductase), III, and IV in humans is coupled to vectorial proton translocation into the IMS, resulting in the establishment of a proton gradient and subsequent membrane potential. The energy from this gradient is then used as the driving force for ion translocation and protein import into mitochondria, as well as for

ATP synthesis, which is catalyzed by complex V, the F,F, ATPase (2.

12).

There are some differences between the mitochondria of yeast and those of humans and many of these differences are the result of HI H+ H+ H+ "extemal" NADH H+ dety dmgenzse H+ H+ 1 MS

NADH kf +

"internai" NADH drhydrognase f FADH2

Fig. 1-2. The mitochondrial respiratory chain of S. cerevisiae. Sketch of the four complexes of the mitochondrial respiratory chain in yeast: (complex II), ubiquinol-cytochrome c oxidoreductase (complex III or bc, complex), and cytochrome oxidase (complex IV), as well as the ATPase (complex V). Arrows indicate the order of electron flow and concurrent proton ejection into the IMS by complex III and the final electron acceptor, complex IV. Protons are transported back across the IMM against the electrochemical gradient by the ATPase, complex V. regulation of the mitochondrial and cytosolic redox balance as expressed by the NAD/NADH and NADP/NADPH redox ratios. For example, unique to yeast mitochondria are the L-lactate:cytochrome c oxidoreductase, cytochrome c peroxidase, and the external and internal NADH dehydrogenases. These NADH dehydrogenases are involved in the oxidation of cytosolic NADH and thus there is no need for shuttle systems which transfer NADH from the cytosol to the mitochondria. Furthermore, aspartate arninotransferase is only present in the cytosol, hence yeast do not have a functional malate- aspartate shuttle; this, also, is probably a result of their ability to oxidize externally added NADH. A fatty acidhalate-citrate shuttle is not necessary, because of the absence of p-oxidation of fatty acids in most yeast mitochondria (2. '6. '7).

Components of the mitochondrial respiratory chain 1. NA DH dehydrogenases In humans, respiratory chain complex I is the largest of al1 the enzyme complexes and consists of at least 42 subunits (18) with many of the subunits having been discovered in recent years. In S. cerevisiae on the other hand, this enzyme complex does not exist. However, yeast have two NADH dehydrogenases which are located in the IMM. One of these is an "external" type of mitochondrial NADH dehydrogenase, which can oxidize externally added NADH. This is a function that mammalian cells do not possess. The other is an "internal" NADH dehydrogenase. This enzyme consists of one subunit (mw=53000) which contains FAD and reacts with ubiquinone. This molecule carries out the oxidation of NADH which is generated in the matrix by the citric acid cycle. Both types of NADH dehydrogenases, unlike the human NADH-ubiquinone reductase, are insensitive to the inhibiton rotenone and piericidin and neither one of them is coupled to proton translocation across the IMM (16, 17). Under conditions of stawation for carbon and nitrogen, however, S. cerevisiae is capable of synthesizing a NADH dehydrogenase that is involved in the establishment of a proton gradient. This dehydrogenase oxidizes intramitochondrial NADH and is insensitive to rotenone (2. 16).

The yeast S. cerevisiae also contains a flavoprotein-containing NADH dehydrogenase that is embedded in the OMM. This enzyme, which is antimycin insensitive, catalyzes the oxidation of cytoplasmic NADH and is overexpressed in a ubiquinone-deficient mutant (1% 20). This NADH dehydrogenase is a component of an outer membrane electron transfer chain in which electrons flow via a .to cytochrome c.

II. Succina te-ubiquinone reduc tase (complex II) Succinate-ubiquinone reductase, also known as complex II, is located on the matrix side of the IMM and catalyzes the oxidation of succinate and the reduction of ubiquinone to ubiquinol (Fig. 1-2). In humans, complex II is composed of four subunits: a 70 kDa flavoprotein that contains covalently bound FAD, a 27 kDa iron- sulfur protein (ISP) with three iron-sulfur clusters, as well as a 15.5 and a 13.5 kDa subunit which are membrane-bound and contain b-type . This enzyme complex, which consists solely of nuclear encoded subunits, does not transfer protons into the IMS and is thus not involved in the establishment of the proton gradient. This is probably why this complex has not been studied as extensively as complexes III and IV in yeast and complexes 1, III, and IV in humans (21).

Ill. Ubiquinol-cytochrome c oxidoreductase (complex 111, bc, complex)

Complex III, a homodimer (1621, accepts electrons from complexes I and II via the electron shuttle ubiquinol and catalyzes the reduction of cytochrome c (Fig. 1-2). In human and beef heart mitochondria, complex III consists of 11 subunits (228 23) and a recent documentation on the preliminary crystallization of the beef heart mitochondrial complex predicts 1 3 transrnembrane helices for each monomer (162). SO far, only 10 subunits have been discovered for yeast complex III (Table 1-1 ) (2. 24). Hence, these complexes contain six to eight additional polypeptides than the homologous enzyme complexes of some other organisms. The and cyanobacterial bsf complexes have four subunits and the complex from P. denitrificans has a mere three polypeptides (25-27). Although the number of subunits varies widely in al1 these comparable enzyme complexes, they al1 contain the same number and kinds of prosthetic groups. There is a b-type cytochrome which contains two hemes, a cytochrome cl or f which contains one , and the Rieske iron- sulfur protein (ISP) which contains a [ZFe-2S] ferredoxin. The subunits containing prosthetic groups are also referred to as the catalytic core. The mode of electron transfer is believed to be according to the Q-cycle mechanism (28-30) (Fig. 1-3). In addition to the subunits containing redox centers (cytochrome b, cytochrome c,, Table 1 -1 . COMPLEX III SUBUNIT COMPOSITION

I - - I YEAST I BEEF HEART Subunit Daltons Subunit Daltons

Core Proteins Core 1 48,225 Core 1 Core 2 38,714 Core 2

Catalytic Su bunits CY~b 43,633 Cyt b CYt Cr 27,419 CYt G ISP 23,349 ISP

Supernumenry Subunits 17 kDa (QCR6) 14,513 Hinge 9,175 14 kDa (QCRT) 14,561 13.4 kDa 13,389 1 1 kDa (QCRB) 1 1,000 9.5 kDa 9,507 DCCD 7,998 7.2 kDa (QCR9) 7,262 7.2 kDa 7,189 8.5 kDa (QCR70) 8,492 6.4 kDa 6,363 Center

Center

Fig. 1-3. Q-cycle rnechanism as adapted from Trumpower (28). The scheme shows the pathway of electron transport through the bc, complex with the reaction order through the four redox centers indicated by numbers (1) through (4). In step (1) ubiquinol is oxidized at center P whereby one electron is transferred from ubiquinol to the iron-sulfur protein (ISP). The newly generated ubisemiquinone anion at center P immediately reduces the cytochrome b-566 heme in what is referred to as oxidant-induced reduction. This first step results in the deposition of two protons on the positive side of the membrane, center P. In step (2) the electron from the ISP is transferred to cytochrome cl which subsequently transfers it to cytochrome c of complex IV. In step (3) the electron from the b-566 heme is transferred to the higher potential b-560 heme against the membrane potential. In step (4a) ubiquinone is reduced to the relatively stable ubisemiquinone anion at center N. Step (4b) occun in the second round of the cycle and in this round the b-560 heme reduces the ubisemiquinone anion to ubiquinol with the consumption of two protons from the matrix side of the membrane. Thus one complete Q-cycle deposits four protons ont0 the positive side of the membrane, and transfers two electrons to complex IV, thereby reducing two molecules of cytochrome c. and ISP), there are two core proteins and five so-called supernumerary subunits (encoded by the genes QCR6, QCR7, QCRB, QCRS and QCR 70 in yeast).

vtochrome b Cytochrome b, the only mitochondrially encoded subunit of complex 111 (34 represents the Iiydrophobic core of the complex with eight predicted membrane-spanning a-helices. Its primary structure is well conserved among various organisms. The two hemes, b566 and b562? are located on opposite sides of the membrane, one at the positive side, center P (also referred to as center "O" for proton output), and one towards the negative matrix side, center N (also called center "i" for proton input). This is in agreement with the Q- cycle mechanism and the eight membrane-spanning helix structure is therefore the accepted model, as opposed to the nine membrane- spanning helix structure proposed in earlier studies (2). The eight membrane helix model is also consistent with a number of studies involving mutants that are resistant to inhibitors (31). Two pairs of conserved histidine residues are the ligands to the two heme groups; each heme is bound to one histidine in the second helix and one in the fourth helix. Mutants that lack cytochrome b are devoid of complex III enzyme activities and display low steady-state levels of the ISP as well as of the 14 kDa and 1 1 kDa supernumerary subunits. The rate of synthesis of these three subunits is not affected in cytochrome b mutants. Rather the low steady-state levels are believed to be the result of a faster turnover rate since cytochrome b, the 14 kDa and the 11 kDa subunits have been shown to form a

subcomplex for which the ISP has a high affinity (2.31-34).

Cytochrome b has been shown to be involved in ubiquinone- binding by photoaffinity labeling of inhibitors. lnhibitors of complex III such as hydroxy quinoline N-oxide (HQNO), diuron, 6-undecyl-5- hydroxy-2,3-dioxobenzothiazole (UHDBT), stigmateIIin, and myxothiazol which are ubiquinone, ubiquinol, or ubisemiquinone analogs bind cytochrome b at center P or N. Cytochrome b by itself is believed to form the binding site for ubiquinone at center N; therefore inhibitors of this site, such as antimycin, block the reoxidation of cytochrome b and destabilize the bound ubisemiquinone. On the other hand, the ubiquinone binding site at center P is formed jointly by cytochrome b and the ISP; hence, inhibitors of this site, such as myxothiazol, block reduction of the ISP and also prevent reduction of cytochrome b in the presence of

antimycin (281 32) (see Fig. 1-3).

çytochrome c, Cytochrome ci, a protein with a molecular weight of 27,419 is another heme-containing subunit of the bc, complex. All cytochrome cl proteins studied so far contain the consensus sequence CXXCH at residues 40-44 of the mature protein. This is a characteristic of protoporphyrin IV proteins in which the heme is liganded to a histidine residue (2. 35). In addition, a conserved methionine at position 164 has been proposed as the sixth ligand to the heme iron. Although cytochrome cl is overall hydrophilic, it has a nonpolar stretch of 15-20 amino acids near the carboxy terminus. It has been suggested that this region forms an a-helix which anchors the protein in the membrane (35). The remaining part of the protein, including the catalytic center, is believed to be located in the IMS

(162). Cytochrome cl is a nuclear encoded subunit that is synthesized with a bipartite signal sequence (two amino-terminal leader sequences arranged in tandem), 61 amino acids long. Two different mechanisms have been suggested to explain the targeting of this protein into its final destination in the IMS (36.37) (see below).

The Rieske iron-sulfur protein The Rieske ISP with a calculated molecular mass of 23,349 daltons and a length of 215 amino acids is the third subunit of complex III containing a redox center. Just like cytochrome cl, the ISP is nuclear encoded and synthesized with a bipartite signal sequence 30 residues long (MLGIRSSVKTCFKPMSLTSKRLISQSLLAS) (381 39). Its catalytic center is located in the aqueous environment of

the IMS (162) and is linked to the inner membrane by a hydrophobic anchor. Proteolytic cleavage at a site just downstream from the putative membrane spanning helix of the ISP in E. coli and R.

sphaeroides renders the protein water-soluble (40).

The ISP contains a [2Fe-ZS] ferredoxin with a very high midpoint potential which is liganded to the two histidines and two of the cysteine residues in the following conserved sequences: CPCH and CTHLGC. Due to its high redox potential the ISP is the primary electron acceptor during hydroquinone oxidation and is thus involved in what is often referted to as "oxidant-induced reduction" of the lower potential heme of cytochrorne b (refer to the Q-cycle mechanism, Fig. 1-3). Like cytochrome b, the ISP binds stigmatellin and ubiquinone analogs. Hence, it has been suggested that both cytochrome b and the ISP interact to form the binding site for these

inhibitors (2, 28,38, 39,41,42).

Results from mutational analyses of the ISP suggest that substituting the conse~edresidues that constitute the ligands to the [ZFe-2S] cluster does not prevent processing of the precursor to the mature polypeptide, although a slight increase of incompletely processed iron-sulfur apoprotein can be detected in the mutants. Mutagenizing the ligands, however, prevents the [ZFe-2S] cluster from being inserted into the apoprotein and, although it does not prevent assembly of the bc, complex, the iron-sulfur apoprotein is more easily lost upon purification of the complex. Earlier studies indicated that when the gene encoding the ISP is deleted the cytochrome b hemes are distorted (39). However, a mutational anaiysis performed by Graham and Trurnpower (42) seems to indicate that the simple presence of the iron-sulfur apoprotein is sufficient to stabilize the environment of the b-hemes resulting in near wild type levels of this holoenzyme. Based on these results it was suggested that the insertion of the [2FeW2S]cluster is the final step in the biosynthesis of complex III. Furthermore, it was suggested that the iron-sulfur apoprotein itself is involved in stabilizing the heme environment of cytochrome b and that the [ZFe-2S] cluster is, in turn, invoived in stabilizing the attachment of the ISP to the

complex (42).

What about the supernurnerarv subunits of com~lexIII? When comparing eukaryotic and prokaryotic bclcomplexes, it becomes evident that some bacterial isoenzymes that only contain the three catalytic subunits (cytochrome b, c,, and the ISP) perform the same function as their eukaryotic counterparts that contain as many as 11 subunits. This raises the question as to the functional importance of the so-called supernumerary subunits. Two models are used to rationalize their presence. In the first model, a11 primordial organisms are believed to have contained supernumerary subunits. Subsequently, as bacteria and mitochondria branched off from one another during the course of evolution, bacteria, being more efficient, lost these subunits whereas mitochondria retained them. From this model it follows that the supernurnerary subunits are redundant and probably do not serve any function. The second model States that none of the primordial organisms contained supernumerary subunits, but rather that they were acquired during the course of evolution by mitochondria as a result of morphological differences between bacteria and mitochondria. This latter model implies that the supernumerary subunits serve a function since why, otherwise, would they have been acquired during the course of evolution? All the yeast genes encoding the supernumerary subunits of complex III have been inactivated and found to be necessary for the integrity and functioning of the compiex (see below). Hence, the knowledge that is available today supports the second model according to which the supernumerary subunits are believed to serve a function.

The SuDernurnerary subunits of com~lexIII abunit 6 may reglate the activity of complex III QCR6 is the yeast gene that encodes the well-conserved, highly acidic polypeptide of 14.5 kDa that is actually referred to as the 17 kDa subunit due to its migration on SDS-PAGE (it is also known as subunit 6). This nuclear encoded subunit is the only polypeptide of the supernumerary subunits that is synthesized with a cleavable, N- terminal targeting sequence, 25 amino acids long (2. 43). The Qcr6 protein contains an acidic stretch of 24 amino acids, is largely a- helical. and has a domain which resembles the Ca2' binding domain of yeast calmodulin (44). However, an involvement of this protein in Ca2' binding has not been demonstrated to date. The only hydrophobic region of the protein is at the carboxy-terminus and this region is postulated to anchor the subunit to the inner membrane. Subunit 6 is homologous to the "hinge protein" of the bcl complex in beef heart which has been implicated in promoting ionic interactions between cytochrome c and cytochrome cl, thereby facilitating electron transfer f rom the bc, cornplex to cytochrome oxidase (45).

Deleting the QCR6 gene gives rise to a strain that has 50% residual ubiquinol-cytochrome c reductase activity and maintains the ability to grow on non-fermentable carbon sources (46). Schmitt et al. have suggested that this deletion causes a conformational change of the complex that results in "silencing" half of the reduction sites of the bel dimer. This led to the belief that the 17 kDa protein is involved in regulating the activity of the complex in vivo during conditions when the requirement for ATP is low. Hence, under such conditions a signal must therefore be present in vivo that rnirnics the action of the deletion. In summary, these gene deletion studies have shown that subunit 6 is not necessary for assembly of

complex 111 (46). On the other hand, studies performed in which the QCR6 gene was disrupted, rather than deleted, resulted in a complex that lacked any detectable enzyme activity and holocytochrome b

(47). This was believed to be due to the existence of an aberrant copy of the 17 kDa protein that was being synthesized. This non- f unctional polypeptide supposedly prevented the complex from assembling by binding either to cytochrome cl or the Qcr9 protein thereby removing these subunits from the assembly pathway and preventing further assembly. Overexpressing the Qcr9 protein could partially correct for this defect by binding the aberrant Qcr6 protein and preventing it from removing cytochrome cl from the assembly pathway. From these results it was concluded that subunits 6 and 9

interact with cytochrome cl to form a subcomplex (47).

Subunit 7: the protein of the current studv QCR7 is the nuclear gene encoding the so-called 14 kDa subunit (also referred to as subunit 7 or Qcr7 protein) which has a molecular weight of 14,561 and is the focus of the current study. Subunit 7 is a largely hydrophilic protein with a hydrophobic stretch from residues 19-38. The QCR7 gene has been cloned and sequenced; the deduced protein was found to be homologous to the 13.4 kDa subunit 6 of the beef heart mitochondrial bc, complex (23s 339 48). The upstream sequence of this gene has elements similar to a CAAT and TATA box, which are important for transcription initiation in humans. However, this promoter region lacks motifs for high level

expression in yeast leading to a low abundance RNA transcript (48).

Previous inactivation of the gene encoding subunit 7 indicated

that the 14 kDa subunit is crucial for complex III function (33). The mutant strain is respiration-deficient and does not grow on non- fermentable carbon sources. The mutant displays low steady-state levels of the 11 kDa and ISP subunits. In addition, spectrophotornetric analyses indicate that holocytochrome b cannot be detected and Western blot analyses show that apocytochrome b levels are far below those in the wild type. As RNA transcripts of the 11 kDa subunit and the ISP are present at wild type levels, the results indicate that the low steady-state levels of the above proteins are likely to be due to a post-transcriptional effect. Hence, the pet- phenotype (the phenotype resulting from a respiratory chain defect) seen in the mutant in which the QCR7 gene has been inactivated, is most iikely due to an inability of the bci complex to assemble, which in turn results in degradation of the subunits associated with the Qcr7 protein. Based on these results Schoppink et al. suggested that the 14 kDa subunit associates with cytochrome b, the 11 kDa subunit, and possibly the ISP to form a subcomplex before further assembling into a functional enzyme complex. A study performed by Hemrika et al. (494 in which the C-terminal 12 residues were replaced by the 3 residue segment L-A-D, resulted in complex III activity that was reduced by approximately 60% when compared to the wild type, whereas the turnover number was normal. This mutant also displayed lowered levels of holocytochrome b, the 11 kDa subunit, and the ISP. In the same study, a mutant was constructed in which the C-terminal 31 residues were replaced by the sequence D-L-Q-P-S-L-L-1-D. This mutant displayed a phenotype similar to the strain in which the QCR7 gene is inactivated. The authors concluded from this study, that the C-terminus of the 14 kDa subunit is involved in the formation of a functional enzyme complex. However, as the authors did not have an antibody that recognized the truncated 14 kDa subunits, it cannot be ruled out from this study that the decreased levels of assembly are in fact a secondary effect resulting from decreased import of the 14 kDa su bunit.

Subunit 8: a ubiauinone-bindina rotei in? QCR8 is the nuclear gene encoding a polypeptide of 11.O kDa which is usually referred to as the 11 kDa subunit (also referred to as subunit 8) of the bc1 complex (50,si). The amino acid sequences of this subunit and its 9.5 kDa beef heart mitochondrial counterpart, which has been implicated in ubiquinone-binding (521, are not significantly similar, however their predicted secondary structures are very similar (53). Mutagenesis of an aromatic region of the 11 kDa subunit has indicated that this region may be involved in the assembly of a functional enzyme and in ubiquinone-binding at center P (50, 54, 55). Deletion of the QCR8 gene leads to low steady-state levels of the 14 kDa subunit as well as the ISP and cytochrome b, the latter being detected by absorption spectra. These results are in agreement with the gene deletion studies of subunit 7 and further strengthen the hypothesis of a subcomplex consisting of the 11 kDa and 14 kDa subunits, and cytochrome b. Since deletion of the gene encoding the ISP does not affect assembly of the bel complex, it is not understood at which stage the ISP associates with the above- mentioned subunits, and whether it is indeed an inherent part of this subcomplex.

Subunit 9 QCR9, the gene encoding subunit 9 has a molecular weight of 7,300 and displays 56% identity to its beef heart counterpart. Deletion of this gene results in a strain that is respiration deficient with a residyal activity of less than 5% although complex III is still assembled (56). Upon further examination, the level of cytochrome cl was found to be normal; holocytochrome b levels were found to be decreased in the mutant strain when compared to the wild type. Mature ISP was present although without the [ZFe-2S] cluster and, as a consequence, the pre-steady-state rate of reduction of cytochrome cl was decreased. Based on these results, it was suggested that subunit 9 interacts with the ISP and the cytochrome b domain involved in binding the lower potential b-566 heme at center P. The absence of subunit 9 is therefore believed to invoke a conformational change of the ISP which prevents the insertion of the [ZFe-ZS] cluster. This conformational change of the ISP would then cause a distortion of the b-566 heme environment and thereby alter the interaction of the ISP with this low potential cytochrorne b heme. This is so far the only described case in which the absence of a supernumerary subunit results in respiration deficiency with the

bel complex still being assembled (44.56).

ubunit 10 The most recent subunit that has been discovered is the 8.5 kDa subunit 10 (8,492 daltons) that is encoded by the nuclear gene

QCRIO (24). This polypeptide is the homologue of the 6.4 kDa subunit 11 in the beef heart bcl complex. The two proteins display similar secondary structures and their amino acid sequences are 51% similar. Deletion of the QCRlO gene alone does not affect growth on non-fermentable carbon sources and the mutant strain has a residual complex III enzyme activity of 40%. However, deleting both QCR 7 O and QCR6 gives rise to a strain with a decreased growth rate on non- fermentable carbon sources. Furthermore, when the bcl complex of the QCRl O deletion mutant is purified, the ISP is lost. From these results it was suggested that subunits 6 and 10 rnay be genetically linked, and that the QCR7O protein may contribute to a tighter

association of the ISP with cytochrorne b (24).

The core proteins Core proteins 1 and 2 are the largest subunits of ubiquinol- cytochrome c oxidoreductase and account for 50% of al1 the protein in the complex. They are both encoded by nuclear genes and have cleavable N-terminal signal sequences 17 and 16 amino acids long, respectively. In contrast to al1 the other subunits of the complex the core proteins are nearly always present at wild type levels in strains in which the other subunits of complex III are absent as a result of gene inactivations. Deletion of the gene encoding core protein 1 results in a pet- phenotype. The mutant strain does not grow on non-fermentable carbon sources. Core protein 1 of Neurospora crassa bc1 complex has been found to be the previously identified processing enhancing protein (PEP) which stimulates the activity of the matrix processing peptidase (MPP) in N. crassa (57). In S. cerevisiae, core protein 1 is homologous, but not identical to the PEP of yeast (58. 59). Deletion of the gene encoding core protein 2 results in a strain that grows slowly on non-fermentable carbon sources. Mutants lacking both core proteins have low residual enzyme activity, but the turnover number is the same as in the wild type. Hence, the core proteins are not involved in the catalytic activity of the bel complex, but rather increase the efficiency of the assembly process (2).

Functional assemblv of complex III All the research that has been performed on the bc, complex to date suggests that three subcomplexes are formed prior to assembly of a functional enzyme complex. One of the subcomplexes is believed to consist of cytochrome b, the 14 kDa and 11 kDa subunits. The reason for the proposed association of these three subunits is their post-translational interdependence, which has been demonstrated by the study of strains in which the respective subunits were missing or mutated (33. 49. 55, 60-63). Although steady- state levels of the ISP are also reduced in mutants lacking either cytochrorne b, the 11 kDa or the 14 kDa subunit, the ISP is not believed to be a component of the cytochrome b subcomplex. This conclusion is based on findings that a mutant strain containing a deletion of the gene encoding the ISP does not contain lowered steady-state levels of any complex III subunits other than of the ISP; in addition, the insertion of the ISP is believed to be the last step in the biosynthesis of complex 111 (56?60, 64). Hence, the lowered steady-state levels of the ISP are more likely to be a secondary effect resulting from degradation of this protein when its neighboring subunits are not present. Two nuclear gene products, encoded by CBP3 and CBP4 in yeast, have been proposed to be involved in assembly of the bc, complex or maturation of cytochrome b (directly or indirectly) (61. 62). More specifically, the Cbp3 and Cbp4 proteins are suggested to stabilize or assemble the cytochrome b subcomplex since cbp3 and cbp4 mutants display reduced levels of cytochrome 6, the ISP, and the 11 kDa and 14 kDa subunits. This might occur by stabilizing cytochrome b, so that it remains in a competent state for the insertion of hemes or the association with the 14 kDa and 11 kDa subunits. This notion is consistent with the belief that maturation of cytochrome b occurs after the formation of the cytochrome b subcomplex or even after the assembly of this subcomplex into a stable quarternary complex of which the composition is unclear at this point (60).

A second subcomplex that is formed is believed to consist of core proteins 1 and 2 (47. 57, 60. 65). Contrary to the cytochrome b subcomplex, this subcomplex is quite stable (2). Hemrika et al. (49) have proposed that the charged C-terminus of the 14 kDa subunit associates with this subcomplex consisting of core proteins 1 and 2 and that this association renders the cytochrome b subcomplex protease resistant. This model is in agreement with previous results which show that steady-state levels of the 14 kDa subunit and cytochrome b are decreased in the absence of core protein 2 due

to an increased sensitivity to proteases (66). The model is furthermore consistent with the more recent prediction of the topology of the 14 kDa subunit, that not only the N-terminus of this

subunit faces the matrix (661, but also the C-terminus (67). This is important because the core proteins are located on the matrix side of the IMM with a large fraction protruding into the matrix (57).

The third subcomplex is believed to consist of cytochrome ci, the 17 kDa and the 7.2 kDa proteins. The prediction of the formation of a subcomplex composed of these three subunits is based on findings that the homologues of the yeast 17 kDa and 7.2 kDa

subunits have been cross-linked to cytochrome c, in beef heart (6s). Further evidence for the formation of this subcomplex is given by studies which indicate that the association of the 17 kDa subunit with cytochrome c, renders the latter resistant to proteases and stimulates the interaction between c, and c (45).

According to Schmitt and Trumpower (47) the 17 kDa and 7.2 kDa subunits associate with each other first, before combining with cytochrome c, into a subcomplex of intermediate stability. This subcomplex then associates with the partially assembled complex III, consisting of the cytochrome b and core protein subcomplexes, to form a stable oligomeric complex without the ISP. Insertion of the ISP completes the assembiy pathway of ubiquinol-cytochrome c oxidoreductase (47,W

Much of the information that led to the prediction of the three subcomplexes discussed above was based on the varying steady- state levels of the supernumerary subunits of complex III in different mutant strains. In most cases, it was concluded that lowered steady-state levels were due to an improperly assembled subcomplex or bc,complex. Hence, the subunits that are lowered are believed to assemble with the mutated or absent protein. This prediction is probably true in most cases. However, it cannot be ruled out that in some cases, in which a protein is mutated rather than absent, the mutated protein is more susceptible to proteolysis and that in these instances the lowered steady-state levels of other subunits are the result of a secondary effect (68-71). As none of the proteases (except the one responsible for the turnover of the F,P subunit of the ATPase) involved in the turnover of the nuclear encoded subunits of the respiratory-chain enzyme complexes are known at the present time, this subject remains open to speculation

(68).

IV. Cflochrome oxidase Cytochrome oxidase (also referred to as complex IV and COX), is the terminal enzyme complex of the mitochondrial electron transport chain (Fig. 1-2). It catalyzes the oxidation of cytochrome c and the irreversible reduction of Oz to HrO. In the crystal structure of cytochrome oxidase from Paracoccus denitrificans (lsg), which contains 22 prirnarily helical membrane-spanning segments, lwata et al. have identified two possible proton transfer pathways. These are for the scalar movement of protons which is involved in the reduction of Oz to H20, and the vectorial movement of protons which is involved in the establishment of the protonmotive force in

the IMS ('59). In the crystal structure of the 13 subunit beef heart

cytochrome oxidase by Tsukihara et ai. (1 681, three networks capable of relaying protons were identified; two for the vectorial movement of protons and one for the scalar movement of protons. Of the 13 subunits of cytochrome oxidase (721, only the three largest ones are encoded by mt DNA and these constitute the catalytic core. These are cox 1, 2, and 3 and they are strikingly similar in beef heart and in the three subunit-containing enzyme complex of P. denitrificans as well as to their counterparts in mitochondria of other organisms. Although cox 1, 2, and 3 are referred to as the catalytic core, only cox 1 and 2 contain redox centers. Cox 1, contains Cu.and hemes a and a3, which in beef heart mitochondria are in perpendicular orientation to the plane of the membrane. Herne a3 and Cu. constitute the binuclear center at which O2 is reduced to H20. Cox 2, which contains Cu., and cox 3, of which the function is unknown, associate with the transmembrane region of cox 1 without any direct contact with each other (168). In the beef heart mitochondrial cytochrome oxidase cox 1 is mainly embedded in the membrane with 12 transmernbrane helices, whereby the N- and C-termini of cox 2 are both located on the cytosolic side. All of the 10 nuclear encoded subunits of beef heart cytochrome oxidase were found to contain transmembrane a-helices, most of which are neither vertical to the plane of the membrane nor parallel to each other (168). In yeast, nuclear encoded subunits 7 and 7a have been implicated in assembly of complex IV (731, while subunits 4 and 8 seem to be involved in the stability of the enzyme (74, 75). The order of electron transfer through cytochrome oxidase is from cytochrome c, the primary redox center, to the Cu, center, then to heme a, and then to the binuclear center.

IL A TP synthase The ATP synthase or complex V (Fig. 1-2) is the enzyme complex that catalyzes the phosphorylation of ADP to ATP (769 77). The complex is composed of three parts: a water-soluble FI portion ("headpiece"), a detergent soluble Fo portion ("basepiece"), and a "stalk". The role of the Fo portion is to direct the electrochemical gradient of protons to the Fi moiety; the role of FI is to bind ADP and Pi and to use this electrochemical gradient to drive the synthesis of ATP. The crystal structure of the FI domain from beef heart mitochondria has been solved at 2.8 angstroms by Abrahams et al.

(160) and was found to be a fiattened sphere 80 angstroms high and 100 angstroms across. In al1 species in which this complex has been studied so far, FI is composed of five subunits: a, P, y, 6, E and in most species the subunit stoichiometry is a3 pg y 6 r. The three a and p subunits are arranged alternately like segments of an orange around a central a-helix which is 90 angstroms long and is formed by the C-terminus of the y subunit. This helical structure protrudes from the main body in a stem and most Iikely constitutes a part of the "stalk" between the FI and Fo moiety (160). The a, P, and y subunits are well conserved with the p displaying 70% similarity from E. coli to humans. These $ subunits contain the three catalytic sites and react with di- and triphosphates of purine and pyrimidine nucleotide substrates. The structures of the three catalytic sites were found to always differ, whereby each one is believed to pass

through a cycle of "open", "loose", and "tight" States (160). This is in agreement with the prediction of an earlier, low resolution X-ray

structure, which showed that the FI domain is asymmetrical (161). The a subunits, which are homologues of the P subunits, do not have a catalytic role, but they bind ADP and ATP. Hence, there are six nucleotide binding sites on the FI moiety, one on each a and P

subunit. In addition to the a, P, y, 6, and E subunits, there is an inhibitor protein, IF,, that associates with FI at low protonmotive force and inhibits ATP hydrolysis.

The subunit composition of the Fo portion varies among species; the human one consists of at least eight different types of subunits: a, b,, ~~(stoichiornetryvaries but does not exceed 12), d, e, OSCP (oligomycin sensitivity conferring protein), F6 (=Factor 6), and A6L. In S. cerevisiae, the ATP synthase consists of only three subunits. In al1 species examined so far, subunit c, which together with the fourth and fifth transmembrane helices of subunit 6 forms the proton channel, is involved in proton conduction via a free carboxyl group (78). Factor 6 is required for FI binding to Fo, and OSCP is involved in energy coupling between FI and Fo. ATP synthesis on the surface of the F1 moiety has been shown to need little input of energy. The reaction step that actually utilizes the energy from the electrochemical gradient is the binding

of substrates and the release of bound ATP from Fi (76. 77). Since the ATPase complex can also work in reverse, catalyzing the hydrolysis of ATP, it is important that some kind of regulation exists in order to prevent the hydrolysis of the newly synthesized ATP. In humans this is achieved in two ways: 1) binding of the inhibitor protein IF1 to sites on the a and p subunits and 2) binding of ADP to sites on the a and p subunits. Hence, by occupying these sites, IF1 and ADP prevent the binding and subsequent hydrolysis of ATP. This model suggests that under high protonmotive force, when IF, or ADP corne in contact with the proton gradient, IF1 will be released from the binding sites whereas ADP will be converted to ATP. Hence, in this model the substrate-binding site will also be the inhibitory site (77).

Nucleo-mitochondrial in teractions As previously mentioned, mitochondria are believed to have evolved from an endosymbiotic relationship. Hence the eukaryotic cell was assembled by a series of symbiotic events. This means that nucleus, mitochondria, and were each derived from a different phylogenetic lineage (1. 3). As a result, mitochondria contain their own genome, but it encodes only a Iimited number of proteins necessary for the biogenesis and function of this organelle. The majority of proteins are encoded by nuclear DNA. Hence, for the bc7complex and the other complexes that are composed jointly of nuclear and mitochondrial gene products, the nuclear encoded subunits have to be targeted for import into mitochondria where they are assembled with their partner subunits to form functional, multisubunit enzyme complexes. For this to occur, there has to be a coordination of the two separate genetic systems.

How do mitochondria and the nucleus communicate? The answer to this question is far from understood, however there are a number of levels at which regulation can occur. These are transcription, translation, import into mitochondria, and assembly. Saccharomyces cerevisiae is a facultative anaerobe that can tailor the level of mitochondrial biogenesis to its specific needs in response to its environment; under different conditions of growth different metabolic pathways are activated ('3). Under conditions of starvation, for example, the enzymes of the gluconeogenesis pathway become operative. On the other hand, when yeast are grown on media containing high levels of glucose, many respiration related proteins suffer from glucose repression, that is the uptake systems of sugars other than glucose are repressed (a phenomenon also referred to as catabolite repression) (2. '5). Expression or repression of certain genes is usually regulated at the level of transcription and is dependent on heme, oxygen, "actively respiring mitochondria", compounds from intermediary metabolism, and other factors. For example, glucose repression of mitochondrial biogenesis is in part attributable to a three- to sixfold decrease in the amount of transcription of mitochondrial genes (79). Another example of regulation at the transcriptional level is the induction of alcohol dehydrogenase II and other enzymes of the gluconeogenic pathway. This induction does not occur in respiratory deficient mutants or in wild type cells grown on minimal media. Conversely, under conditions when yeast need oxidative metabolism, growth and division of the cell are matched by an increase in mitochondrial mass (13). Although yeast seem to "prefer" the anaerobic growth on glucose, they cannot grow in the complete absence of oxygen since it is needed for the synthesis of heme, egosterol, and unsaturated fatty acids. A mutation resulting in a respiration-deficient phenotype or addition of an inhibitor to the respiratory chain in combination with depletion of ATP in the mitochondria is lethal to S. cerevisiae.

There are several types of lactate dehydrogenases, specific for either L- or D- lactate, that can be synthesized depending on the growth conditions of S. cerevisiae. On high concentrations of glucose, whether yeast are grown aerobically or anaerobically, an NAD-linked dehydrogenase is induced. This lactate dehydrogenase reduces pyruvate to lactate and thus regenerates NAD, thereby keeping the NAD/NADH redox couple balanced (2. 80. 81). However, this lactate dehydrogenase can also be induced at low glucose concentrations when the mitochondrial respiratory chain or mitochondrial protein synthesis are inhibited. There are two other types of lactate dehydrogenases that are synthesized when yeast are grown aerobically on lactate: a D-lactate:cytochrome c oxidoreductase and a L-lactate:cytochrome c oxidoreductase. These enzymes catalyze the oxidation of lactate to pyruvate and the electrons from lactate reduce cytochrome c of complex IV in the mitochondrial respiratory chain. Thus growth on lactate, unlike growth on ethanol or pyruvate, can often still occur in the presence of antirnycin since the proton gradient established by cytochrome oxidase generates enough ATP to sustain slow growth (2). When yeast are grown on galactose the majority of mitochondrial enzymes are derepressed and ATP is derived from the respiratory chain.

The ex~ressionof manv cornplex III subunits is dependent on thg need for oxidative metabolism Steady-state levels of the subunits of the bel complex and cytochrome c of complex IV Vary in response to the need for oxidative meta bolism. For cytochrome c, this response is primarily governed by the rate of transcription (82-85). The nuclear gene encoding cytochrome c, CYC 7, has two upstream activation sequences, UAS1 and UASZ. An activator protein called HAP1 binds to UAS1 and increases RNA synthesis 300-fold under aerobic conditions; this binding of HAP1 to UAS1 is stimulated by the presence of heme. Oxidative phosphorylation is therefore regulated by the intracellular level of heme, which is logical, since the final stages of heme synthesis occur in the rnitochondrion and are dependent on the level of oxygen. On the other hand, CYC1 is affected by glucose repression (86, 87). There is evidence that HAP1 control is also exerted on genes that encode proteins without hemes, such as core protein 1 of complex 111 (44).

Transcription of the nuclear genes QCRG, QCR7, QCRB, QCR9, and the genes encoding core protein 2, cytochrome cl, and the ISP are al1 repressed by glucose (4% 44). Most of these genes contain the consensus sequence element of UASZ, but it is not known whether this is involved in the catabolite repression. The QCR9 gene might also be regulated at the level of mRNA splicing. This gene has an intron, and since introns are rare in yeast genes, they often serve a function, such as the intron of the mitochondrial cytochrome b gene. The intron of the QCR9 gene has some identical sequence elements to the intron within the COX4 gene; these sequences might regulate the coordinate expression of the bc, complex and the cytochrome oxidase complex (44).

Coordination of the expression of nuclear and mitochondrial genes encoding subunits of the mitochondrial respiratory chain can involve the direct regulation of expression of these genes, or the regulation of genes whose products are involved in the synthesis of individual mitochondrial products. Examples of the latter include gene products involved in the S'-end processing and intron excision of the pre-mRNA of cytochrome b and in its translation (83-85). Another example is the CBP6 gene, a nuclear gene coding for a protein that stimulates translation of cytochrome b mRNA; by suppressing this gene, synthesis of cytochrome b could be decreased

(88). Furthermore, cytochrome oxidase subunits 1 and 2 require nuclear gene products for the processing and translation of their transcripts (15). An example of a nuclear encoded protein that regulates mitochondrial function at the level of assembly is provided by the RCA 7 gene product. If this gene is mutated, assembly of the ATPase subunits into a functional complex V is prevented (89). As can be seen, there are rnany nuclear encoded enzymes that are necessary for transcription of mt DNA's, processing of precursor RNA's, and translation of mitochondrial messages. In addition, RNA polymerase and almost al1 the proteins that constitute the mitochondrial translational machinery are encoded by nuclear DNA. An example of possible regulation by the proton gradient in the IMS is given by the Qcr6 protein of the bc7 complex. In experiments where the QCRG gene was deleted, half of the reduction sites of the dimeric bc7 complex were silenced. Results of these studies suggested that under physiological conditions when concentrations of ADP are low and ATP is abundant, the increase in the positive charge surrounding subunit 6 may cause it to regulate the complex so as to silence half of the reduction sites and thereby downregulate the activity of the bel complex (46).

Do mitochondria exert reaulation on nuclear aene products? All the above examples include regulation exerted by nuclear genes on mitochondrial function and biogenesis. What about the regulation of expression of nuclear genes by mitochondria? An argument against the existence of such a regulation is that the synthesis of PET products, nuclear genes required for the morphogenesis of respiratory competent mitochondria, is not dependent on the presence of an intact mitochondrial genome (15). Expression of many PET gene products is unaltered in p- and po strains, strains with large mitochondrial deletions or no mt DNA at all, respectively. Mitochondria of po cells can therefore be very similar to wild type cells and can contain many functional enzyme complexes. Although not abundant, there is some non-specific evidence for the regulation exerted by mitochondria on the nuclear genorne. Experiments performed in N. massa suggested that treatment of cells with chlorarnphenicol, an inhibitor of the mitochondrial protein synthesis apparatus, increases the synthesis of some nuclear encoded proteins that are part of the mitochondrial transcription and translation apparatus. This study may suggest that nit DNA

encodes a repressor protein that regulates nuclear genes (90). This conclusion seems far-fetched and it is more probable that the increase in synthesis of nuclear encoded proteins is the reponse to the lowered ATP levels in the cell. Another study ernployed subtractive hybridization and indicated that some transcripts of the nuclear genome Vary in abundance by a factor of five or more among derepressed isochromosomal cells depending on whether they were part of a p -, po, or mit- cell (91).

Pro tein import in to mitochondria Localization of a protein into its correct compartment of the cell is essential since mislocalization can lead to severe diseases. Lysosomal storage disease, for example, in which degradative enzymes are erroneously secreted into the bloodstream, results in the accumulation of undegraded products in the lysosome. The accumulation of glycosaminoglycans or glycolipids in the lysosome is the cause for 1-cell disease (92). Another example of a disease that can be caused by a targeting defect is primary hyperoxaluria. This disease can sometimes be the result of erroneous targeting of the peroxisomal enzyme L-a1anine:glyoxylate aminotransferase into mitochondria.

As already mentioned earlier, only a srnall percentage of mitochondrial proteins are encoded for by the mitochondrial gemme. Hence, the vast majority of mitochondrial proteins are synthesized on cytoplasmic ribosomes and must therefore be transported into one of the four compartments of mitochondria, namely the OMM, IMM, IMS, or matrix. Of the four final destinations in the mitochondrion, import into the matrix is the best characterized. However, virtually al1 mitochondrial precursors are initially imported by the same machinery (Fig. 1-4; please refer to this figure throughout this section). jm~oftinto the mitochondrial matrix lmport into the matrix is the best characterized pathway. It constitutes the route into mitochondria for many precursors. Even precursors with final destinations other than the matrix are often imported into this cornpartment first, before being further transported to their final destinations. The sequence of events is as follows: 1. Precursors usually have an amino-terminal targeting sequence (also referred to as signal-, leader-, transit-, or presequence) that is recognized by cytosolic factors which escort the protein to recepton in the OMM. Fig. 1-4. lmport of proteins into the mitochondrial matrix, as adapted from Schatz (93). The precursor protein may bind to a cytosolic chaperone (MSF, mitochondrial import-stimulating factor, or hsp70) and then to the receptor (R) in the OMM which transports it to the protein import channel. Subsequently, the signal sequence of the precursor inserts itself into the protein import channel (which consists of TOM42, 8, and 6 in the OMM and TIM17, 23, and 6 in the IMM) and is pulled across the membrane by the mhsp70-GrpEp-TIM44 complex; translocation across the IMM requires an electrical potential across this membrane, AT. The mhsp70-GrpEp-TIM44 complex resides on the matrix side of the membrane and interacts loosely with the import channel in the IMM. Once in the matrix, the targeting sequence of the precursor is cleaved by the matrix processing peptidase (MPP). The precursor is most Iikely aided by the rnitochondrial chaperone hsp60-cpn10 complex in refolding and assembly in to a rnultimeric enzyme complex, if appropriate. Precursor Protein

IMM +++ y"'

ATP" "y& 2. Precunors bind proteinaceous receptors on the surface of the OMM. 3. Receptors transport precursors to translocation channels which are opened by the signal sequence. 4. The precursor is translocated across the IMM by a protein that is located on the matrix side of the membrane. Transport across the IMM requires an electrical potential, acp, across the membrane. 5. The protein folds on the matrix side of membrane with the help of chaperones and its signal sequence is cleaved off. Proteins that are part of a multimeric enzyme complex are aided in their assembly by chaperones.

The taroetino seauence Many, but not al1 mitochondrial proteins have an amino- terminal targeting sequence that is cleaved on the trans side of the

membrane (94). Targeting sequences lack a consensus and up to 25% of randomly generated peptides can function as mitochondrial signal sequences (95). Nonetheless, there are some general features that most matrix targeting signals have in common; they are 15-35 residues long, rich in hydroxylated and basic amino acids, and capable of folding into an amphipathic a-helix (9% 94). Hence, signal sequences display highly degenerate prirnary sequences, but their common features result in a similar distribution of charged/polar and hydrophobic residues. This common distribution of residues results in the formation of an amphipathic helix which is essential for the function of signal sequences and renders them more effective in targeting than randomly generated peptides. Experiments in E. coli have shown that targeting sequences have two functions; to retard folding of the precursor, and to recognize the receptor on the membrane. Removal of the signal sequence was

found to inhibit targeting by a factor of 100 to 1000 fold (96.97).

Some proteins of the IMS contain composite or bipartite signal sequences. Cytochrome cl and the Rieske iron-sulfur protein of the bc7 complex are two such examples. In these proteins the amino- terminal part of the leader sequence resembles the hydrophilic matrix targeting sequence. The carboxyl half of the leader sequence resembles a bacterial export sequence and is more hydrophobic than its import counterpart (94).

osolic chaperones Molecular chaperones are proteins that interact with non- native conformations of other proteins such as mitochondrial precursors. This interaction prevents aggregation of the (often newly synthesized) proteins until these have either folded correctly or been transferred to a membrane receptor. There are two groups of precursors, classified according to their requirement for extramitochondrial ATP (7. 9*v 99). The first group does not need ATP for import into mitochondria and includes precursors of cytochrome br, holocytochrome c synthase, mitochondrial hsp60, and dihydrofolate reductase fusion proteins. Hence, this group is unlikely to interact with any ATP-requiring chaperones. The second group of precursors is ATP-dependent and therefore is likely to bind to chaperones. This group includes alcohol dehydrogenase III, cytochrome cl, the Flp subunit of the ATPase complex V, and the ADPIATP carrier. Two types of ATP-dependent cytosolic chaperones have been identified to date. MSF, or mitochondrial import- stimulating factor, binds to the signal sequence of a subset of

precursor proteins that have been released from the ribosome (7#94). Since this chaperone interacts specifically with mitochondrial precursors, it most likely functions in escorting precursors to the receptors in the OMM as well as in preventing aggregation. The second type of ATP-requiring cytosolic chaperone is the cytosolic

heat-shock protein 70, hsp70 (98. 99). There are at least two closely related hsp701s in the cytosol of S. cerevisiae and they function together with another protein called MAS5 or YDJ1 which stimulates the release of precunor proteins from hsp70 (100). Hsp70 interacts not only with mitochondrial precursors, but with a wide spectrurn of non-native proteins targeted to different organelles and its function is therefore not to target but rather to prevent aggregation and misfolding (7.94). Chaperones are important for maintaining proteins in a partly unfolded state since most membrane systems can only translocate proteins that are loosely folded (8. 101). Certain small proteins that can spontaneously adopt a loose conformation, such as the coat protein of the Ml3 phage, do not need the aid of chaperones to maintain them in a transport competent state. However, most large proteins and especially those with hydrophobic interiors require the assistance of cytosolic factors (102). Recentors in the OMM Receptors in the OMM are mobile molecules that move within the plane of the membrane and interact dynamically with the translocation channel. Hence, one translocation channel could be served by several different receptors whereby the function of the receptors is most probably to "trapu the precursor protein and to transport it to a translocation channel. The receptors of the OMM are integral membrane proteins with cytosolically exposed domains. Depending on the type of receptor, it recognizes either the signal sequence of a precursor protein or the precursor bound to a cytosolic

chaperone (94). So far, four integral OMM proteins have been identified in yeast that function as import receptors; these are termed MASZO, MAS22, MAS37, and MAS70, consistent with their molecular weights. MAS20 and MAS22 form a subcomplex that contains a highly acidic region referred to as acid bristles. These acid bristles are believed to bind the basic and amphipathic signal sequence of precursor proteins in an ATP-independent manner. MAS37 and MAS70 form a subcomplex that recognizes precursors bound to the cytosolic chaperone MSF. Hence, this subcomplex most likely recognizes the same features in the mature part of the protein as the chaperone and this interaction also requires ATP. Due to the dependency of MAS37-MAS70 on ATP, the group of precursors that is preferentially imported by this subcomplex is prone to aggregation and thus bound to cytosolic chaperones. As already mentioned above, this group includes ADHIII, cytochrome cl,F$, and the ADP/ATP carrier (7, 9, 103, 104). It is not known whether the two subcomplexes function separately or together and there are currently two different rnodels regarding the association of the two receptor subcomplexes MASZO- MAS22, and MAS37-MAS70. The first model suggests that they act independently; hence for a given precursor the majority of that precursor will bind preferentially to one of the subcomplexes. The second model States that the two subcomplexes function in a joint fashion whereby al1 precursors use the same pathway and several of the receptor subunits are necessary for the recognition of one precursor. Examples to substantiate the second model include the irnport of ADHlll and FIP. Studies with these precursors found that they are bound to both subcomplexes; the signal sequence is bound to the MAS20-MAS22 subcomplex and the mature part of the protein is bound to MAS37-MAS70. Another example is given by the ADP/ATP carrier. Although this protein does not have an amino-terminal signal sequence, but rather contains the targeting information within its mature region, it was also found to bind to both receptor subcomplexes. Since the MAS20-MAS22 subcomplex binds precursors in an ATP-independent fashion, only precursors that are imported in a manner that does not require extramitochondrial ATP interact with this subcomplex. The two-hybrid systern was used to show that the two subcomplexes associate loosely with one another

(los) and that the cytosolic domains of MASZO and MAS70 interact (7).

Deleting the genes encoding the receptors prevents transport of different precurson to varying extents. Hence, a given precursor may be presented to the membrane system by different routes although it may have a preference for a certain receptor or receptor subcomplex. Deleting the genes encoding receptor subunits MAS20, MAS37, and MAS70, respectively, results in strains that are still viable; however, deleting the genes encoding both MAS37 and MAS70 is lethal. MAS22 is the only subunit that is essential for viability since deletion of this subunit alone is lethal. This finding may reflect the fact that the acidic domain may bind the presequence, and the fact that MAS22 spans the OMM and thus constitutes part of the transport system across the OMM (7,941.

The translocation channel and the mitochondrial chaoerones W hen conside ring the exposed charges of the mitochondrial signal sequence and the hydrophilic side chahs of the partially unfolded polypeptide, one may wonder how the protein can cross the hydrophobic phospholipid bilayer. The answer is that the translocating protein passes through a hydrophilic, hetero- oligomeric transmembrane channel that is composed of integral membrane proteins. How exactly this translocation takes place is not understood, however, the channel is opened across the membrane by the signal sequence. Lateral movement through the membrane is mediated by the stop-transfer of signal-anchor sequences. lmport channels span both the IMM and OMM and they are situated at "contact sites" where the two membranes are in close contact with each other. The channel in the OMM consists of approximately three subunits; TOM42 (formerly called ISP42), an essential protein, as well as TOM6 and TOM8. The channel in the IMM consists of TIM17 (formerly MIM17), TIM23, TIM6 (formerly MASG), and TIM44 (formerly MIM44 or ISP45). TIM44 is bound to mhsp70 on the trans

side of the membrane, and the latter in turn is bound to its CO- chaperone GrpEp. The mhsp70-GrpEp-TIM44 complex interacts loosely with the import channel in the IMM and operates as a "translocation motor" that binds the emerging signal sequence and "pulls" the precursor across the membranes. Translocation across the IMM membrane requires intramitochondrial ATP and is dependent on the electrochemical potential for most precursors (1 06-1 0).

Precursors that have been translocated into the matrix are partially unfolded and have to be refolded in order to be able to assume their biological functions. Some small, monomeric proteins have the ability to fold rapidly by themselves. However, others require the help of mitochondrial chaperones (yllp 112). Hsp60 and cpnl0 forrn a complex that operates in an ATP-dependent manner to refold translocated proteins and assemble them into multimeric enzyme complexes where appropriate. In addition, mhsp70 also aids in the folding of proteins as well as in binding misfolded proteins and delive ring them for degradation (1 3-1 6).

1lm The import pathway into the IMS, which can be reached by several possible routes, is not well understood. Cytochrome c of the cytochrome oxidase complex, for example, spontaneously inserts itself into the OMM (l17) and is then pulled across the OMM by the enzyme cytochrome c heme lyase which catalyzes the covalent attachment of heme to cytochrome c in the IMS (6. 117). This protein does not require the aid of chaperones or receptors and is therefore imported in a manner that requires neither ATP nor a membrane potential.

A different route is taken by the precursors of cytochrome cl and cytochrome bt which contain bipartite signal sequences, cieaved

in two steps (118, 119). There are currently two views concerning how these proteins reach their final destination. In the first one, a stop-transfer pathway, the proteins are transported across the OMM and then become anchored to the outer face of the IMM by their signal sequence which is subsequently cleaved off. After rernoval of the amino-terminal part of the leader sequence the proteins remain bound to the outer face of the IMM or are released into the IMS. The carboxy half of the signal sequence thus functions as a sorting signal that prevents transport of the intermediates into the matrix

and it is cleaved off by a peptidase in the IMS (118. lzo). The second pathway is referred to as the conservative route in which the precursors are first irnported into the matrix where the signal sequence is cleaved for the first time and where they interact with

hsp60 (1219 122) and mhsp70 (123). In this pathway, the carboxy- terminal end of the signal sequence functions as an export signal that resembles that of bacteria and it targets the intermediates from the matrix across the IMM into the IMS. Additionai proteins that are conservativeiy sorted include the ISP of the bcl cornpiex and the ATPase subunit 9. Cytochrome c heme lyase (CCHL) is sorted in yet a different manner from cytochromes cl and b2. This protein does not contain a presequence and is translocated selectively across the OMM by using

the import channel (124). CCHL is believed to be loosely folded after synthesis since it does not require ATP or a membrane potential for import and therefore does not interact with cytosolic chaperones.

lm-wrt into the IMM There are two known routes for a protein if its destination is

the IMM, the "direct" route and the "detour" route (101). In the direct route the protein is transported across the OMM and directly inserted into the IMM from the IMS side. An example for this import pathway is the ADP/ATP carrier (125). The import of the ADP/ATP carrier requires extramitochondrial ATP, hence it probably interacts with a cytosolic chaperone. lmport across the OMM is dependent on a proteinaceous component on the surface of this membrane, most probably a receptor, and the protein import channel. In addition, insertion into the IMM requires a membrane potential. There is no requirement for matrix ATP, an indication that the protein does not pass through the matrix to reach its final destination in the IMM.

The detour route resembles the import pathway into the matrix or the conservative pathway taken by some proteins of the IMS. In this pathway, proteins are imported into the matrix first where the matrix signal sequence is cleaved off; this import into the matrix occurs in a manner that is dependent on receptor interaction, extra- and intramitochondrial ATP and the membrane potential. Proteins are then assembled with their partner subunits, if appropriate, and inserted into the IMM from the matrix side. Proteins that follow this indirect route include cytochrome oxidase subunit IV in yeast,

ATPase subunit 9, and the Rieske ISP of the bel complex (101, 1269

127). jmwn into the OMM lmport into the OMM is the simplest pathway, however the mechanism of import is not well understood. Transport into the OMM is receptor dependent for most proteins and occurs by direct insertion of the protein into the membrane. Thus import into the OMM requires extra-, but not intramitochondrial ATP; neither a membrane potential nor intramitochondrial components of the import machinery are required (101. 128). It is therefore not understood how transport into the OMM is energized. There is some preliminary evidence to suggest that proteins are driven across the membrane by a two-step sequential binding. In the first step the presequence associates with a receptor on the surface of the OMM and in the second step, which drives the translocation of the protein across the membrane, the precursor binds on the trans side of the OMM (lin129). Details about the signals that encode the pathway into the OMM are unknown.

Objectives of the research Abundant research has been performed on those subunits of complex III that contain catalytic centers. On the other hand, knowledge about the functions of the supernumerary subunits is still limited. The Qcr7 protein was chosen as the focus of this study based on the knowledge that subunit 7 is crucial for the functioning of complex 111 (33). In addition, study of this subunit was appealing because it constitutes the homolog to the fourth polypeptide of prokaryotic bci complexes which consist of only four subunits: the three catalytic subunits cytochrome b, cytochrome c or f, the Rieske ISP, and the homolog to the Qcr7 protein in yeast.

In this study I focused on the role of the amino-terminal region of subunit 7. The N-terminus of subunit 7 is thought to face the matrix side of the IMM in yeast (1301, and in the homologous subunit of beef heart mitochondria (131). Because proteolysis of the N-terminal seven amino acids of the beef heart mitochondrial counterpart leads to a decreased H'/e- ratio, Cocco et al. (131) suggested that this subunit is involved in proton uptake from the matrix with subsequent transfer of these protons to the hypothetical ubiquinone binding pocket at center N. Since proteolysis also resulted in a small amount of cleavage from the Rieske ISP, it cannot be ruled out that the ISP is in fact responsible for the decreased H+/e- ratio. Nevertheless, based on the available information of subunit 7, 1 decided to investigate the function of the Qcr7 protein with an emphasis on the amino-terminal region. CHAPTER 2

Inactivation of the QCR7 Gene

A synopsis of the work presented in this dissertation has been accepted for publication in The Journal of Biologieal Chemistry. CHAPTER 2 Inactivation of the QCR7 Gene

2.1 . INTRODUCTION

The yeast Saccharomyces cerevisiae is an ideal eukaryotic microorganism for the study of biochemical pathways and is frequently used as a model system for investigating the function of proteins. Yeast have greater genetic complexity than bacteria. However, they share many of the technical advantages that have permitted rapid progress in the understanding of the molecular genetics of . Some of these advantages include the availability of strains with multiple auxotrophic markers, rapid growth, a well-def ined genetic system, a highly versatile DNA transformation system, and the ability of the organism to exist both in the diploid and haploid state. In addition, replica plating in yeast can be used as a quick preliminary identification of mutants (i32). The ability of wild type yeast to grow aerobically and anaerobically allows for the rapid preliminary identification of mutants defective in aerobic growth and makes this organism very attractive for the study of mitochondrial assembly and function.

Many genes have been inactivated in yeast and the resulting phenotypes have contributed significantly toward understanding the functions of proteins in vivo. All the genes encoding nuclear subunits of ubiquinol-cytochrome c oxidoreductase (complex III or bc, complex), including the QCR7 gene for subunit 7, have been In this study I attempted to determine the precise function of subunit 7 (the Qcr7 protein) by the expression of a series of mutated Qcr7 proteins in the yeast S. cerevisiae. To accomplish this, I had to first create a strain in which the chromosomal copy of the QCR7 gene was inactivated so that a Qcr7 protein is not synthesized. This chapter describes the disruption of the QCR7 gene to create the qcr7A:LEUZ allele-containing respiration deficient strain, YSM- qcr7A, which was used for the expression studies outlined in the following chapten. 2.2. MATERIALS AND METHODS

Ma terials Saccharomyces cerevisiae strain W303-1 B (MA Ta, ade2- 7, his3-71, 15, ura3-7, leu2-3, 712, trp7-7, can7-700), which was used as the parent strain throughout this study, and the pJJ250 plasmid were kind gifts from Dr. Jim Friesen's laboratory (Hospital for Sick Children, Toronto). The rho- strain (MATa, ural [rho-] [CX1 R ES 1401) was purchased from ATCC (strain number 42209; donated by Dr. L. A. Grivell, University of Amsterdam). Escherichia coli strain JMED3 was obtained from Promega (Madison, WI). Restriction enzymes, the T7 sequencing kit, and NlCK gel filtration columns were purchased from Pharmacia (Montreal, PQ). All materials for the preparation of yeast and bacterial media were obtained frorn Difco (Toronto, ON) or ICN (Costa Mesa, CA). The pCR II vector was purchased from lnvitrogen (San Diego, CA). All remaining chernicals were purchased from Sigma (St. Louis, MO) or BDH (Toronto, ON). Alkaline phosphatase-conjugated AffiniPure rabbit anti-chicken IgY (IgG) (H+L) was obtained from Jackson lmmuno Research Laboratories, Inc. (West Grove, PA). Random primed labeling kits were from Boehringer Mannheim (Mannheim, Germany), and Hybond-N' nylon membranes and radionucleotides were from Amersham (Arlington Heights, IL). BiomaxTM MS x-ray film was purchased from Kodak (Rochester, NY). Nitrocellulose membranes were purchased from Mandel Scientific Company Ltd. Serocluster vinyl plates used for ELISA assays were obtained from Costar (Cambridge, MA). NBT (p-nitro blue tetrazolium chloride) and BClP (5-bromo-4-chloro-3-indolyl phosphate-toluidine) were purchased from BioRad.

2.2.1. Disruption of the chromosomal copy of the QCR7 gene

The QCR7 gene was amplified from genomic yeast DNA (136) using primers corresponding to sequences upstream (5'- ctgtaattaaacgttccagaaag-3') and downstream (5'- cgggttgtgtgttcgtggtga-3') of the coding sequence. Amplification was perforrned for 35 cycles with a denaturation time of 1 min at 94<, an annealing time of 1 min at 65oC, and a 1 min extension at 720C. The PCR product was subcloned into the pCR II cloning vector by using the 3' adenylic acid overhang created by Taq DNA polymerase and giving pCR II-QCR7. Eight clones were used to confirm the sequence and orientation of the insert by dideoxy chain termination sequencing.

A Pvull fragment (2300 bp) containing the LEU2 gene was purified from plasmid pJJ250 and blunt-end ligated into the Hincll site of pCR II-QCR7 to give pCR Il-Aqcr7 in which the qcr7 gene is disrupted by the LEU2-containing fragment. The vector pCR Il-dqcr7 was digested with Bgll and Apal to liberate the DNA fragment containing the disrupted qcr7 gene (Fig. 2-1). Ten micrograms of digested DNA was used to transform yeast strain W303-1B (137). Appropriate integrative transformation was confirmed by phenotypic analyses and by Southern blot analysis of genomic DNA from Leu+ transformants. 1acr7, LEM qcr7 C

KR II vector a qcr7codng region vector pJJ250 LEU-containing fragment

Fig. 2-1. DNA fragment used for disruption of the chromosomaI QCR7 gene. The triangles represent the direction of transcription. 2.2.2. Confirming inactivation of the QCR7 gene

Selection of diploids Diploids were obtained by inoculating YPD medium simultaneously with cells from a rho- strain and cells from a Leu+ transformant containing the putative qcr7 gene disruption. The culture was grown overnight, cells were washed twice with water, diluted, and diploids were selected on SD medium lacking leucine and containing glycerol and ethanol as the sole carbon sources.

Northern and Southern blotting Total yeast RNA was prepared by growing a 25 mL culture overnight, harvesting the cells and resuspending the pellet in 2 mL of AE buffer (50 mM NaOAc, pH 4.8; 10 mM EDTA). The suspension was vortexed after the addition of 200 pL of 10% SDS, and again after the addition of 450 pL of phenol saturated with AE buffer. The suspension was vortexed three times during a five minute incubation at 650C. The mixture was extracted with phenol-chloroform (1:l) and RNA was precipitated from the aequeous phase with 2.5 volumes of ethanol. The pellet was washed with 70% ethanol, dried, and resuspended in TE buffer (pH 7.0). For the Northern analysis, 30 pg of RNA was precipitated with 2.5 volumes of ethanol-0.1 volumes of 5 M LiCI. The RNA pellet was dissolved in 5 IL of 25 mM EDTA-0.1 % SDS. A DNA fragment encompassing the entire QCR7 coding region

(48) was amplified by PCR and subsequently radiolabeled with [a- WPI~CTPusing a random primed labeling kit; unincorporated nucleotides were removed from the labeled probe by gel filtration through a NlCK column. Northern blotting was performed as

described by Fourney et al. (1 38). Following hybridization, the membranes were washed twice at 650C for 30 min in 2x SSPE (20xSSPE: 3 M NaCI, 0.2 M NaH,PO., 0.02 M EDTA, adjusted to pH 7.4 with Na0H)-0.1% SDS and subsequently exposed to x-ray film.

Ten micrograms of yeast genomic DNA, isolated as described by Strathern and Higgins (136), was digested with appropriate restriction endonucleases and electrophoresed through a 0.6% agarose gel. The DNA was denatured in 1 M NaOH for one hour and subsequently transferred in this solution to a Hybond-Ne nylon membrane as outlined by Sambrook et al. (139). Prehybridization was performed at 420C for a minimum of four hours in 40% formamide- 1% SDS-Sx SSPE-0.5% skim milk powder and 250 rng/mL of denatured, sonicated salmon sperm DNA. A DNA fragment containing the QCR7 coding region was radiolabeled with [a-IPI~CTP as described previously. Hybridization was carried out at 42oC overnight in a solution containing 40% formamide-1 % SDS-5x SSPE- 0.5% skim milk powder-10% dextran sulfate. Following hybridization, membranes were washed twice at 650C for 30 min in 2x SSPE-O. 1 % SDS and were exposed to x-ray film.

Isolation of yeast mitochondria Cultures were grown for two days in SD media containing different carbon sources. Mitochondria were isolated essentially as outlined by Guthrie and Fink (140) with the substitution of breaking buffer (0.6 M sucrose, 20 mM HEPES-KOH, pH 6.5, 0.1% BSA, 1 mM PMSF) for mitochondrial isolation buffer. In short, cells were pelleted, washed with water, and then prepared for cell wall digestion by incubation at 30C in 0.1 M Tris-SOI (pH 9.4)-10 mM DTT at a concentration of (0.5 g cell wet weight)/mL. After this preliminary incubation, cells were washed with 1.2 M sorbitol and incubated again at 30.C for cell wall digestion in spheroplasting buffer (1.2 M sorbitol, 20 mM KPi, pH 7.4) containing zymolyase (Img/g cell wet weight) and PMSF (1 mM). Spheroplasts were disrupted by homogenization with a Dounce steel homogenizer in breaking buffer (0.6 M sucrose, 20 mM HEPES-KOH, pH 6.5, 0.1% BSA, 1 mM PMSF). Following disruption of the spheroplasts, mitochondria were isolated by differential centrifugation (two rounds of 3,000~9 followed by 10,000xg whereby mitochondria are pelleted at the high speed spin). The purity of the mitochondria was determined by measuring the specific activity of complex IV.

Western analysis Following SDS-PAGE using gels containing 16% polyacrylamide, proteins were transferred to nitrocellulose membranes for two hours at 55 volts at 40C in lx running buffer (0.025 M Tris, 0.1 9 M glycine) containing 0.1 % SDS and 20% methanol. Biots were subsequently blocked for one hour with Blotto (10 mM Tris-CI, pH 7.5; 150 mM NaCI; 0.05% Tween 20) containing 2% gelatin, and then incubated overnight with primary antibody in Blotto containing 1% gelatin. Membranes were washed 4x30 min in Blotto and then incubated in Blotto containing 1% gelatin for one to two hours with a rabbit anti-chicken antibody coupled to alkaline phosphatase. Membranes were washed 4x1 5 min in Blotto and proteins were visualized using NBT and BClP as substrates in 0.1 M NaHC0,-1 mM MgCl,.

2.2.3. Raising an antibody against the Qcr7 protein in chicken

The following peptide from the carboxy-terminus of the Qcr7 protein was chosen as antigen and synthesized by the Alberta Peptide Institute:

keyhole limpet -AAKEKDELDN IEVSK-COOH

Isolation of anti-Qcr7 antibody from rabbit and chicken The above peptide which is conjugated to keyhole limpet hernocyanin at the amino-terminus, was used to raise an antibody in rabbit (performed by the Centers of Excellence, Canada) and in chicken. To raise the antibody in chicken, 250 pg of the peptide was solubilized in 500 pl PBS, mixed with an equal volume of Freund's Complete adjuvant and injected into the chest muscle of a chicken. Booster shots containing 250 pg of peptide solubilized in 500 pL PBS and mixed with an equal volume of Freund's lncomplete adjuvant were given one and two weeks after the initial injection. Antibodies were purified from egg yolks starting two weeks after the second booster injection.

Three egg yolks (50 mL) were aspirated through a syringe (without needle) and diluted in three volumes of 0.1 M sodium phosphate buffer, pH 7.6. One volume of a PEG8000 solution (0.1 75 g PEG8000/mL of 0.1 M sodium phosphate buffer) was added. After 10 min the mixture was centrifuged at 5000xg for 25 min at RT. The resultant supernatant was filtered through 3 mm filter paper and protein was precipitated by the adding PEG8000 (0.085 g/mL) and incubating for 10 min at RT. This suspension was centrifuged at 5000xg for 25 min at RT. The pellet was dissolved in 2.5 volumes of 0.1 M phosphate buffer and proteins were precipitated by adding PEG8000 (0.1 2 g/mL) and incubating for 10 min at RT. The precipitate was recovered by centrifugation at 5000xg for 25 min at RT and the pellet was dissolved in 0.25 volumes of 0.1 M phosphate buffer and cooled to OoC on ice. After the addition of an equal volume of cold 50% ethanol, the mixture was left on ice for approximately four hours and then centrifuged at 10,000xg for 25 min at 4oC. The final pellet was dissolved in 0.25 volumes of 0.1 M phosphate buffer and dialyzed overnight at 40C against 0.1 M phosphate-0.1 5 M NaCl (pH 7.6). After the addition of 0.02% NaN3 as preservative, the antibody-containing fraction was aliquoted and stored at -700C. The immune reactivity was tested by performing an enzyme-linked immunoadsorbent assay (ELISA procedure was devised by the Alberta Peptide lnstitute at the Department of Biochemistry, University of Alberta, Edmonton, Alberta). 2.3. RESULTS

Disruption of the QCR7 gene As a first step in the study of mutant Qcr7 proteins, I constructed a yeast strain in which the chromosomal QCR7 gene was inactivated. The chromosomal QCR7 gene was replaced by integrative transformation of a DNA fragment containing the qcr7 gene interrupted by the LEU2 gene (Fig. 2-1). lntegrative transformation in yeast generally occurs by homologous recombination, hence Leu' transformants could result not only from replacement of the QCR7 gene by the qcr7:LEUZ sequence, but also by recombination of the transforming DNA at the chromosomal leu2 gene; in addition, Leu+ transformants could result from random recombination elsewhere in the genome. To eliminate the seléction of a strain in which the qcr7:LEUZ fragment has recombined at a site other than the genomic QCR7 locus, I first carried out a phenotypic analysis of the Leu+ transformants.

The QCR7 gene is required for respiration-dependent growth, that is growth on non-fermentable carbon sources (33). 1 therefore patched Leu' transformants ont0 medium containing glycerol and ethanol as carbon sources. Transformants that failed to grow on this medium were pet- and therefore candidates for containing the qcr7 gene disruption. However, mutations in the mitochondrial genome can also lead to a pet- phenotype, hence I had to ascertain that the respiration-deficient Leu+ transformants had a wild type mitochondrial genome. This was performed by mating the Leu4 transforrnants with a rho- strain (a strain which contains large deletions in its mt DNA) and testing the resulting diploids for their ability to grow on ethanoVglycerol medium. Diploids that were able to grow on this medium had to contain an intact mitochondrial genome.

Finally Leu+, respiration-deficient transformants that contained intact mitochondrial DNA were subjected to Southern blot analysis to confirm that the QCR7 gene had been replaced by the qcr7A:LEUP allele. A blot of genomic DNA was probed with an [a- "PIdCTP-labeled DNA fragment encompassing the QCR7 coding region. The hybridization pattern of DNA that had been digested with Hindlll confirmed that a 2600 bp fragment containing a Hindlll site had replaced the chromosomal QCR7 gene. Only one fragment containing the uninterrupted QCR7 gene is seen in the lane containing DNA from the parental strain, W303-1 B (Fig. 2-2, panel B, lane 1). This is as expected since the QCR7 coding region does not have a Hindlll recognition site (Fig. 2-2, panel A). On the other hand, in the lane containing DNA from the mutant strain the large fragment is replaced by two smaller fragments since the coding region of the qcr7 gene was interrupted by the LEU2-containing fragment which introduced a new Hindlll site (Fig. 2-2, panel B, lane 2). In summary, the phenotypic and Southern analyses confirmed that I had obtained a strain in which the chromosomal QCR7 gene had been replaced by the qcr7d:LEUZ allele, thus creating the new strain YSM-qcr7A. Fig. 2-2. Southern blot analysis of the parental strain W303-1 B and strain YSM-qcr7~with the qcf7d:LEU2 allele. Yeast genomic DNA (1 0 pg) was digested with Hindlll (refer to panel A) and electrophoresed through a 0.6% agarose gel. The DNA was denatured in 1 M NaOH and transferred to a Hybond-N* nylon membrane, followed by fixation of the DNA to the membrane by treatment with ultraviolet light (254 nm) for 10 min. Hybridization was carried out at 420C with a fragment encompassing the QCR7 coding region that had been labeled with [a-"PIdCTP by a random priming method. Panel B: Lane 1; DNA from the parental strain, W303-1 B; lane 2; DNA from the strain containing the qcr7A:LEUP allele, YSM-qcr7A. Hind II Qm7 1 . 4 wiid type lows

Hin d lIl 2575 bp Hindi I I

0 no~codngregions iqstiearnanddownstrearn of chrmasomal QCR7gene 0 QCR7obding region vector pJJ250 LEU'-containing fragment Confirmation of the qcr7 gene inactivation The DNA construct (Fig. 2-1) used to disrupt the QCR? gene included the entire coding region of this gene. Hence, it is conceivable that in the strain YSM-qcr7~ a fusion transcript consisting of the 5' end of the qcr7 gene and the LEU2 gene is synthesized which results in a fusion protein. Such a protein might have a dominant negative effect. To rule out the presence of such a fusion transcript, Northern blotting was performed. From Fig. 2-3 it is evident that no such transcript is present in the lane containing RNA from strain YSM-qcr7A (lane l), whereas a transcript of expected size (approximately 680 bases) is present in the wild type parental strain (lane 2). This indicates that a stable fusion transcript is not synthesized and further substantiates the QCR7 gene inactivation.

An antibody was raised in chickens to a carboxy-terminal peptide of the Qcr7 protein. The antibody was purified from egg yolks and the titer was compared to that of pre-immune egg yolks by means of an ELISA. The preimmune egg yolks did not react with the peptide (the peptide being the same one that was used to raise the antibody) used in the ELISA, whereas a substantial reaction of the post-immune egg yolks could be seen with the peptide. The antibody was authenticated by immunoblotting of SDS-PAGE fractionated mitochondrial proteins (Fig. 2-4). A band of expected size corresponding to the 14.5 kDa Qcr7 protein is seen in the wild type parental strain (lane 2). As anticipated, a Qcr7 protein is not present in strain YSM-qcr7~ (lane l), which confirms that the protein recognized &y the antibody is the Qcr7 protein rather than another protein of similar size. Fig. 2-3. Northern blot analysis of RNA isolated from the parental strain W303-1 B and YSM-qcr7~. Northern blot of total RNA (30 pg) isolated from the parental strain W303-1B (lane 2) and strain YSM-qcr7a (lane 1). Total RNA was separated on a 1% agarose gel containing 1.8% formamide and subsequently transferred to a nylon membrane. The DNA probe consisted of the QCR7 coding region which was radiolabeled with [a-"PIdCTP by using a random prirned labeling kit. Fig. 2-4. Western blot analysis of mitochondrial protein from YSM-qcr7a and the parental strain W303-1 B. Mitochondrial proteins (1 00pg) were dissolved in SDS-PAGE buffer containing D7T and heated for three minutes at 950C. Samples were run on a 16% polyacrylamide gel and then transferred to a nitrocellulose membrane. Blots were probed with a polyclonal antibody (dilution 1 :100) raised against a c-terminal peptide of the 1 4 kDa subunit. Lane 1 : protein from YSM-qcr7~;lane 2: protein from the wild type parental strain W303-1 B. 2.4. DISCUSSION

This chapter describes the inactivation of the QCR7 gene by replacement of the functional chromosomal copy with a version of the gene that was disrupted by the LEU2 marker. Since the qcr7 gene was disrupted rather than replaced, I had to ensure that a fusion protein was not synthesized. Northern blot analysis (Fig. 2-3) demonstrated that a stable transcript or fusion transcript is not synthesized. Western blot analysis indicates that a fusion protein which contains an in frame fused Qcr7 protein C-terminus is not synthesized either. Whether or not a fusion protein is synthesized, that contains an out of frame fused C-terminal end of the Qcr7 protein, cannot be determined as the antibody epitope is located in the C-terminus. However, it is extremely unlikely that such a fusion protein would be functional and/or stable. Hence, the gene encoding subunit 7 of the bci complex has successfully inactivated and the newly created strain, YSM-qcr7A, which contains the qcf7A:L EU2 allele, was used for further analysis of the Qcr7 protein by site- directed mutagenesis of plasmid-borne qcr7 genes.

Since recombination and gene conversion could occur between the chromosomal gene and a plasmid-borne version of the gene, the mutant strain was always propagated in a selective manner (lacking leucine) when transformed with a version of the qcr7 gene on an expression plasmid. This ensured that the chromosomal gene was qcr7A:LEUZ. Reversion of YSM-qcr7~back to wild type has not been found to be a cause for concern. CHAPTER 3

Mutagenesis of the QCR7 Gene CHAPTER 3 Mutagenesis of the QCR7 Gene

3.1 . INTRODUCTION

With the QCR7 gene inactivation verified it was now possible to proceed with in vitro mutagenesis of the gene and express the encoded mutant Qcr7 proteins in the newly created strain, YSM- qcr7A. This study focused on the amino-terminus of the Qcr7 protein and three functions were investigated. The first was based on studies performed on the bc, complex of beef heart mitochondria in which the purified complex was solubilized and subjected to protease digestion. Proteolysis resulted in partial cleavage of some or al1 of the following subunits, depending on the experimental conditions: core protein 2, the Rieske iron-sulfur protein, the 6.4 kDa subunit, the 9.2 kDa subunit, and 7 to 11 residues from the N- terminus of the 13.4 kDa subunit (the homologue of the yeast 14.5 kDa Qcr7 protein). When complex III was reconstituted into phospholipid vesicles after proteolysis and tested for redox and protonmotive activities, it was found that there was a significant decrease in the H'/e- ratio. This decrease in the H+/e- ratio was still present under conditions where the 13.4 kDa subunit had been cleaved but, of the other subunits, only marginal cleavage of the iron-sulfur protein had occurred. The authors concluded from this observation that the amino-terminus of the 13.4 kDa subunit is involved in binding protons from the matrix phase and in promoting conduction of these protons to the ubiquinone-binding pocket (131. 741). It was furthermore concluded that the amino-terminus of the 13.4 kDa subunit protrudes into the inner space of the phospholipid vesicles since there was no proteolytic cleavage of this subunit under conditions where the complex was inserted into phospholipid vesicles prior to digestion. This finding correlates with the predicted topology of the 13.4 kDa subunit in vivo where it is believed to be a monotopic protein that is bound to the surface of the IMM on the matrix side where it presumably interacts with the membrane and other subunits of complex III (Fig. 3-1 ) (23. 65). Similar to its beef heart rnitochondrial homologue, the N-terminus of the yeast Qcr7 protein is also believed to be located at the matrix side (66,130, 142).

The second putative role for the Qcr7 protein that I investigated dealt with mitochondrial protein targeting and import. The rnechanisms for mitochondrial targeting of the Qcr7 protein are unknown. Of the nine nuclear encoded subunits of the ber complex, five have been shown to contain cleavable mitochondrial signal sequences (2. 36-39, 43) of varying lengths: core proteins 1 and 2 have cleavable signal sequences 1 7 and 16 residues long, respectively; cytochrome cl and the ISP have bipartite signal sequences of 61 and 30 amino acids, respectively; subunit 6 has a presequence of 25 amino acids. Subunits 7, 8, and 9 only undergo cleavage of the initial methionine and this is presumably also the case for subunit 10. Although the mechanisms of import of several of the components of complex III in yeast have been studied extensively, I M S

IMM

Fig. 3-1. Helical wheelplots and folding pattern of the 13.4 kDa subunit of beef heart mitochondrial complex III; as predicted by Link et al. (23). Hydrophilic regions are located in the matrix while helices are predicted to be located in the IMM. The amphipathic helix at the immediate N-terminus is predicted to be located on the matrix side of the IMM. nothing is known about how subunits 7, 8, and 9 are imported into mitochondria.

Core protein 2 follows the general energy-dependent import

pathway into the matrix where its signal sequence is cleaved off (2). It is not known how core protein 1 is imported, however, it most probably follows the same pathway since it also contains a cleavable signal sequence and its final destination is in the rnatrix with a small fraction of the protein bound to the IMM. The ISP is irnported into the matrix in a receptor and membrane potential- dependent manner where its signal sequence is cleaved for the first time. The protein is then exported to the outer face of the IMM in an energy-dependent manner and cleaved by a protease for the second time (2. 42). There are currently two theories on the import pathway of cytochrome cl: In the first one, a stop-transfer pathway, the precursor is transported across the OMM and then becomes anchored to the outer face of the IMM by its signal sequence which is subsequently cleaved. After removal of the amino-terminal part of the leader sequence the protein remains bound to the outer face of the IMM or is released into the IMS. The carboxy half of the leader sequence thus functions as a sorting signal in this pathway and prevents passage of the intermediate into the matrix. Cytochrome ci is cleaved for the second time by a peptidase in the IMS (118). The second pathway is referred to as the conservative route and the precursor is first imported into the matrix according to the general pathway where it interacts with hsp60 (ils) and mhsp70 (tzi). In this pathway the carboxy terminal end of the signal sequence functions as an export signal that resembles the bacterial export signal and targets the intermediates from the matrix across the IMM into the IMS.

As already mentioned, the Qcr7 protein does not contain a cleavable amino-terminal signal sequence and it is not known which pathway it follows to reach its final destination in the IMM. However, it probably follows the general matrix pathway, since it requires extra- and intramitochondrial ATP as well as the membrane potential for import (1 42). Furthermore, there is circumstantial evidence that the Qcr7 protein does not follow the import pathway of proteins without a cleavable signal sequence such as the

ADP/ATP carrier (143). In addition, subunit 7 is believed to assemble into a subcomplex with cytochrome b and the 1 1 kDa subunit (33. 60). Although it is not certain at present whether this subcomplex assembles directly in the membrane or first in the matrix before insertion into the IMM, the latter hypothesis seems to be favored according to the findings of a recent study. Japa et al. (142) have shown that the Qcr7 protein is not bound to the membrane when performing in vitro import studies with mitochondria from a strain lacking cytochrome b. When performing the same experiment with mitochondria from wild type yeast, however, the Qcr7 protein was bound to the IMM through protein-protein interactions. Hence, the formation of a subcomplex containing cytochrome b, the 14 kDa and the 11 kDa subunits probably occun in the matrix and a targeting sequence must therefore be contained in the Qcr7 protein. However, it cannot be ruled out completely that the Qcr7 protein is imported into the IMM without passing through the matrix and, in the absence of cytochrome b, passes on to the matrix.

The third possible function for the Qcr7 protein that I investigated was the role of the subunit 7 N-terminus in the assembly of ubiquinol-cytochrome c oxidoreductase. Previous studies indicated that deletion of the QCR7 gene resulted in low steady-state levels of the ISP, cytochrome b, and the 1 1 kDa protein

(33. 60). Due to a post-translational interdependence of cytochrome b, the 14 kDa subunit, and the 11 kDa subunit, which has been demonstrated a number of times, it is believed that a subcomplex consisting of these three components is formed prior to assembly of these subunits into a functional complex 111 (33. 60-63). The ISP does not seem to be a component of this subcomplex since a mutant strain containing a deletion of the gene encoding the ISP does not contain lowered steady-state levels of complex III subunits other than the ISP itself (60).

To test the involvement of the Qcr7 protein N-terminus in proton pumping, rnitochondrial targeting, and assembly of complex 111, 1 mutated the QCR7 gene in regions corresponding to the following residues (refer to Fig. 3-2). A previous study by lmoto et al. suggested that a number of serine and threonine residues of the nicotinic acetylcholine receptor probably form a ring or hydrophilic pore structure that comes into close contact with permeating cations and may determine the selectivity of the channel (165). In addition, a study performed by Yool and Schwarz also implicated a role for serine and threonine residues in the formation of a K+

channel (166). Similarly, in solving the crystal structure of the beef heart mitochondrial cytochrome oxidase Tsukihara et al. found that one of the channels involved in H+ pumping terminated at two serine

residues that may be involved in hydrogen bonding (168). Based on these studies and the knowledge that mitochondrial signal sequences are enriched with hydroxylated residues (939 941, such residues in the N-terminus were targeted for mutagenesis to investigate their role in import and in the possible construction of a H+ channel. Since, however, mutating Ser-4 and Thr-6 did not affect mitochondrial import or H+ translocation, and since a protein with an N-terminal truncation of 20 residues is imported into the mitochondria to the same extent as a protein with a truncation of 7 residues, no additional hydroxylated residues were mutated (see Chapter 4).

Four mutants with Qcr7 proteins truncated by 7, 10, 14, and 20 residues from the mature protein (that lacks Met-1, due to cleavage) N-terminus were also tested for their role in H+ translocation, in accordance with the results from the studies in beef heart mitochondria. Since the yeast 14 kDa subunit contains a longer N- terminal extension than its beef heart homologue, deletion of 20 residues was comparable to the proteolytic cleavage (7 to 11 residues) that occurred from the N-terminus of the beef heart subunit (131, 141) (Fig. 3-2). Contrary to the studies performed in beef heart mitochondria, the introduction of deletions into the plasmid-borne QCR7 gene enabled me to study the involvement of the .L L* .L * -L* + A Y:EQSF~SIARIGDYILKSPVLSKLCVPVANQFI B: AGRPAV S ASSRWLEGIRKWYY

Y:NLAGYKKLGLEFDDLLAEEuPIMQTALRRLPED B:NAAGFNKLGLMRDDTI HENDDVKEAIRRLPEN

* A** * * **+ ** X -ic Y: ESYARAYRI IRAHQTELTHALLPRNQWIKFFQE B:LYDDRVFRIKRALDLsMRQQILPKEQWTKYEE

Fig. 3-2. Alignment of yeast Qcr7 protein and its 13.4 kDa homologue from beef heart mitochondrial cornplex III; adapted from Link et al. (23). Y = yeast Qcr7 protein (1 4.5 kDa sobunit 7); 8 = beef heart 13.4 kDa subunit. * = conserved residues; + and - indicate conserved charges, and A indicates an exchange of one aromatic residue for another. Truncations of 7, 10, 14, and 20 residues from the N-terminus of the mature Qcr7 protein (which lacks Met-1) are indicated by arrows. Individual residues that were substituted as a result of mutagenesis are underlined. Qcr7 protein N-terminus (in events prior to and involving the assembly of the complex) in vivo rather than in vitro.

The Qcr7p-~7displays a phenotype that is comparable to the wild type at 30°C. Hence, a number of point mutations were introduced into the QCR7 gene in the context of a a7 deletion. Arginine-10 was targeted for two reasons. The first one, is to test its involvement in mitochondrial targeting since arginines are typical for mitochondrial signal sequences. The other reason for mutating Arg-1 O, which was also the reason for mutating Asp-13, was to find out whether these amino acids might be important for assernbly by providing contact with another subunit through salt- bridge formation. Full-length mutants containing Qcr7 proteins with substitutions for Arg-1 O and Asp-13 were created to investigate the possible role of these residues in import and assembly of the Qcr7 protein.

Of the other substitutions shown in Fig. 3-2, substitutions in Gln-31 were created to test the earlier hypothesis that the Qcr7 protein is involved in ubiquinone-binding. Although this hypothesis was later corrected in the literature, as it was based on a mistake, mutants with Qcr7 proteins containing residue substitutions for Gln-31 had already been tested for complex Ill-linked activities and ATP synthesis and found to be normal. Hence. Gln-31 is most likely not involved in ubiquinone-binding. Full-length mutants with Qcr7p- N53S and Qcr7p-N53D were created to investigate the pet- phenotype of the mutant with the truncated protein Qcr7p- ~7(N53S/E116G). Residue Glu-1 16 was not assessed as a cause for the pet- phenotype as a previous study, in which a segment containing this residue was replaced by three amino acids encoding STOP codons in al1 reading frames, did not result in a respiration-

deficient mutant (49). However, the levels of apo-cytochrorne b, the ISP, and the 11 kDa subunit are reduced in this mutant. In addition, complex Ill-linked enzyme activities are reduced by approximately 40%, whereby the mutant enzyme retains a normal turnover number.

These findings of the study by Hemrika et al. (49) implicate the Qcr7 protein C-terminus in assembly. All the other residue substitutions arose fortuitously during mutagenesis and were also investigated for causing phenotypes relevant to this study.

The current chapter describes the mutagenesis of the QCR7 gene followed by a prelirninary characterization of the mutant strains according to growth and enzyme activities. In total, 22 different residues were substituted and they were changed to 38 different amino acids. In addition, four truncated proteins were constructed which were missing the N-terminal 7, 10, 14, and 20 residues, respectively. The protein with the N-terminal truncation of 7 residues was further subjected to point substitutions, whereby a total of six such proteins were constructed, four of which were multiple mutants. In total, 38 individual mutant proteins were constructed and the strains expressing these were further analyzed. A detailed analysis of the role of the Qcr7 protein N-terminus with respect to proton pumping, mitochondrial import, and assembly of complex III is given in Chapter 4. 3.2. MATERIALS AND METHODS

Ma terials Oligodeoxynucleotides were synthesized by the Department of Clinical Biochemistry at the University of Toronto. Restriction enzymes were purchased from Pharmacia. The T7 GEN in vitro mutagenesis kit was obtained from United States Biochemical

(Cleveland, OH). The pG-3 expression vector (il*) was a kind gift from Dr. Jacqueline Segall at the University of Toronto. This vector contains a 2p origin for replication in yeast as well as pUC18 sequences for replication in bacteria and ampicillin resistance. Selection in yeast occurs by the TRP-1 gene marker. Expression of the insert is driven by the glyceraldehyde-3-phosphate dehydrogenase promoter and termination as well as polyadenylation signals are from the phosphoglycerokinase gene.

Mutagenesis by PCR Oligodeoxynucleotides were synthesized containing mutations in codons for the N-terminal portion of the Qcr7 protein (Table 3-1) and a Kpnl restriction site at the 5' end. Together with a wild type oligodeoxynucleotide downstream of the QCR 7 coding region to which a Sall restriction site was added, mutant genes were amplified using PCR as described in Chapter 2. The original pG-3 expression plasmid was modified by digestion with Sacl to excise a 1700 bp fragment in the polylinker region and subsequently self- ligated to create the new pG-3~vector. PCR products were digested with Safi and Kpnl and then subcloned into the respective sites of Table 3-1. Oligodeoxynucleotide sequences used for mutagenesis of the QCR7 gene. OIigodeoxynucleotides were designed with deletions or base substitutions in the QCR7 gene. Following PCR amplification or Ml3 mutagenesis, the wild type codon sequence was substituted by a sequence containing a deletion or a missense mutation. Abbreviations used are: n=a, c, g, t; z=a, c, t; x=a, c, g, and "/" stands for "or1'. Table 3-1. Primen Used for Mutagenesis

Primer Sequence (5'03') Mutation/s I Encoded wild type for 3'end A7 A1 O A1 4 A20 ccggtaccatggcgazaattgg tgacP R101, K, T (67) gtcaccaattg/atcgcaatagacgu RI 01, T ccggtaccatggcgagaattggtga/tgtatattttgaagtcacccP Dl 3E, V (~7) ggtgacttcaaaatatactc/taccaattctcgM D13E, K S4A, C, DlG, H, L, Y T6P, Q R, S Q31L, S, WQ N34SQ N53iY tctgcatgatgggagattcctctgcaM N53Sç Oligonucleotides used for mutagenesis by PCR. Oligonucieotides used for Ml 3 mutagenesis. These mutations were introduced to test an earlier hypothesis according to which the Qcr7 protein was believed to be a ubiquinone-binding protein. However, this was a mistake in the literature which has since been corrected. This residue was mutated to investigate the pet- mutant expressing Qcr7p ~7(N53S/E116G). the pG-3a vector. lnserts were sequenced as described in Chapter 2. Yeast strain YSM-qcr7~was transformed as described by Hill et al.

(137) with pG-3A plasmids containing mutated qcr7 genes.

Site-directed mutagenesis using the Ml 3 phage Oligodeoxynucleotides were synthesized containing various nucleotide changes (Table 3-1). Ml3 mutagenesis was performed using the T7 GEN in vitro mutagenesis kit and al1 manipulations were performed as suggested by the manufacturer. For this technique the QCR7 gene was subcloned into the double stranded, replicative form RFM13, of the Ml 3 phage. The QCR7 insert was subsequently used in the single stranded form of the Ml3 phage as a template for mutagenesis. In the in vitro mutagenesis, primers were annealed to the template and extended by T7 DNA polymerase to synthesize a second strand of the QCRP gene-containing Ml3 phage. During the second strand synthesis, 5'-methyl dCTP was incorporated into the new strand. This ensured that the newly synthesized, methylated strand was preserved whereas the original strand was nicked by treatment with Mspl and Mal and subsequently degraded by exonuclease III. Following mutagenesis, single stranded phage DNA was prepared and sequenced to identify mutated qcr7 genes. Mutated genes were subsequently isolated from the RFM13 phage (139) and transferred to the modified yeast expression vector pG-3A. The pG- 3~-gcr7plasmids were transformed into strain YSM-qcr7A (1 37). Enzyme assays Mitochondria were isolated frorn glucose-grown cells. NADH- cytochrome c reductase (complexes I+III) and succinate-cytochrome c reductase (complexes II+III) activities were assayed in 0.1 M potassium phosphate buffer (pH 7.0)-94 pM cytochrome el mM sodium azide containing between 20 and 100 pg of mitochondria. To start the reaction, 34.2 pM NADH or 10 mM sodiumsuccinate were added as substrates, respectively, and the reduction of cytochrome c was monitored spectrophotometrically at 5 50 nm. Cytochrome oxidase activities were assayed in 0.1 M KPi buffer (pH 7.0) with reduced cytochrome c as substrate (94 PM). Reduced cytochrome c was prepared by the addition of 3.2 mM ascorbate to a suspension of oxidized cytochrome c (40 mg/mL) followed by dialysis against 0.1 M KPi for 48 h during which the buffer was changed twice. Mitochondria (20-50pg) were added to start the reaction and the oxidation of cytochrome c was followed at 550 nm. A number of ubiquinol-cytochrome c reductase assays (complex III) were performed to show that the observed succinate-cytochrome c reductase and NADH-cytochrome c reductase activities were a true reflection of the technically more difficult complex III assays. For these assays decylubiquinol, which was prepared from decylubiquinone by reduction with HCI, was used as the substrate in

0.1 M KPi-94 p M cytochrome c-1 mM azide (144).

Growth studies Growth was tested on solid medium at RT (20-230C),30oC, and 370C. To identify respiration deficient mutants, cells were grown on SD medium containing ethanol (4%) and glycerol (3%) as the main carbon sources with the addition of a small amount of glucose (0.1 46). Mutant strains that were pet at 300C were picked and rnated with a rho- strain for further selection by growth on SD medium containing only the non-fermentable carbon sources glycerol and ethanol. Respiration deficient mutants that contained mutations in the mitochondrial genome failed to grow under these conditions and could thus be eliminated from the selection. 3.3. RESULTS

Characterization of mutants by gro wth Mutant versions of the Qcr7 protein were assessed for their ability to support respiration-dependent growth. This was performed by site-directed mutagenesis of the QCR7 gene using either the PCR or M 13 mutagenesis technique (see Materials and Methods) and the oligodeoxynucleotides listed in Table 3-1 . Plasmid-borne versions of the QCR7 gene were introduced into the strain containing the chromosomal qcr7A:LEUZ allele, YSM-qcr7A, and characterized according to their growth patterns at RT (20- 23oC), 30C, and 37oC on two types of solid media. Synthetic deficient medium containing ethanol and glycerol as main carbon sources with the addition of 0.1% glucose is typically used to identify respiration deficient strains in yeast. Hence, respiration deficient strains arising from mutated Qcr7 proteins that affect complex III function grow on this medium until the small amount of glucose is depleted, but only small colonies form which are referred to as pet- mutants. Strains containing mutations in the qcr7 gene which do not affect function, on the other hand, will grow to wild type size colonies. Such strains can continue to grow by catabolizing ethanol and glycerol once glucose has been depleted. Similarly, respiration deficient strains remain white when grown on SD medium containing glucose as the sole carbon source, while cells with a functional respiratory chain assume a red phenotype. This red phenotype is due to the ade2 mutation that is present in the parental strain W303-1B and causes a red pigment to accumulate (14s). In respiration deficient strains, however, this pigment does not accumulate and the cells retain a white phenotype. Hence, pet strains can be clearly distinguished from wild type strains on the basis of their growth characteristics on these types of media. The growth characteristics of selected strains are shown in Fig. 3-3 and the results of the analyses of al1 38 mutant strains are summarized in Table 3-2. The strain expressing Qcr7p-67 had wild type growth characteristics at RT and 30oC, but was pet- at 370C. This conclusion was based on the observations that at RT and 300C this strain formed wild type shed colonies on SD medium containing ethanol and glycerol as the main carbon sources (top panel) and the colonies were red on SD medium containing glucose as the sole carbon source (bottom panel). In contrast, the strains expressing Qcr7p-~10, Qcr7p-dl4 and Qcr7p-A20 were respiration deficient at al1 temperatures. These strains formed small colonies on the non- fermentable carbon sources (Fig. 3-3, top) and the colonies were white on glucose (Fig. 3-3, bottom). Strains containing Qcr7p- ~7(R10K) and Qcr7p-~7(A9V/R1OT/Y14N/N53D) behaved as did Qcr7p-a7; that is, they were pet- at 370C but not at RT or 300C. In contrast, Qcr7p-~7(RIOT/K44N), Qcr7p-~7(RIOI/G1 ZV), Qcr7p- ~7(D13V),and Qcr7p-~7(N53S/E116G) were pet- at al1 temperatures tested. At 370C, al1 mutants including proteins with an amino- terminal deletion, even those which displayed wild type characteristics at RT and 300C, were respiration deficient. None of the full-length genes containing point mutations were found to cause deficiencies at any of the three temperatures. Fig. 3-3. Growth on SD agar plate with ethanol/glycerol and 0.1% glucose, or SD media plate containing glucose. Top: when grown primarily on the non-fermentable carbon sources ethanol and glycerol, the mutant strain containing the Qcr7p-~7is comparable in size to the wild type. Mutant strains expressing Qcr7 proteins truncated by 10, 14, or 20 amino acids are pet- mutants, indicative of a respiratory chain defect. Bottom: when grown on SD medium containing glucose as the sole carbon source, respiration competent strains turn red due to the ade 2 mutation which causes a red pigment to accumulate. Strains expressing Qcr7 proteins truncated by 10, 14, and 20 residues remain white, confirming severe respiration defects.

Enzyme activities I next tested whether the strains expressing mutated qcr7 genes had functionai deficiencies of ubiquinol-cytochrome c oxidoreductase. For this experiment, I had planned to grow cells on a carbon source such as galactose, raffinose, or maltose, since these sugars do not lead to catabolite repression. I discovered, however, that pet- mutants did not grow well on minimal medium containing these sugars, presumably because they were too compromised. Hence, I performed enzyme assays with mitochondria from cells grown in glucose. This was less than ideal, since glucose resulted in significant catabolite repression of the QCR7 gene and most likely the genes encoding the other subunits of the bci complex. The activity of complex III was drastically lower in glucose grown cells relative to cells grown in galactose. Nonetheless, it was possible to compare complex Ill-linked activities in mitochondria from glucose- grown cells.

Complex III-lin ked enzyme activities were measured with mitochondria purified from each of the mutants grown at 300C. Complex III was either measured in combination with complex 1, using NADH as substrate, or in combination with complex il, using succinate as substrate. The results obtained from the enzyme assays correlated with the results from the growth studies, i.e., al1 mutants that were characterized as pet- mutants in the growth studies, displayed a severe decrease in complex Ill-linked enzyme activities. Cytochrome c oxidase activities were measured as a control to determine the integrity of the mitochondria. I found that in al1 pet- mutants cytochrome c oxidase activities were decreased by about 40% when compared to the wild type. This decrease in complex IV activity can be explained by a downregulation of the expression of mitochondrial respiratory chain proteins due to a respiratory chain defect. Results are summarized in Table 3-2. Table 3-2. Summary. Characterization of mutants according to growth and enzyme activities. Growth was monitored at three temperatures: RT, 30°C, and 37OC. Mutant strains carrying plasmid-borne versions of qcr7 are named according to their mutations in the Qcr7 protein and are classified with regard to whether they cause a pet- phenotype (pet) or not (+). Complex III- Iinked enzyme activities were measured and the mutants were divided into two classes. One class of mutants had wild type level enzyme activity (wt), and the activity of the other class was as low as in YSM-qcr7~ (0). Complex IV enzyme activities from mutants were divided into two classes. One class had wild type activity (wt) and the other class had activity of about 60% that of wild type. Typical enzyme rates are: complex Ill-linked wild type, 30-35 +/-5 nmol min-' mg"; complex Ill-linked activities for YSM-qcr7A, O nmol min-' mg-'; complex IV activity of wild type, 125 +/- 11 nmol min-' mg-'. - MUTANT RT 30OC Complex III Complex IV linked activity - - activity A7 + + wt R1 OK (~7) + + wt R1 OI/G72V (~7) Pet Pet trace R1 01 + + wt RIOT/K44N (~7) Pet pet - A9V/R1 OT/ Y74N/N53D (~7) + + wt RIOT + + wt Dl3V (~7) Pet Pet - Dl3K + + wt Dl3E + + wt N53S/E 1 7 6G (~7) Pet Pet - N53S + + wt N53D + + wt 110 Pet Pet - $1 4 Pet pet - 120 Pet Pet - s4c + + wt 340 + + wt 54G + + wt 34H + + wt 34Y + + wt 54WP2T + + wt 54G/E 7 O9G + + wt 54NL35P + + wt ;4y/17 7 v + + wt ;4G/P2Q + + wt r6P + + wt r6Q + + wt -6R + + wt -6s + + wt 231 L + + wt 13 1s + + wt 13 1W + + wt C7 7N + + wt WS/I 7 07V + + wt 49v + + wt !78s + + wt 199N + + wt IO plasmid (strain YSM-qcr7a) pet pet - Italicized mutations arose for tousiyand were also tested for causing a respiratory chain defect. 3.4. DISCUSSION

This chapter describes the introduction of a number of point and deletion mutations into various regions of the QCR7 gene with an emphasis on the portion corresponding to the Qcr7 protein amino- terminus. These mutant genes, contained in an expression vector, were subsequently expressed in yeast and the phenotype of the resulting strains was assessed. Classification of al1 mutants according to growth and enzyme activities divided them into three categories: mutants with wild type characteristics, mutants that resemble strain YSM-qcr7~, a nd mutants that had wild type characteristics at RT and 30oC and characteristics like YSM-qcr7~at 37°C (Table 3-2). Notably, neither the strain expressing Qcr7p-~7, nor any of the strains containing full-length Qcr7 proteins with point mutations were deficient at 3OoC. However, strains expressing deletion proteins Qcr7p-Al O, Qcr7pA14, and Qcr7p-a20 as well as Qcr7p-A 7(Rl OT/K44N), Qcr7p-b7(R1 OI/G12V), Qcr7p- ~7(D13V),and Qcr7p-b7(N 5 3 S/E1 1 6G) were respiration-deficient at al1 temperatures tested.

Many mutants displayed the inability to grow in media containing non-repressive carbon sources such as galactose, raffinose, or maltose. When yeast are grown on high levels of glucose, many respiratory chain related proteins suffer catabolite repression; this means that the uptake systems of sugars other than glucose are repressed (Z 15). ln order to compare results between wild type and mutants, al1 strains had to be grown in the same medium, hence this medium had to contain glucose as the carbon source. Unfortunately, I found that growing the cells on glucose resulted in a significant catabolite repression of the QCR7 gene. This was evidenced by a lower level of the QCR7 RNA transcript in glucose-grown cells as opposed to galactose-grown cells (results not shown). Catabolite repression of the QCR7 gene also led to lowered complex Ill-linked enzyme activities; this might indicate that al1 or some of the genes encoding the other subunits of the bci complex are also repressed.

As can be seen from the results summarized in this chapter, despite 50 mutations that were introduced into the QCR7 gene only very few resulted in a respiration defect. In addition, none of the full-length Qcr7 proteins containing point mutations resulted in a deficiency. It might therefore be more instructive to target highly conserved residues in the future, especially with the use of a recent cornparison of homologues to the Qcr7 protein from a wide variety of organisms (146). Alternatively, it might be more successful to "work backwards"; for example, one could select for respiration-deficient mutant strains on non-fermentable carbon sources following random mutagenesis of the QCR7 gene. A random mutagenesis approach was not used for this study as I was particularly interested in determining the function of the Qcr7 protein amino-terminus with respect to proton translocation, mitochondrial import, and assembly of complex III (see Introduction of this Chapter). In addition, truncation of seven residues from the N-terminus of the Qcr7 protein resulted in the same phenotype, but a different profile of subunits from the wild type and indicated that the N-terminus is essential. This prornpted me to concentrate on the amino-terminus.

lmmunoblotting established that deleting as many as 20 residues from the Qcr7 protein N-terminus did not prevent this subunit from being imported into mitochondria (results shown in Chapter 4). Hence, the preliminary analysis of the respiration- deficient mutants described in this chapter indicates that the amino-terminus of the Qcr7 protein is essential for the functioning of ubiquinol-cytochrome c oxidoreductase. A detailed characterization of the role of the Qcr7 protein amino-terminus in assembly of complex III, proton pumping, and mitochondrial import is given in Chapter 4. CHAPTER 4

The Role of the Amino-terminus

of the Qcr7 Protein in

Mitochondrial Targeting,

Complex III Assembly,

and

Proton Pumping

Contribution to this chapter: Joses Jones perforrned the immunoblotting of figure 4-7. CHAPTER 4 The Role of the Amino-terminus of the Qcr7 Protein in Mitochondrial Targeting, Complex III Assembly, and Proton Pumping

4.1 . INTRODUCTION

The mutational analysis described in Chapter 3 indicated that the amino-terminus of the Qcr7 protein is essential for formation of a functional complex III. Hence, with some pet- mutants now identified I proceeded to test whether the amino-terminus of the Qcr7 protein is involved in mitochondrial targeting and/or assembly of ubiquinolsytochrome c oxidoreductase.

The involvement of the Qcr7 protein amino-terminus was also investigated with respect to proton translocation. This hypothesis was tested by indirectly measuring the integrity of the proton gradient which is only established under conditions where electron transport is functional. Hence, only strains which contained a fully or partially functional enzyme cornplex were tested; these included strains expressing Qcr7 proteins with mutations in Pro-2, Ser-4, Thr-6, and Ile-11, as well as the strain expressing Qcr7pA7. 4.2. MATERIALS AND METHODS

Ma terials Chemicals were purchased from Sigma (St. Louis, MO), BDH (Toronto, ON), or BioRad (Hercules, CA). Dodecylmaltoside was purchased from Boehringer Mannheim (Mannheim, Germany), and dithiobis(succinimidyl propionate) (DSP) from Pierce (Rockford, CT). Restriction enzymes and protein A sepharose were obtained from Pharmacia (Montreal, PQ). Anti-Qcr7 protein antibodies used for immunoprecipitation were raised in chickens and rabbits against a carboxy-terminal peptide of the protein (refer to Chapter 2). Antibodies raised against subunits of the bel complex were a kind gift from Dr. Bernard L. Trumpower at Dartmouth Medical School. An antibody to yeast S-acetyl coenzyme A synthetase was raised in rabbit by the Centers of Excellence (Canada). Goat anti-mouse IgG (H+L) coupled to alkaline phosphatase and goat anti-rabbit IgG (H+L) coupled to alkaline phosphatase were purchased from BioRad. Alkaline phosphatase-conjugated AffiniPure rabbit anti-chicken IgY (IgG) (H+L) was purchased from Jackson lmmuno Research Laboratories, Inc. (Westgrove, CT). BioMag magnetic beads were obtained f rom Perseptive Diagnostics (Cambridge, MA).

Isolation of yeast mitochondria and Western analyses Mitochondrial isolations and Western analyses were performed as outlined in Chapter 2. A TP synthesis assays Freshly prepared mitochondria (approximately 50 pg) were resuspended in 200 PL breaking buffer (0.6 M sucrose, 20 mM HEPES- KOH, pH 6.5, 0.1% BSA) without PMSF. To start the reaction, the mitochondria were added to 5 mM KPi (pH 7.41-1 mM ADP-5 mM succinate. The mixture was incubated at 370C for 45 min and the reaction was stopped with 80 mM perchloric acid. Proteins were pelleted and the supernatant was assayed for the amount of ATP synthesized by using a hexokinase/gIucose-6-phosphate

dehydrogenase coupled assay (147) in which NADPH is generated. NADPH was quantitated in an Eppendorf fluorimeter.

CWarDichroism spectra Peptides were chosen from the N-terminus of the Qcr7 protein in yeast and its homolog in beef heart and synthesized by the Alberta Peptide Institute: AGRPAVSASSRWLEG (residues 2-1 6, beef heart subunit 6; peptide 1); AGRPAVSASSRWLEGIRKWYYNAAG (residues 2-26, beef heart subunit 6; peptide 2); PQSFTSIARIGDY (residues 2-1 4, Qcr7 protein; peptide 3); PQSFTSIARIGDYILKSPVLSKL (residues 2-24, Qcr7 protein; peptide 4). For recording CD spectra, peptides were dissolved at 1 mg/mL in either 10 mM NaCI-10 mM NaH2P04, methanol, or SDS (30-fold molar excess) diluted in the above buffer. Spectra were recorded on a Jasco J-720A spectropolarimeter, each with two to three scans from 250 to 190 nrn at 250C. Baseline spectra for each solvent were subtracted from the peptide spectra.

Spectra of the cytochromes For spectral analyses of the cytochromes, rnitochondria were resuspended in 0.1 M potassium phosphate (pH 7.4)-0.25 M sucrose-

0.5% cholic acid (33). TO obtain a spectrum containing cytochromes c and cl, cytochrome b, and cytochromes a and a,, a ferricyanide- oxidized spectrum was subtracted from a dithionite-reduced spectrum. To obtain a spectrum containing cytochrome b only, dithionite-reduced minus ascorbate-TMPD (0.2 mM) reduced samples were run. Spectra were recorded on a DW-2a Aminco spectrophotometer from 520 to 620 nm.

Co-immunoprecipita tion Different immunoprecipitation protocols were performed with various combinations of the buffers and procedures listed below. When the anti-Qcr7 antibody raised in chicken was used as the primary antibody, magnetic beads had to be employed since Protein A Sepharose does not bind chicken lgG. On the other hand, when imrnunoprecipitations were performed with the anti-Qcr7 antibody raised in rabbit, protein A sepharose was employed as the matrix to bind the primary antibody. lmmunoprecipitated proteins were analyzed by immunoblotting under both reducing and non-reducing conditions. Buffen: a) NET-gel buffer (50 mM Tris-CI pH 7.5, 150 mM NaCI, 0.1 % Nonidet P-40, 0.25% gelatin, 1mM EDTA pH 8.0, 0.02% sodium azide), containing 0.05% to 1% Nonidet P-40 (139) b) 50 mM Tris-HCI (pH 8), 1mM MgS04, 0.5 mM PMSF, 0.8 g dodecylmaltoside/g protein c) 250 mM sucrose, 1 mM EDTA (pH 8), 10 mM MOPS-KOH (pH 7.2), 60 mM KCI, 0.5 mM PMSF, 0.7% digitonin d) 50 mM Tris-HCI (pH 8), 1 50 mM NaCI, 1 mM EDTA (pH 8), 0.25% gelatin, 0.5 mM PMSF, 0.8 g dodecylmaltoside/g protein e) 50 mM Tris-HCI (pH 8), 1 50 mM NaCI, 5 mM EDTA, 0.5 mM PMSF, 0.8 g dodecylmaltoside/g protein

Procedures; A) Preclearing: mitochondria (100-200 pg) solubilized in one of the above buffers, were incubated with a) pre-immune serum, or b) rabbit-anti-yeast S-acetyl coenzyme A synthetase at 0°C for approxirnately 60 min. Subsequently, protein A sepharose or magnetic beads were added and the suspension was incubated for another 30 min at O°C. Beads were pelleted, and the supernatant fraction was used further for imrnunoprecipitation.

Immunoprecipitation: mitochondria-containing supernatant (from above) was rotated at 0°C with varying concentrations of anti-Qcr7 protein antibody. Subsequently, protein A sepharose or magnetic beads (coupled to rabbit-anti-chicken antibody), were added and the suspension was rotated at 4°C for 1 h. The beads were pelleted and washed three times with buffer by rotating for 20 min during each wash. After the final wash, al1 of the liquid was removed from the beads which were subsequently resuspended in 30 pL of SDS-PAGE buffer with or without DlT, heated for 3 min at 95°C and analyzed by immunoblotting.

B) Mitochondria solubilized in one of the above buffers (5 rng/mL) were incubated on ice for 10 min, centrifuged at 4*C for 10 min in a microcentrifuge to remove insoluble materials, and transferred to Protein A Sepharose beads/BioMag magnetic beads. Subsequently, the volume was increased to 1 mL and the mitochondria-bead suspension was rotated for 1 h at 4°C to reduce the concentration of proteins in the suspension which bind the matrix in a nonspecific manner. The beads were pelleted and the supernatant was transferred to a new bead suspension, after which the primary antibody was added and the suspension was incubated overnight with rotation at 4°C. Samples were washed three times in buffer, beads were resuspended in 2x SDS-PAGE buffer not containing DTT and analyzed by immunoblotting.

Cross-linkina:- Mitochondria (200 pg) were resuspended in 450 pL breaking buffer (pH 8.0) containing 0.5 mM PMSF and 10 mM iodoacetamide. The cross-linking agent DSP was added from a 500 mM stock in dry DMSO to a final concentration of 0.28 mM, the suspension was incubated on ice for 20 to 30 min, and the reaction was terminated with 50 pL of 100 mM glycine containing 10% aprotinin. Mitochondria were pelleted and washed once with breaking buffer containing 0.5 mM PMSF. lmmunoprecipitations using procedure (B) with various buffers from the list above followed the cross-linking procedure devised by Dr. David B. Williams at the University of Toronto. lmrnunoprecipitated proteins were analyzed by immunoblotting (as described in Chapter 2) under reducing and non- reducing conditions. 4.3. RESULTS

Circular Dichroism spectra of amino-terminal peptides Link et al. (23) have previously suggested that the N-terminus of the 13.4 kDa subunit 6 of complex III from beef heart mitochondria forms an amphipathic a-helix. To confirm this helicity, and to compare the yeast N-terminus of the Qcr? protein to its homologue in beef heart mitochondria, two peptides corresponding to amino acids 2-16 and 2-26, respectively, for the bovine sequence and 2-1 4 and 2-24, respectively, for the yeast sequence were synthesized (see Methods section).

CD spectra were obtained for al1 four peptides in diluted saline buffer, methanol, and SDS micelle suspension in saline buffer (Fig. 4-1). All peptides formed only a Iimited amount of secondary structure in aqueous buffer. However, as shown in Fig. 4-1 (middle panels, top and bottom) by the minima at 208 nrn and 222 nrn which are characteristic for a-helix formation, peptides 2 (long beef heart peptide) and 4 (long yeast peptide) display considerable a- he lix content in methanol. In SDS micelles, the secondary structure of peptides 2 and 4 are similarly a-helical. Spectra and helical content of peptide 4 strongly resemble that of peptide 1 (shon beef heart peptide, spectra not shown). This indicates that although the N-terminal regions of the yeast and beef heart proteins are not very similar (due to the increased length of the yeast N-terminus), they nevertheless display similar secondary structures. Peptide 3 (short yeast peptide), on the other hand, did not display any a-helix Fig. 4-1. Circular Dichroism spectra. Peptides corresponding to amino acids 2-26 of the beef heart 13.4 kDa subunit (peptide 2) and amino acids 2-24 of the yeast 14.5 kDa subunit (peptide 4) were assayed for secondary structure by CD. Peptides were dissolved at a concentration of 1 mg/mL in buffer (1 0 mM NaCI-10 mM NaH2P04), methanol, or SDS (to a 30-fold molar excess diluted in above buffer). Spectra were recorded at 250C on a Jasco J-720A spectropolarimeter. The y-axis represents the mean residue molar ellipticity which contains the units [deg cm2 dmoïl]. Top three panels, beef heart peptide 2; bottom three panels, yeast peptide 4. 24' A 4 iu ô gggbb 000000~ T'TYY'++T' OOOOOOO ONNNNOW formation in water, methanol, or SDS (not shown). This indicates that although many peptides display helical spectra in SDS independent of their role in vivo, one can at Ieast conclude from these experiments that the peptides involved possess the intrinsic capability ("threshold hydrophobicity" coupled with residue- dependent helical propensity) to penetrate membranes and form stable secondary structures (1 48).

The amino-terminus of the Qu7 protein may facilita te import into mitochondria Many nuclear encoded mitochondrial proteins possess targeting sequences, 15-70 amino acids long, that are usually located at the N-terminus. Some proteins such as cytochromes c, and b2 even have bipartite signal sequences that are cleaved in two steps (12& 149). Mitochondrial targeting sequences have no obvious homology, but are generally rich in hydrophobic and hydroxylated a mino acids, have a net positive charge, and are able to forrn amphipathic a-helices when in contact with the lipid bilayer (6. 50. 'si 1.

The Qu7 protein does not contain a cleavable N-terminal mitochondrial targeting sequence (21, but according to Sirrenberg et al., it is also unlikely to follow the import pathway used by proteins without a cleavable amino-terminal signal sequence such as the

ADP/ATP carrier (143). Since the Qcr7 protein N-terminus contains many residues that are typical of mitochondrial signal sequences, and since CD spectrophotornetry of an N-terminal peptide has shown that there is a potential for forming an a -helix (Fig. 4-1) with Fig. 4-2. Helical wheel projections. The amino-terminal 18 residues (starting after Met-1) are plotted. a) Wild type; b) Qcr7 protein truncated by residues 2-8 from the N-terminus. A helical wheel projection of the wild type shows that al1 the charged and most of the polar residues are located on one face of the helix, whereas the majority of the hydrophobic residues are located on the opposing face. A helical wheel projection of the Qcr7p-a7 shows that charged residues are located on one face of the helix whereas hydrophobic and polar residues are interspersed throughout. amphipathic character for the wild type (Fig. 4-2), 1 decided to investigate whether this region contains any information for the mitochondrial localization of the protein. Accordingly, I tested the mutants expressing Qcr7p-A7, Qcr7p-Al O, Qcr7p~14,and Qcr7pa20 (see Chapter 3, Fig. 3-2) for the presence of their respective truncated Qcr7 proteins in the mitochondria. Mitochondria were purified from YSM-qcr7A strains grown at 30°C, each of which expressed one of the above truncated proteins. Western blot analyses of these mitochondrial proteins revealed that Qcr7 proteins lacking 7, 14, and 20 N-terminal amino acids are synthesized and transporteci into mitochondria (Fig. 4-3, lanes 1, 2, and 4). However, it was consistently found that the levels of truncated Qcr7 proteins were reduced by approximately 60% when compared to the Qcr7 protein from the strain overexpressing the wild type gene (Figs. 4-3 and 4-4). These lower steady-state levels of the Qcr7 proteins truncated by 7, 14, and 20 residues might be the result of a-helix formation (if indeed they form a-helices) with compromised amphipathicity as shown for Qcr7p-A7 (Fig. 4-2). Amphipathic helices contain charged and polar residues on one face of the helix and hydrophobic residues on the other. While this rule holds true for the first 18 residues (after Met-1) of the wild type N-terminus, the amphipathicity of Qcr7p-~7(the N-terminus corresponds to residues 9 to 26 of the wild type sequence) is compromised with hydrophobic and charged residues interspersed throughout the helix (Fig. 4-2). In addition, deletion of seven or more residues from the Qcr7 protein N-terminus (after Met-1) results in the loss of the three hydroxylated residues Ser-4, Thr-6, and Ser-7. Decreasing the 320 ~7 KO WT Core 1 -0 .- - - - Core 2

Fig. 4-3. Western blot analyses of mitochondrial proteins from YSM-qcr7~strains overexpressing wild type and N- terminally truncated proteins Qcr7p-~7, Qcr7p-~lO, Qcr7p 61 4, Qcr7p-820. Mitochondrial proteins (100pg) were dissolved in SDS-PAGE buffer containing DlT and heated for 3 min at 950C. Samples were run on 16% polyacrylamide gels and then transferred to nitrocellulose membranes. The blots on the top and middle panels were probed with an antibody recognizing core proteins 1 and 2, intermediate (i-ISP) and mature ISP (ISP), the 11 kDa and 14 kDa subunits. The blot containing cytochrome cl,on the bottom panel, was probed with a monoclonal antibody raised against this subunit. Top panel: Lane 1 : protein from YSM-qcr7~ overexpressing Qcr7p- ~20;Lane 2: protein from YSM-qcr7A overexpressing Qcr7pd7; Lane 3: protein from YSM-qcr7A; Lane 4: protein from YSM-qcr7A overexpressing wild type Qcr7 protein. Middle and bottom panels: Lane 1: protein from YSM-qcr7A overexpressing Qcr7p-A 14; lane 2: protein from YSM-qcr7~overexpressing Qcr7p-A20; lane 3: protein from YSM-qcr7~ overexpressing Qcr7p-Al O; lane 4: protein from YSM-qcr7a overexpressing Qcr7p-~7; lane 5: protein from YSM- qcrïA; lane 6: protein from YSM-qcr7~overexpressing wild type Qcr7 protein. 9) % WILD TYPE % WILD TYPE -

w % WILD TYPE % WILD TYPE w Panel II. Number of blots used for histogram determinations

Subunit 14 kDa 11 kDa al1 ISP m-ISP a11 cyt cl m- cyt cl core 1 core-- - 2 1 - - * Due to faint bands on some of these blots, accurate quantifications could not be performed for these samples by densitometry. The numbers indicated reflect the number of blots inspected visually. The levels of 11 kDa subunit in the various mutant strains are as follows: WT, ~7,RIOK > AI 4, 420 > Dl3V > RlOVG12V > AI 0, KO. # These nurnbers reflect the number of blots inspected visuatly (due to uneven running of the gel).

Fig. 4-4. Quantification of Complex III subunits in various Qcr7 mutant strains by densitometry. Panel 1: Histograms were constructed by averaging the density of each subunit from a number of blots (see table on panel II). The density is expressed as a percentage of the respective subunit compared to the overexpressed wild type of each blot. a) Histogram representing quantitation for the 14 kDa and 1 1 kDa subunits. Error bars correspond to the standard error. Where error bars are not shown, the standard error is too small to be visible. b) Histogram representing quantitation for the mature ISP (m-ISP) and combined intermediate and mature ISP (al1 ISP). Error bars correspond to the standard error. Where error bars are not shown, the standard error is too small to be visible. c) Histograrn representing quantitation for mature cytochrome c, (m-cyt c,) and combined intermediate and mature cytochrorne c, (al1 cyt c,). d) Histogram representing quantitation for core protein 1 (core 1 ) and core protein 2 (core 2). amphipathicity and eliminating these hydroxylated residues removes some of the important features that are typical of mitochondrial signal sequences. This could account for the lowered steady-state levels of Qcr7 proteins seen in mitochondria isolated from strains expressing the truncated versions of this subunit. Western blot analyses of mitochondria which were treated with proteinase K revealed that the mutant proteins were properly located inside the mitochondria. It cannot be ruled out, however, that the observed lowered levels of mutated Qcr7 proteins are due to degradation of these proteins as a result of misfolding or a defect in assembly (see "Future Directions" in Chapter 5).

Yeast overexpressing Qcr7p-al O do not contain any detectable levels of the 14 kDa protein in the mitochondria although a band migrating slower than the expected Qcr7 protein can be seen (Fig. 4- 3, lane 3). It is unlikely that this band corresponds to an aberrantly running Qcr7p-al O, however, since a faint band of the same size can be visualized in lanes 6 and 7. In addition, blots performed with a different antibody, which among other subunits also recognizes the Qu7 protein, do not show the presence of this band. Hence, it may be speculated that the Qcr7p-Al O is not imported into mitochondria to detectable levels because, in addition to its compromised amphipathicity and elimination of hydroxylated residues Ser-4, Thr- 6, and Ser-7, it contains a negatively-charged aspartate as the second residue from the N-terminus. If indeed the Qcr7 protein N- terminus is involved in import, this residue might repel the protein from the negatively-c harged phospholipid backbone of the membrane and decrease the eff~ciencyof import to levels below detection.

lmmunoblotting of the cytosolic protein fraction from the mutant with QcrTp-Al O did not show an accumulation of Qcr7p-Al O precursor in the cytosol (results not shown). This is not unexpected as impon of rnitochondrial proteins is tightly coupled to synthesis and it is rare that mitochondrial precursor proteins can be detected in the cytoplasm (16% 164). Hence, Qcr7p-A10 is either imported quickly after synthesis, or CO-translationally or, alternatively, it is degraded rapidly due to impaired import. Further experiments need to be performed to resolve the issue of whether Qcr7p-A~Ois not imported and degraded in the cytoplasm or whether it is degraded in the mitochondria due to an unassembled, partially assernbled, or misfolded mutant protein.

The amino-terminus of the Qcr7 protein is essential for assembiy of the bel complex Examination of mutants with truncated proteins Qcr7p-A?, Qcr7p-Al O, Qcr7p-A14, and Qcr7p-~20with respect to the levels of the other subunits (refer to Figs. 4-3, 4-4, and 4-5) of complex III indicates that the content of core protein 1 does not Vary significantly. Core protein 2 seems to be slightly reduced in al1 respiration-deficient mutants except in that with Qcr7p- ~7(RlOl/G12V). Intermediate ISP (i-ISP) whose bipartite signal sequence has been processed once, is present at lower arnounts in the strain expressing the Qcr7p-~7than in the strain with wild type Qcr7 protein. The strain carrying Qcr7p-~14,however, contains only trace amounts of this intermediate and i-ISP is undetectable in strains with Qcr7p-a20 and Qcr7p-al0 and in YSM-qcr7A. Despite the decreased leveis or absence of this intermediate, mature ISP is present in al1 the mutant strains, showing decreased levels to about 75% of wild type in mutants with Qcr7p-A14 and Qcr7p-~20. Yeast expressing wild type Qcr7 protein and Qcr7p-A7 contain comparable levels of mature ISP, whereas the remaining mutants have lower amounts. The strain with Qcr7p-A? contains levels of 11 kDa subunit comparable to the wild type, the strains expressing Qcr7p- A 14 and Qcr7p-A 2 0 have intermediate levels of this subunit (approxirnately 60% of wild type), and the lowest levels of 11 kDa subunit are seen in the strain expressing Qcr7pal O and in YSM- qcr7A. Examination of the levels of cytochrome clsuggests that combined intermediate and mature cytochrome clare reduced approximately by 40% in the strain with Qcr7p-AI 4, and by over 60% in strains with Qcr7p-A20 and Qcr7p-~lO and in YSM-qcr7A. In summary, this initial analysis of the truncated Qcr7 proteins indicates that deletion of seven or more residues from the amino- terminus of the Qcr7 protein leads to levels of this protein that are reduced by approximately 55%; truncation of seven residues leads to lowered levels of intermediate ISP and intermediate cytochrome cl. In addition, truncation of 10 or more residues results in lowered steady-state levels of the 11 kDa subunit, as well as intermediate and mature iron-sulfur and cytochrome cl proteins. These results implicate the amino-terminus of the Qcr7 protein in the functional assembly of the bel complex (33.47,49, 55,60,133.152). Because the strain expressing Qcrfp-a7 was the least compromised of the mutants in assembly of a functional complex III, I decided to analyze the effect of point mutations in the context of a A7 deletion. The mutations targeted two charged residues, Arg-1 O (mutated to Lys and Ile) and Asp-13 (mutated to Val). Three strains were studied: a strain with Qcr7p-d7(RI OK), a strain with Qcr7p- A7(D13V), and a strain containing Qcr7p-a7(R1 OVG12V). The analysis presented in Chapter 3 indicated that strains expressing Qcr7p-~7and Qcr7p-a7(RI OK) were respiration-deficient only at 3 7OC. Cornplex Ill-linked activities measured in mitochondria from these strains grown at 300C were comparable to the activities of wild type cells. Strains expressing Qcr7p-A 7(Dl3V) and Qcr7p- A7(R 10VG12V) were respiration-deficient at RT, 300C, and 370C. W hereas the strain containing Qcr7p-A~(D1 3V) lacked complex III- linked enzyme activity, the strain with Qcrip-a7(R1 OVG12V) contained a trace of complex Ill-linked activity.

Examination of the subunit composition of complex III in mutant strains (Fig. 4-5) indicated that, of al1 the mutants studied in detail, only the respiration-deficient mutant with Qcr7p- 87(D13V) contained levels of 14 kDa subunit comparable to that of the strain overexpressing the wild type QCR7 gene. In strains containing Qcr7p-d7(R1 OK) and Qcr?p-~7(RlOI/G12V) the amount of 14 kDa subunit is reduced to levels similar to that of yeast strains expressing Qcr7p-~7,Qcr7p-~14, and Qcr7p-~20(Figs. 4-3 and 4-4). All the other subunits of the bc, complex in the yeast containing Fig. 4-5. Western blot analyses of mitochondrial proteins from YSM-qcr7a strains overexpressing wild type Qcr7, Qcr7p-a7(D13V), Qcr7p-A 7(R1 OU), and Qcr7p- A 7(R 1 01/G 1 2V), respectively. Mitochondrial proteins (1 00pg) isolated from the above strains were dissolved in SDS-PAGE buffer containing DTT and heated for 3 min at 95oC. Samples were run on a 1 6% polyacrylamide gel and then transferred to a nitrocellulose membrane. The blot containing core proteins 1 and 2, the ISP, the 11 kDa and 14 kDa subunits was probed with an antibody detecting al1 those subunits. The blot containing cytochrome c, was probed with a monoclonal antibody raised against this subunit. Lane 1: protein from YSM-qcr7A overexpressing wild type; lane 2: protein from YSM- qc r7A overexpressing RIOK; lane 3: protein from YSM-qcr7A overexpressing Dl3V; lane 4: protein from YSM-qcr7~overexpressing RIOVG12V. Qcr7p-~7(RlOK) are present in amounts comparable to the wild type. Strains expressing Qcr7p-h7(D13V) and Qcr7p-~7(RlOI/G12V), have core protein 1 levels comparable to the wild type, whereas densitometry indicated that the level of core protein 2 is reduced in the strain with Qcr7p-A i(Dl3V), similar as in the respiration- deficient strains Qcr7p-A 1O, Qcr7p-~14,Qcr7p-A20, and YSM-qcr7~ (Figs. 4-4 and 4-5). Mature ISP and cytochrome c, levels are sirnilar to the wild type in yeast with Qcr7p-~7(RlOVG1 ZV), but combined intermediate and mature cytochrome cl and ISP are reduced by approximately 30% in this mutant. The mutant containing Qcr7p- ~7(D13V)has lowered levels of both intermediate and mature ISP and cytochrome c,. Although only mature ISP and cytochrome c, contain catalytic activity, the reduced levels of their intermediates are indicative of a higher turnover rate due to an unassembled or weakly assembled complex. As for the 11 kDa subunit, it is severely reduced in the mutants with Qcr7p-h7(D13V) and Qcr7p- a7(R1 OI/G12V) with the lower apparent level being in the latter.

Since an antibody was not available to monitor cytochrome b, I assessed the presence of holo-cytochrome b in mitochondria by spectrophotometric analyses (Fig. 4-6). Some samples could not be assayed for cytochrome b content as they did not grow on non- repressive carbon sources. Panel (a) in Figure 4-6 shows typical spectra from the strain expressing the wild type QCR7 gene. In the top part of panel (a) reduced minus oxidized spectra are seen corresponding to cytochromes cic, at 550 nm and cytochromes a+a, at 605 nm. In the bottom part of panel (a), reduced minus oxidized spectra are seen corresponding to cytochrome b at 560 nm and cytochromes a+a, at 605 nm. With respect to the other strains, the difference spectra show that the strain carrying the conservatively substituted Qcr7p-~7(R1OK) is comparable to that of the mutant with Qcr7p-~7. The levels of holocytochrome b from these two mutants are comparable to the wild type, whereas the levels of holocytochrome c+c, are somewhat reduced. In contrast, strains expressing Qcr7p-~?(RlO!/G12V) and Qcr7p-~7(D13V) displayed significantly lowered and undetectable amounts of holocytochrome b, respectively, by this technique. The levels of holocytochrome cic, were comparable in mutants with Qcr7p-A7 and Qcr7p- ~7(R101/G1ZV), whereas the mutant with Qcr7p-b7(D13V) displayed slightly reduced levels (Fig. 4-6).

The reduced levels of cytochrome b in strains expressing Qcr7p-a7(RI OI/G12V) and Qcr7p-~7(D13V), correlate with the decreased levels of overall ISP and cytochrome c,, as identified by immunoblotting (Fig. 4-4). Although, as identified by immunoblotting, mature cytochrome c, is comparable to the wild type in the mutant with Qcr7p-b7(RlOI/G12V) and only slightly reduced in the mutant with Qcr7p-b7(D13V), the lower relative levels of holocytochrome c+c,, as seen by spectrophotometry, may be due to decreased maturation of apocytochrome c, to holocytochrome c, as a result of an unstable or a weakly assembled complex. In summary, immunoblotting and spectrophotometric analyses have shown that combined intermediate and mature ISP, as well as holocytochromes b and c+c, are reduced in the mutant with Qcr7p- ~7(D13V)when compared to the mutant with Qcr7p-~7(RlOI/G12V). Keeping in mind that the mutant with Qcr7p-A7(D13V) has slightly higher levels of 11 kDa subunit and significantly higher levels of Qcr7 protein in the mitochondria than the mutant with Qcr7p- a7(RlOI/G12V), and considering the results from the enzyme activities (Chapter 3), the varying levels of complex III subunits implicate a role for the Qcr7 protein amino-terminus in assembly of a subcornplex/bc, complex. Fig. 4-6. Difference spectra of the cytochromes. For spectral analyses of the cytochromes, mitochondria (2mg/mL) were resuspended in 0.1 M potassium phosphate (pH 7.4)-0.25 M sucrose- 0.5% cholic acid (33). TO obtain a spectrum containing cytochromes c+c, and cytochromes aia,, a ferricyanide oxidized spectrum was subtracted from a dithionite reduced spectrum (top panel). To obtain a spectrum containing cytochrome b only, dithionite reduced minus ascorbate-0.2 mM TMPD reduced samples were run (bottom panel). Spectra were recorded on a DW-Za Aminco spectrophotometer from 520 to 620 nm. cl- -/-- Co-immunoprecipitatlons Since the N-terminus of the Qcr7 protein seemed to play a role in assembly of the bel complex, it was of interest to determine which subunits the N-terminus associates with and in which order. These issues were approached by attempting to CO-precipitate the subunit/s surrounding the 14 kDa protein. lmmunoprecipitations were carried out under a variety of conditions using an anti-Qcr7p antibody raised in chicken and in rabbit to a carboxy-terminal peptide of the protein. The immune response is unique for each animal, hence the two anti-Qcr7 antibodies obtained varied with regard to their specificities. The antibody raised in chicken did not immunoprecipitate the Qcr7 protein, hence the anti-Qcr7p antibody raised in rabbit was employed for all imrnunoprecipitation procedures. The results proved to be inconclusive (results are not shown). Three secondary antibodies were used in Western blot analyses to determine which complex III subunits had CO- precipitated with the 14 kDa protein. The first one, raised in rabbit, recognizes core proteins 1 and 2, the ISP, and subunits 7 and 8; the other two were monoclonal antibodies raised in mice against the ISP and cytochrome c I, respectively. When the rabbit antibody was employed to detect immunoprecipitated proteins on the immunoblot there was a significant amount of cross-reaction with the anti- Qcr7p antibody since both were raised in the same animal species. Hence, the large molecular weight core proteins could not be detected due to interference from the IgG heavy chains (about 50 kDa in size). Even under non-reducing conditions there was sufficient background on the immunoblot corresponding to the heavy chains to obscure the potentially CO-precipitated core proteins. Similarly, the ISP could not be detected with this antibody due to interference from the IgG light chains (approximately 25 kDa in size). However, when ernploying the mouse monoclonal anti-cytochrome cl and anti- ISP antibodies, there was minimal interference frorn the IgG (H+L) chains of the anti-Qcr7 antibody used in the immunoprecipitation. Blots visualized with these secondary antibodies seemed to indicate that cytochrome cl and the ISP did not CO-precipitate. This notion was substantiated by the identical band profile seen in the two immunoblots. That is, since the bands that could be seen in the two blots were identical, they could not correspond to cytochrome cl and the iron-sulfur protein, but rather to the minimal interference of the IgG light chains which migrate at approximately the same rate as cytochrome cl and the ISP.

Under some conditions employed, a protein band that migrates in the region of the 11 kDa protein could be visualized in the wild type, but not in the strain YSM-qcr7~. On the other hand, under different conditions there appeared to be such a protein band even in the disruption strain suggesting that the protein migrating in the region of 11 kDa seen following immunoprecipitation is not the 11 kDa protein of the bci complex.

Results from immunoprecipitations following cross-linking were inconclusive as well. No matter which of the three antibodies described above were used to detect the proteins on the immunoblots, the profile seen for al1 the mutant strains tested and the wild type (whether under reducing or non-reducing conditions) was the same. That is, the same band pattern was seen for al1 the mutant strains, including YSM-qcr7A, and for the wild type. Since the immunoprecipitations were carried out with an anti-Qcr7 antibody, this indicates that the observed bands are not relevant to subunits from complex III. A new antibody raised against the 14 kDa subunit or the 11 kDa subunit could perhaps be tested with better results in the future (see "Future Directions", Chapter 5).

Deletion of seven residues from the amino-terminus does not impair pro ton pumping 1 found that truncation of the amino-terminal seven amino acids did not impair electron transport (that is complex Ill-linked enzyme activity was not affected), hence I tested whether this segment might contribute to proton translocation. Generation of ATP can be used as an indirect measure of the proton gradient. With succinate as substrate, ATP synthesis in mitochondria is a measure of the integrity of the proton gradient that is established by complexes III and III+IV. When ferricyanide is used as electron acceptor with succinate as substrate, the ATP produced is solely due to the electrochemical gradient generated by complex III. As already mentioned, to establish a proton gradient electron transport has to be intact; hence, as expected, of al1 the strains containing truncated Qcr7 proteins that were tested, only Qcr7p-~7produced ATP in the assay system. The amount of ATP produced in mitochondria from the A7 protein-containing mutant, however, was no different from that in mitochondria from the wild type irrespective of whether the ATP was generated by the activity of complex Ili alone or complexes III+IV. Similarly, mutant strains expressing Qcr7 proteins with residue substitutions in Ser-4 and Thr-6 did not show a decrease in the amount of ATP produced when compared to the wild type (Table 4-1). As the yeast Qcr7 protein has a longer N-terminal extension than its beef heart homologue, Qcr7 proteins with N-terminal truncations of 14 and 20 residues (after Met-1) were synthesized to test for the amount of ATP synthesized. Unfortunately, mutants expressing Qcr7p-AI 4 and Qcr7p-A 20 completely lack complex III- linked enzyme activities (see Table 3-2) and could therefore not produce any ATP.

The strain expressing Qcr7p-d 7 is temperature sensitive The growth of the yeast strain expressing the Qcr7p-~7at 370C (Fig. 4-7) displays a different profile from the phenotype at 30oC (refer to Fig. 4-3). At 370C this strain is a pet- mutant with undetectable NADH-cytochrome c reductase activity. In this case immunoblotting (Fig. 4-7) showed that the A7 protein was not present in the mitochondria (lane 1) although it was readily detectable in YSM-qcr7~overexpressing the wild type Qcr7 protein (lane 2). This result may implicate involvement of the N-terminus in mitochondrial targeting and import in a temperature sensitive manner. Alternatively, the absence of Qcr7p-A7 in the mitochondria rnay simply be the result of an intrinsically unstable protein and a higher rate of degradation at 370 C. Table4-1: ATPS nt hesis Mutation Complex III+IV Complex III

(- Ferricyanide) (+ Ferricyanide) II episomal wild type 41 9 (8) +/- 73 1 12 (8) +/- 27 ~7 374 (2) 107 (2) S4G 366 - S4Y/11 1V 516 - S4G 554 - S4WE109G 540 - P2T/S4L 412 (2) 122 T6P - 85 T6R - 85 v The numbers correspond to the total arnount of ATP synthesized per milligram of protein (nmol of ATP/mg of protein) in 45 minutes in the presence or absence of femcyanide (for details see Material and Methods). Fig. 4-7. Western blot analysis performed with mitochondrial membranes prepared from YSM-qcr7~(g rown at 370C) overexpressing Qcr7p-~7and the wild type Qcr7 protein. Mitochondrial proteins (1 00 pg, from cells grown at 370C) were dissolved in SDS-PAGE buffer and heated for 3 min at 950C. Samples were run on a 16% polyacrylamide gel and then transferred to a nitrocellulose membrane. The blot was probed with a polyclonal antibody that recognizes core proteins 1 and 2, the iron-sulfur protein, the 14 kDa and 1 1 kDa subunits. Lane 1: protein from the strain YSM-qcr7~overexpressing Qcr7p-A7; iane 2: protein from strain YSM-qcr7~overexpressing the wild type Qcr7 protein. 4.4. DISCUSSION

From the analyses of the mutant strains presented in Chapters 3 and 4 it is evident that despite the 50 mutations that were introduced into the QCR7 gene, only few caused a respiration deficiency. Of 28 full-length Qcr7 proteins containing point mutations that were examined, none caused any deficiencies at RT, 30°C, or 37OC when expressed in the strain containing the qcr7 gene disruption, YSM-qcr7A. On the other hand, some of the mutant strains expressing Qcr7 proteins with point mutations in the context of a a7 deletion were respiration-deficient even though the strain expressing Qcr7p-~7by itself did not display such a defect at 30°C. These results rnay indicate that while individual residues alone are not crucial for the function of the Qcr7 protein amino-terminus, the destabilized, but still functional Qcr7p-~7can be disruptive with respect to cornplex III function when additional residues are mutated. This theory would explain why substitution of residues without an added deletion, as in the mutants containing full-length proteins, did not result in a deficiency. Furthermore, it explains why the strain expressing Qcr7p-A7 displays a phenotype equivalent to the wild type at 30°C, whereas some of the strains expressing point mutations in the context of a ~7 deletion are pet-. A deletion of seven residues rnay be tolerated, but any additional unconserved substitutions in critical regions apparently exceed the threshold. If, for example, the Qcr7 protein N-terminus is involved in assembly, truncating this region could weaken the interaction with the other subunith. Hence, truncation of the N-terminal seven amino acids still allows assembly, but the complex is less stable. If, then, additional deletions or point mutations are introduced into the N- terminal region of the Qcr7p-A7, the secondary structure of the protein may be further altered especially if the amino acid substitutions are not conservative. This could cause the already weakened interactions to be disrupted. Construction of mutants with full-length Qcr7 proteins showing normal activity, particularly Qcr7pG12V and Qcr7pD13V, would strengthen this argument.

The results outlined in this chapter are consistent with this model. The strain expressing Qcr7p-a7(R1 OK) with the conservative amino acid substitution RlOK displays a phenotype identical to the strain containing the Qcr7p-A7; however, the strain containing the protein with the non-conservative substitution, Dl 3V, is a pet- mutant. The hypothesis that the preservation of the entire N- terminal region and its inherent conformation are crucial rather than the individual residues alone, is further substantiated by strains expressing Qcr7p-A ï(Rl OT/K44N) and Qcr7p- A~(A~V/RIOT/Y14N/N53D) (see Chapter 3). The former of these mutants is respiration-deficient, whereas the latter has a phenotype comparable to strains expressing Qcr7p-a7 and Qcr7p-a7(RlOK). This observation rnay argue against a functional involvernent of Arg- 10, but on the other hand, one has to be cautious in drawing conclusions from the strain with the quadruply mutated Qcr7p- a7(A9V/R1 OT/Y14N/N53D), as the substitution of Arg-1 O by threonine could potentially be compensated for by the other amino acid substitutions. The wild type phenotype of the strains expressing full-length Qcr7p-R 1OT, Qcr7p-R 101, Qcr7p-D13K, and Qcr7p-Dl SE further confirm the notion that preservation of the N- terminus and its inherent conformation are important. Since, however, full-length proteins Qcr7p-K44N, Qcr7p-G1 ZV, and Qcr7p- Dl 3V were not constructed, it cannot be ruled out cornpletely that these individual residues are essential. This seems improbable however, since none of the strains expressing full-length Qcr7 proteins with point mutations caused a respiratory chain defect, no matter whether the charged residues were substituted for by a polar residue (RlOT), a residue of opposite charge (Dl 3K), or a hydrophobic residue (RI 01). Furthermore, the mutant containing the truncated protein Qcr7p-~7(N53S/EI1 6G) is pet-, whereas expression of neither of the full-length proteins Qcr7p-N53S and Qcr7p-N53D resulted in a respiration-deficient phenotype. In this case, the pet- phenotype cannot be attributed to the mutation at residue Glu-1 16, since Hemrika et al. (49) have truncated a region including this residue and found that the mutant containing this protein retains approximately 40% of complex Ill-linked activity and is not respiration-deficient. Hence, the pet- phenotype of the strain expressing Qcr7p-~7(D13V)is probably the combined result of an unconserved substitution of a critical residue, and the N-terminal truncation of seven residues.

Recent results obtained from preliminary growth analyses of newly generated mutants indicate the following: al1 mutants containing Qcr7 proteins with residue substitutions in the context of the truncated protein Qcr7p-A7 are respiration-deficient. These include mutants with proteins Qcr7p-~?(D13Y), -(Dl 3A), -(Dl 3G), -(Dl 3N), -(G12V), -(RI OI), and -(A9V/G12E). In contrast, the only new mutant containing a protein with a residue substitution in the context of the full-length protein, Qcr7pG1 ZV, did not display a pet- phenotype. This confirms the prediction that truncation of seven residues from the amino-terminus is dominant and that any additional, non-consewative residue substitutions of important amino acids are disruptive. These results also make it less likely that a full-length Qcr7p-Di 3V would cause a respiratory chain defect (So-Young Lee, personal communications).

Co-precipitation of proteins was atternpted by employing an anti-Qcr7 antibody in order to reveal the identity of any subunit cornplexed with the 14 kDa protein. Although the antibody used was capable of immunoprecipitation, under conditions where subunits were not cross-linked, CO-precipitation could not be achieved. It is thus probable that the anti-Qcr7 protein antibody cannot access the 14 kDa subunit without disrupting the interactions with the surrounding subunits. Hence, the yield of only the 14 kDa subunit following immunoprecipitation. This speculation is in agreement with previous studies which suggested that the 14 kDa subunit is located in the interior of the enzyme complex (66). CHAPTER 5

Discussion and Future Directions CHAPTER 5 Discussion

Previous inactivation of the QCR7 gene has shown that the Qcr7 protein is an essential component of ubiquinol-cytochrome c oxidoreductase (33). The disrupted strain displays a pet- phenotype and does not grow on non-fermentable carbon sources. Previous studies have implicated the Qcr7 protein in the functional assembly of ubiquinol-cytochrome c reductase (33, 4% 55, 60-63). The N- terminus of the Qcr7 protein is believed to be oriented towards the matrix in yeast (66, 130) and in its homologue of beef heart mitochondria (*3# 31 1. Because proteolysis of the N-terminal seven amino acids of the beef heart mitochondrial counterpart leads to a decreased H+/e- ratio, Cocco et al. (131 ) suggested that this subunit is involved in proton uptake from the rnatrix with subsequent transfer of these protons to the hypothetical ubiquinone binding pocket at center N. Since, however, proteolysis also resulted in a small amount of cleavage from the Rieske ISP, it cannot be ruled out that the ISP is in fact responsible for the decreased H+/e- ratio.

In the current work, the role of the Qcr7 protein amino- terminus was investigated with respect to three putative functions; (1) proton translocation, (2) mitochondrial targeting, and (3) assembly of the bcl complex. Although the Qcr7 protein does not contain a cleavable N-terminal targeting sequence, the Qcr7 protein is unlikely to follow the import pathway used by mitochondrial proteins without cleavable N-terminal leader sequences, such as the ADP/ATP carrier (143). In addition, the N-terminal amino acids display features characteristic of signal sequences, such as net positive charge, and a number of hydroxylated and hydrophobic residues. The N-terminus of the beef heart mitochondrial counterpart has been postulated to form an amphipathic a-helix (231, a feature that is typical of mitochondrial targeting sequences. The above issues were approached by inactivating the QCR7 gene and complernenting the resulting mutant strain, YSM-qcr7~, with a number of qcr7 genes containing point and deletion mutations. In addition, the secondary structures of selected amino-terminal peptides were studied by CD spectroscopy and by cornparison to their beef heart homologues.

Circular dichroism studies of a 23 residue peptide from the amino-terminus of the Qcr7 protein (peptide 4) indicated that this region displays a-helical spectra in SDS and in methanol (Fig. 4-1). The extent of a-helix formation of this peptide was comparable to that of beef heart peptide 1 (residues 2-1 6, results not shown) and similar to beef heart peptide 2 (residues 2-26). These results indicate that even though the primary sequences of the yeast and beef heart N-termini do not show significant similarity, their secondary structures are nevertheless similar. In addition, these findings rnay point towards the importance of the conformation of the N-terminus as a whole. Helical wheel plots of the wild type N- terminus show that the predicted a-helix is amphipathic in nature (Fig. 4-2) in agreement with one of the typical features of mitochondrial signal sequences. Overexpression of the genes encoding Qcr7p-87 and Qcr7p- ~7(R1OK) in YSM-qcr7~ restored complex Ill-linked enzyme activities to levels comparable to the strain overexpressing the wild type QCR7 gene at 30°C. However, overexpressing the genes encoding Qcr7p-A 1 O, Qcr7p-A14, Qcr7p-~20,Qcr?p-~7(RlOl/G1 ZV), and Qcr7p-~7(D13V) did not restore a respiration-competent phenotype. This prompted me to investigate whether the truncated proteins are in fact transported into mitochondria. From Figs. 4-3, 4-4, and 4-5 it is evident that al1 the above mutated proteins with the exception of Qcr7p-A~Oand Qcr7p-a7(D13V) are present in the mitochondria at levels comparable to the functional Qcr7p-~7. Althoug h these steady-state levels are signif icantly reduced in cornparison to the wild type Qcr7 protein in the strain overexpressing QCR7, the levels are nonetheless comparable to those of Qcr7 protein in the parental strain W303-1 B.

Respiration-deficient mutants with Qcr7p-Al0 and Qcr7p ~7(D13V)displayed a different profile from the other Qcr7 proteins (Figs. 4-3, 4-4, and 4-5). The strain containing Qcr7p-~7(D13V)is the only mutant with Qcr7 protein steady-state levels comparable to the strain overexpressing the wild type QCR7 gene. One can argue that the elimination of the negatively-charged Asp-13, an untypical residue for rnitochondrial signal sequences, compensates for the truncation of the seven N-terminal residues and restores protein levels back to those comparable to the strain overexpressing the QCR7 gene. However, one cannot eliminate the possibility that this protein is present at wild type levels because it is less susceptible to proteolysis in the mitochondria than the other truncated Qcr7 proteins. In contrast to Qcr7p-A 7(Dl3V), Qcr7p-Al O is not detectable in the mitochondria. Western blotting of the cytosolic protein fraction from the strain with Qcr7p-Al O did not show an accumulation of this subunit in the cytosol. Hence, it is not clear whether the absence of detectable Qcr7p-Al0 is the result of an unstable protein which is degraded in the cytoplasm or the mitochondria to levels below detection, or whether Qcr7p-~lO is degraded in the cytoplasm because it is not imported. It is conceivable, that Qcr7p-al O is not imported into mitochondria due to the negative charge of Asp-13 which, in this truncated protein, is exposed at the amino-terminus and might repel the protein from the negatively charged phopholipid backbone. Negatively charged

residues are uncommon in mitochondrial signal sequences (isol '531, but they may not always be as detrimental to the irnport process depending on their location or orientation. Further experiments are clearly needed to resolve the issue between import and degradation (see "Future Directions").

If the Qcr7 protein was to follow the same import pathway as proteins with a cleavable amino-terminal signal sequence, then deletion of the amino-terminus could conceivably result in

decreased import (1 25). For example, truncated proteins may not interact with cytosolic chaperones as efficiently or, alternatively, they rnay not bind as tightly to the OMM receptors. The absence of Qcr7p-~7at 370C could thus be due to a higher degradation rate at that temperature resulting from the impaired binding to cytosolic chaperones. Roise et ai. (154) have suggested that part of the mechanism of protein import is the perturbation of the phospholipid bilayer by the surface active amphipathic helix of the presequence. This ability of the presequence to cause a local defect in the membranes would then create a route for the rest of the protein to follow. It is thus conceivable that the truncated proteins of this study cannot enter the membrane as efficiently, since their amphipathic character and inherent lytic properties have been decreased by shortening the presequence. Presequences must be able to form amphipathic structures with the charged and hydrophilic residues located on one side of the helix and the hydrophobic residues on the other side (1W 155). As indicated by the CD spectra, the wild type N-terminus is capable of forming an a-helix in a membrane-mimetic environment (Fig. 4-1 ). In addition, helical wheel plots of the wild type N-terminus show that this helix is amphipathic. In contrast, helical wheel plots of Qcr7p-A7 (Fig. 4-2) and Qcr7pdl O, Qcr7p-AI 4, and Qcr7pA20 (not shown) show that the amphipathicity of these helices, if indeed they form helices, is compromised. Baker and Schatz (95) have shown that 25% of randomly generated peptides are capable of targeting proteins into mitochondria, although at lower efficiency than authentic signal sequences. Hence, if the Qcr7 protein amino-terminus does in fact play a role in mitochondrial import, then elimination of some of the features typical of mitochondrial signal sequences, as seen here in the truncated Qcr7 proteins, does not have to prevent irnport into mitochondria, but merely reduce its efficiency. To make a definite statement on the efficiency of mitochondrial import, further experiments can be performed (see "Future Directions"). Such experirnents were not atternpted as a part of this study, as the question whether the Qcr7 protein amino-terminus is essential for import into mitochondria was answered by the presence of Qcr7p- a20 in the mitochondria.

Since transformation of the gene encoding Qcr7p-A7 into the strain YSM-qcr7a restored complex Ill-linked enzyme activities to levels comparable to the strain expressing the episomal wild type Qcr7 protein at 300C (Table 3-Z), 1 proceeded to test whether the N- terminal seven amino acids play a role in proton transfer by assaying the amount of ATP synthesized in mitochondria. As ATP synthesis is driven by the protonmotive force, the amount of ATP produced can be used as an indirect assessment of the integrity of the proton gradient. I found that concentrations of ATP produced were comparable in strains expressing wild type Qcr7 protein, Qcr7p-A7, Qcr7p-S4G, Qcr7p-S4Y/IIlV, Qcr7p-S4Y, Qcr7p- S4G/E109G, Qcr7p-P2T/S4L, Qcr7p-T6P, and Qcr7p-T6R (Table 4-1 ). From this it can be concluded that in yeast, the involvement of the N-terminal seven amino acids in proton translocation and ATP synthesis is unlikely to be critical. Unfortunately, it was not possible to test the N-terminal region in yeast that is relevant to the region found to be critical in the beef hem homologue, as yeast with truncated proteins Qcr7p-a14 and Qcr7p-a20 did not contain any complex Ill-linked activitieç. Although these findings may seem to contradict the studies performed in beef heart mitochondria (1 311, one has to remember that the yeast homologue contains a longer N- terminal extension. Furthermore, one has to keep in mind that Cocco et al. truncated the 13.4 kDa subunit (and others) by subjecting the already assembled complex to proteases, thereby circumventing a potential assembly problem. Hence, it cannot be ruled out that the region between residues 9 and 21 of the yeast Qcr7 protein are in fact involved in proton translocation.

Although the N-terminal seven amino acids (after Met-1) of subunit 7 may facilitate import into mitochondria or confer stability to the complex, the results of this work suggest that the amino-terminus of the Qcr7 protein, especially the region between residues 8 and 20 (after Met-1), is essential for the formation of a functional bc, complex. Steady-state levels of the majority of truncated Qcr7 proteins are reduced when compared to the overexpressed wild type (Fig. 4-4), but the pet- phenotype of the respiration-deficient mutants cannot be attributed to these reduced levels as they are comparable to the functional Qcr7pa7. Hence, the pet- character of the strains expressing Qcr7 proteins with truncations (and point mutations) is more likely to be the result of the decreased levels of some of the other subunits of complex III.

lmmunoblotting shows that the steady-state level of the 11 kDa subunit is decreased in al1 respiration-deficient mutants. Combined intermediate and mature cytochrome c, and ISP are also decreased in al1 respiration-deficient mutants tested except for the mutant with Qcr7p-~7(RlOI/G12V); this mutant contains wild type levels of mature cytochrome c, and ISP, but lowered overall levels of these proteins. In addition, spectrophotometric analyses of the respiration-deficient strain containing Qcr7p-a7(D13V) show that holocytochrome cl is sig nificantly decreased in this mutant when compared to the wild type, in agreement with the observations from the immunoblotting. Furthermore, holocytochrome cl seems to be reduced slightly in the respiration-deficient mutant with Qcr7p- ~7(R101/G12V) and the respiration-competent mutants with Qcr7p- A7 and Qcr7p-a7(Rl OK). Nonetheless, the strain with Qcr7p-A7, which has complex Ill-linked activities indistinguishable from the wild type at 30°C, contains lowered intermediate cytochrome cl and ISP.

Although the N-terminal seven residues of the Qcr7 protein are not essential for assembly, the complex seems to be less stable without them. This is indicated by the reduced intermediates of cytochrome c, and ISP which are the result of a higher turnover rate of the complex in the mutant containing Qcr7pWA7. When comparing mutants with Qcr7p-A7, Qcr7pa14, and Qcr7p-A20, one can see that with increasing N-terminal truncation the levels of intermediate cytochrome c, and ISP decrease to be virtually absent in the mutant containing Qcr7p-~20. This makes sense since the larger truncations presumably result in more compromised assembly and since processing of intermediate ISP into mature ISP is predicted not to occur until after insertion of this protein into the assembled

complex (133. 156). Hence, even though mature ISP and mature cytochrome c, are present, the reduced levels of these proteins as seen in some mutants and the absence of their intermediate forms are a typical indication of an assembly defect (33, 47, 49. 55. 60. l33, 152). For example, mature ISP is reduced and intermediate ISP is undetectable in the study performed by Schoppink et al. (331, which describes the previous inactivation of the 14 kDa subunit and in which the authors conclude that the obsewed defect is due to compromised assembly of the complex. This profile is similar to the profiles seen for YSM-qcr7~, and the strains expressing Qcr7p-~10, and Qcr7p-~20,respectively. In addition, in the study by Hemrika et al. (55) who have concluded that the aromatic nature of residue 66 of the 11 kDa subunit is important for assembly of a functional complex III, intermediate and mature ISP are characteristically reduced in a mutant in which residues 66 to 70 (YWYWW) have been replaced by the sequence SASAA. A revertant of the SASAA mutant with growth rates approximately equal to the wild type was sequenced and found to contain the sequence FASAA. Analysis of this revertant revealed that mature ISP is present at comparable levels to the wild type, whereas intermediate ISP is reduced. This profile is similar to the profile seen for the ISP in the mutant with

Qcr7p-~7. Furthermore, in the study in which Hemrika et al. (49) have shown that the C-terminus of the 14 kDa subunit is involved in assembly of a functional enzyme, intermediate and mature ISP are characteristically reduced. Also, a study by Crivellone et al. (60) has shown that intermediate and mature ISP are reduced in al1 mutants in which the genes encoding complex III subunits have been deleted and, as a result, the complex does not assemble into a functional enzyme. Thus, numerous mutational studies of complex III subunits have been performed, where reduction of intermediate and mature (or interrnediate only) forms of ISP and cytochrome c, have been interpreted as being indicative of an impairment of the assembly process. The levels of ISP and cytochrome c,, as documented by previous studies and by this study, faIl between the wild type levels and the drastically lowered levels seen in strains in which the QCR7 gene has been deleted. However, in spite of the interpretation of lack of intermediate ISP and cytochrome c, proteins in complex III mutant analysis as being indicative of lack of complex III assembly, there is a paucity of experimental work in the literature to substantiate this assurnption.

The hypothesis that truncation of the N-terminal seven residues (of the mature protein) destabilizes the assembly of the bc, complex, but that any further truncation or non-conservative substitution of important residues is detrimental to the assembly process is substantiated by the following observations: i) truncation of seven residues from the amino-terminus by itself results in lowered levels of cytochrome c, and ISP intermediates; ii) any truncation beyond seven residues of the Qcr7 protein amino- terminus results in lowered levels of intermediate and mature cytochrome c, and ISP, as well as the 11 kDa subunit, despite levels of 14 kDa subunit comparable to the strain with Qcr7p-A7; iii) the substitution of valine for Asp-13 in the context of a A7 truncation results in severely reduced levels of the 11 kDa subunit, intermediate cytochrome c, and ISP, mature ISP, holocytochrome c, and holocytochrome b; the mutant containing Qcr7p-~7(RlOI/G12V) has severely reduced levels of the 11 kDa subunit, reduced levels of intermediate cytochrome c, and ISP, as well as reduced holocytochrome b and holocytochrome c,; iv) none of the mutants with full-length Qcr7 proteins containing only point mutations display either a respiratory chain defect or lowered levels of intermediate or mature cytochrome c, and ISP (not shown). This is the case, no matter whether the charged residues were substituted for by a polar residue (RI OT), a residue of opposite charge (Dl 3K), or a hydrophobic residue (RI 01). Furthermore, the mutant containing the truncated protein Qcr7p-~7(N53S/E116G) is pet-, whereas expression of neither of the full-length proteins Qcr7p453S and Qcr7p-N53D resulted in a respiration-deficient phenotype. In this case, the pet- phenotype is less likely due to the mutation at residue

Glu4 16, since Hemrika et al. (49) have truncated a region including this residue and found that the mutant containing this protein retains approximately 40% of complex III-linked activity and is not respiration-deficient. Hence, the evidence from al1 the mutants constructed in the context of Qcr7p-A7 and the new finding that the mutant with the full-length Qcr7p-G1ZV is comparable to the wild type according to growth, speaks for the dominant effect of the N- terminal truncation of seven residues.

The observed decrease in the levels of complex III subunits other than the Qu7 protein cannot be explained by an unstable Qcr7 protein which is misfolded and subsequently degraded. In addition, the presence of most mutated Qcr7 proteins (except Qcr7p-al O and Qcr7p-~7(D13V))in the mitochondria at comparable levels which are also similar to, or higher than the level of Qcr7 protein in the parental strain W303-1 B, speaks against their lowered levels as a result of degradation. If the mutated Qcr7 proteins were unstable, one would expect them to be degraded to levels below detection or

to varying levels as shown in the study by Crivellone et al. (60). In addition, the fact that the majority of mutated Qcr7 proteins are present in the mitochondria at comparable levels, despite their different mutations, speaks against their instability as a result of mutation. The fact that so many mutants with wild type phenotype (Table 3-2) could be isolated speaks for the stability of the protein and indicates that overall configuration is perhaps more important than the individual residues. A precedent of this type of phenornenon is seen in some of the subunits of complex I from a variety of

where few amino acid residues are consewed (18).

Nevertheless, the comparable levels of Qcr7 protein seen in most mutants by themselves do not rule out the possibility that this protein is partially degraded and that the assembly defect of complex III follows from that. However, the levels of the other subunits of the complex speak against an assembly defect as a result of a misfolded Qcr7 protein. When comparing the degree of assembly to that in the mutant strain YSM-qcr7A, in which a Qcr7 protein is not synthesized, the following observations speak for an increase in the assembly of a subcomplex or cornplex III itself (Fig. 4-4): i) elevated levels of 11 kDa subunit in al1 mutants when compared to YSM-qcr7a; ii) elevated levels of mature ISP in the respiration-deficient mutants containing Qcr7p-~7(D13V), Qcr7p- A14, and Qcr7p~20when compared to YSM-qcr7A; iii) elevated levels of mature cytochrome c, in some of the respiration-deficient mutants when compared to YSM-qcr7A. Taken together, these results speak against a mechanism involving total misfolding followed by degradation independent of the other subunits of the complex, as this would give levels of 11 kDa subunit, ISP and cytochrome c, identicai to those seen in YSM-qcr7A.

One could possibly argue that truncation of the Qcr7 protein N- terminus alters the state of acetylation of the truncated proteins as opposed to the wild type and that this could influence the assembly process. This seems unlikely, however. According to Persson et a/.

(167) Ser and Ala are the most common residues at position 1 in acetylated proteins, followed by Met. Furthermore, Asp, Glu, Ser, and Thr are common at position 2 and Lys is generally overrepresented in the N-terminal 10 residues of the 250 acetylated proteins that were characterized. When applying these guidelines to the wild type and truncated Qcr7 proteins, the wild type Qcr7 protein and Qcr7p-A14 are not candidates for acetylation; Qcr7p-~7, 410, and -A20 could potentially be acetylated, with Qcr7p-A7 being the most likely candidate. Since Qcr7p-~7is similarly functional in assembly to the wild type whereas Qcr7p-AI 4 and Qcr7p-A20 are similarly disruptive with respect to assembly, it is highly improbable that the potentially different states of acetylation are responsible for the observed assembly defect. In addition, it is not clear whether the Qcr7 protein is acetylated in the first place as mitochondrial proteins are less frequently acetylated than their non-mitochondrial isozyme counterparts. This makes sense since acetylation is dependent on the availability of acetyl Co-A which in turn is decreased when the tricarboxylic acid cycle is active or when yeast are grown under aerobic conditions (169).

Now, that the implication of the Qcr7 protein N-terminus in assembly has been established, a model is proposed on the basis of these findings which is compatible with the observations. Consider the results obtained for the 11 kDa subunit: it is present at wild type levels in the mutant expressing Qcr7pd7, at intermediate levels in mutants containing Qcr7p-AI 4 and Qcr7p-AZO, but this subunit is drastically lowered in strains with Qcr7p-a7(R101/GlZV) and Qcr7p-~7(D13V). This observation suggests that the putative interaction between the Qcr7 protein and the 11 kDa subunit occurs beyond residue 8. In light of the proposed formation of a subcomplex between cytochrome b, the Qcr7 protein, and the 11 kDa subunit (339

4% 559 60-631, this may suggest that the potentially destabilized complex that is seen for the mutant containing Qcr7pa7, is the result of a disruption of the putative interaction between cytochrome b and the 14 kDa subunit. Examination of the data obtained for the mutant containing Qcr7pd7(D13V) confirms the above model. In this mutant the levels of 11 kDa subunit are significantly lowered which can be explained by the substitution of Val-13 for Asp-13 which could lower the extent of the putative interaction of the Qcr7 protein and the 11 kDa subunit. Because of the potential repulsion between the Qcr7p-~7(D13V)and the 1 1 kDa subunit in addition to the potentially destabilized interaction between the Qcr7p-~7(D13V)protein and cytochrome b as a result of the N-terminal truncation, further assembly into a multimeric complex III is presumably unstable and the degree of assembly is therefore decreased. This notion is conf irmed by the spectrophotometric analyses of holocytochrome b, which indicate that cytochrorne b maturation does not occur to detectable levels in this mutant, and by the lowered levels of total apocytochrome cl, holocytochrome cl, and mature ISP. Now consider the data obtained for the mutant containing the Qcr7p-~7(RlOI/G12V) in light of the postulated hypothesis. Of al1 mutants with detectable Qcr7 proteins in the mitochondria, this mutant has the lowest levels of 11 kDa subunit, but has intermediate levels of cytochrome b. The more severe putative inhibitory effect between Qcr7p-~7(RlOl/GlZV) and the 1 1 kDa subunit, as compared to that between Qcr7p-~7(D13V) and the 11 kDa subunit, may be explained by a "double" severe negative effect due to the creation of two hydrophobic residues. The higher level of holocytochrome b seen in the mutant with Qcr7p- ~7(R101/G12V) as opposed to that with Qcr7p-~7(D13V),might be explained by a closer proximity of Arg-1 O tu cytochrome b than Asp- 13. Hence, in this mutant, as opposed to the mutant with Qcr7p- ~7(D13V),the newly created lie-1 0 (and perhaps Val-1 2) may repel the 11 kDa subunit, but, on the other hand, may strengthen the putative hydrophobic interaction with cytochrome b. This more stable interaction with cytochrome b in the mutant containing Qcr7p-d7(Rl OI/G1 ZV), as opposed to the mutant containing Qcr7p b7(D13V), results in a higher degree of assembly of a multimeric complex III and cytochrome b maturation, despite the relatively lower abundance of the 11 kDa subunit. The higher levels of cytochrome c, and ISP in the mutant containing Qcr7p- ~7(R101/G12V) as compared to YSM-qcr7~are in agreement with the hypothesis of an increase in the degree of assembly. The finding that cytochrome c, is present at relatively higher levels than the 11 kDa subunit when compared to the wild type may seem to contradict

the hypothesis of Hemrika et al. (49) who suggest that the C- terminus of the 11 kDa subunit anchors the cytochrome c,

subcomplex (45. 47, 65). However, the 11 kDa subunit does not constitute the only contact site of the cytochrome c, subcomplex with the bc, complex.

So how does the above model explain the findings of mutants expressing Qcr7p~14and Qcr7p-AZO? These mutants display an intermediate levei (approximately 60%) of 11 kDa protein. This fact reinforces the notion stated earlier, that the contact site between the Qcr7 protein and the 11 kDa subunit is beyond residue 8. One may wonder why the interaction between Qcr7pA14, Qcr7p-A20 and the 11 kDa subunit is stronger than that between the 11 kDa subunit and Qcr7p-~7(D13V)or Qcr7p-~7(RlOVG1 ZV), since Arg-1 O, Gly-12, and Asp-13 are absent. Firstly, this could be taken as evidence for, and could be explained by, the existence of another contact site (beyond residue 15) between the Qcr7 protein and the 11 kDa subunit. Secondly, the putative repulsive effect that is present between the 11 kDa subunit and the mutant proteins Qcr7p-A7(RlOI/G12V) and Qcr7p-a7(D13V), de facto, is absent in Qcr7p-A14 and Qcr7p-~20. The observation that mutants with Qcr7p-~14and Qcr7pA20 have significant levels of 11 kDa subunit, speaks for an association of the Qcr7 protein and the 11 kDa subunit and against total misfolding of Qcr7p-a14 and Qcr7pa20. The observation that the mutant with Qcr7p-a20 has ISP and cytochrome c, levels comparable to YSM- qcriA, whereas the level of 11 kDa subunit is significantly higher, speaks for a direct interaction of the Qcr7 protein with the 11 kDa subunit. The levels of ISP and cytochrome c, which are as low as in YSM-qcr7~, furthermore constitute circumstantial evidence that holocytochrome b is also undetectable in this mutant as it is in YSM- QC~~A(33). Hence, taken together al1 the evidence suggests that the mutant containing Qcr7p-~20is presumably the most compromised of al1 the mutants that contain detectable Qcr7 protein in the mitochondria. This argument is indirectly supported by the inability of this mutant to grow on non-repressive carbon sources, such as galactose.

The results of the current work suggest that the N-terminus of the Qcr7 protein is involved in the assembly of complex III. Although residues 1 to 7 (of the mature protein) seem tu play a role in the assembly process, they are not crucial for the formation of a functional bc,complex. In contrast, the region between residues 8 and 20 of the mature Qcr7 protein is essential for the functional assembly of ubiquinol-cytochrome c oxidoreductase. In addition to the role of the Qcr7 protein N-terminus in assembly, residues 2 to 8 (of the mature protein) may facilitate import into mitochondria or confer stability to the protein. The elucidation of the function of the subunit 7 N-terminus with respect to assembly may explain why this region is retained as part of the mature protein rather than being cleaved upon import into the mitochondria (if indeed it is involved in import). Future Directions

The current study indicates that the amino-terminus of subunit 7 of the yeast bc, complex is essential for the formation of a subcomplex consisting of cytochrome b, the 14 kDa and the 11 kDa subunits. Without the formation of this subcomplex a functional multimeric enzyme complex is not formed and mutant yeast display a respiration-deficient phenotype. In addition to the proposed role in assembly, the N-terminus of the Qcr7 protein is suggested to increase the efficiency of the localization of subunit 7 into mitochondria. Further studies can be perforrned to confirm the predicted functions of the Qcr7 protein amino-terminus.

Confirmation of assembly of the Qcr7 protein N-terminus with cytochrome b and/or the 17 kDa subunit and identification of contact sites Confirmation of the putative interaction between cytochrome b and the Qcr7 protein, as well as between the 11 kDa subunit and the Qcr7 protein can be performed by CO-immunoprecipitation procedures. The use of different conditions could not only confirm the CO-precipitation of the components of the cytochrome b subcomplex, but could potentially identify other subunits of complex III that are in contact with either cytochrome b, the 14 kDa subunit, or the 11 kDa subunit, respectively. For successful immunoprecipitation it is important to obtain a good antibody. Although obtaining a good antibody requires some luck, the investigator should take into account the following information when choosing the peptide that will be used to obtain the antibody. The anti-Qcr7 protein antibody used in the immonoprecipitation procedures of this study did not seem to CO-precipitate any other subunits. This might be because the C-terminus (which contains the epitope for the antibody) of the 14 kDa protein is buried in the interior of complex III and/or because the C-terminus is believed to

anchor the core protein subcomplex (49. 66). Since the middle part of the Qcr7 protein might be involved in forming a membrane-spanning

segment (481, raising an antibody against a region immediately following the Qcr7 protein N-terminus would be a plausible possibility. Alternatively, it might be more useful to raise an antibody against cytochrome b or the N-terminus of the 11 kDa subunit. The N-terminus of the 11 kDa subunit would probably be a better choice than the C-terminus of the 11 kDa subunit, which has been suggested to be located either on the matrix side of the IMM (5%

65) or on the IMS side of the IMM (66. 671, because the C-terminus has

been proposed to anchor the cytochrome c, subcomplex (49). Raising an antibody against cytochrome b is difficult owing to the hydrophobicity of this protein and was unsuccessfully attempted during this study. An antibody against cytochrome b would open up a multitude of experimental possibilities (see below).

The residues involved in the interaction between the Qcr7 protein N-terminus and cytochrome b and/or the 11 kDa subunit might be identified by the yeast "two-hybrid system". In this experimental system, the Qcr7 protein amino-terminus can be used to assay for the interaction with various regions of the 11 kDa subunit and cytochrome b. The advantage of this system is the inherent HA epitope tagging that can be used to detect fusion proteins by Western blot analysis with commercially available antibody. Hence, antibodies against the subcloned fragments are not required. The disadvantage of the two-hybrid system is that it is based on the solubility of proteins in aqueous environment. This could be problematic for membrane proteins or for protein-protein interactions that occur in a hydrophobic environment.

An anti-cytochrome b antibody can be used to further clarify some of the results obtained in this study. Assaying for the levels of apocytochrome b by Western blot analysis, versus holocytochrome b by spectral analysis, could be used to gain further insight into the mechanism of assembly. If results indicate that the levels of apocytochrome b are low in strains expressing the N-terminally truncated proteins of this study, then the respective truncated regions of the Qcr7 protein could be implicated in the direct assembly with cytochrome b. If, however the results showed that apocytochrome b is present at significantly higher steady-state levels than holocytochrome b, then this would implicate the Qcr7 protein N-terminus in assembly at the level of cytochrome b maturation. In this case the Qcr7 protein N-terminus might be involved in the stabilization of the cytochrome b heme environment so as to maintain it in a state that is competent for the insertion of heme/s. Testing for the involvement of the Qu7 protein N-terminus in mitochondrial irnport Based on the results of this study, the N-terminal seven amino acids (after Met-1) of the Qcr7 protein have been implicated in mitochondrial import of this subunit. The truncation of seven or more residues from the N-terminus resulted in decreased mitochondrial steady-state levels of the mutated Qcr7 proteins from strains overexpressing the mutated genes as compared to the strain overexpressing the wild type QCR7 gene. The functional deficiencies observed in some of these strains expressing mutated proteins cannot be attributed to the decreased levels of subunit 7 in the mitochondria, as they are no lower than the levels observed in the parental strain in which the QCR7 gene is not overexpressed. Nevertheless, it is interesting to investigate the cause for the observed lowered steady-state levels.

To determine whether any of the lowered levels of the mutated Qcr7 proteins are due to a Iowered efficiency of mitochondrial import or to increased degradation, one could radioactively label cytoplasmic proteins in vivo with [3SS]-methionine. After chasing proteins for various times with cold medium, immunoprecipitation, and SDS PAGE, one could distinguish whether the mutated Qcr7 proteins are accumulating in the cytoplasm and subsequently degraded due to a decreased rate of import, or whether the mutant proteins are imported at equal rates to the wild type, but degraded faster inside the mitochondria due to an instability as a result of mutation. An alternative to determining the efficiency of import in vivo, is to radiolabel proteins by in vitro translation in the presence of reticulocyte lysate. Purified precursor protein can then be added to mitochondria at increasing concentrations in a standard import

mixture according to Millar and Shore (171). Following SDS PAGE, import into mitochondria can then be tested for by resistance to externally added proteases and the extent of import can be determined by fluorography. To test whether the N-terminus of the Qcr7 protein contains a mitochondrial targeting sequence, one could fuse the N-terminal 21 amino acids of subunit 7 to a non- mitochondrial passenger protein such as the cytosolic mouse dihydrofolate reductase (DHFR) (37). AS above, this import assay could also be performed by in vitro synthesis of the chimeric Qcr7- DHFR protein. Again, localization into mitochondria can then be tested for by the resistance to externally added proteases.

An in vitro assay system can also be used to determine whether the mutated Qcr7 proteins that are localized in the mitochondria reach their final destination in the IMM or whether they are accumulating in the matrix. Depending on whether mitochondria or mitoplasts (mitochondria which lack the OMM) are used in the in vitro assay in combination with protease resistance, one can determine whether the Qcr7 proteins are associated with the IMM or present in the matrix (142). According to Japa et al (142) cytochrome b is required for the association of the Qcr7 protein with the membrane. Hence, if the mutated Qcr7 proteins are shown to be localized in the matrix rather than in the membrane, then this would further implicate the N-terminus in the direct assembly with cytochrome b. If, however, the mutated Qcr7 proteins are localized to the IMM, the obsewed assembly defect would more likely be at the level of cytochrome b maturation as discussed in the assembly section above.

Further mutagenesis of the Qcr7 protein Further mutational analysis of the Qcr7 protein in the context of the full-length protein or in the context of a a7 truncation, can be performed to confirm sorne of the predictions from this study. For example, to identify which of the two residues, if not both, are responsible for the pet- phenotype in mutants with Qcr7p- ~7(R101/G12V) and Qcr7p-A7(RlOT/K44N), one would construct mutants with full-length proteins Qcr7p-G1 ZV and Qcr7p-K44N. Should these mutants display a wild type phenotype, then it would be of interest to construct two mutants with the same residue substitutions in the context of Qcr7p-~7. Should these mutants compare to the wild type as well, then the issue of whether substitutions for Arg-1 O or both Arg-1 O and Gly-lZ/Lys-44 together are causing the pet- phenotype could be resolved by constructing mutants with Qcr7p-~7containing substitutions for Arg-10. In addition to point substitutions at the above residues, it would also be of interest to construct a mutant with the full-length Qcr7p-D'i3V. This will resolve the question as to whether point mutations are only informative in the context of Qcr7p-A7, or whether they can disrupt assembly in the context of a full-length protein. In addition, this will identify potentially important residues.

If further analysis of the Qcr7 protein is desired, it might be more informative in the future to perform random mutagenesis of the QCR7 gene rather than site-directed mutagenesis, since very few of the mutants in this study displayed a respiration-deficient phenotype. This can be performed by a number of methods, including

UV irradiation, PCR, and alanine scanning (1 57# 58). Alternatively, further site-directed mutagenesis can be employed to mutate consewed residues as determined by the comparison of homologues of the Qcr7 protein (146). A preliminary screen for respiration- deficient mutants can be performed by growth analysis on non- fermentable carbon sources. Deficient mutants can then be further characterized by the various methods outlined in the results chapters and in the Future Directions of this dissertation. In order to circumvent the issue of glucose repression which results in a significant decrease of complex Ill-linked enzyme activities and in the inability to perform spectra of the cytochromes (see Chapter 4), mutant genes can be permanently integrated into the genome by homologous recombination (as described in Chapter 2). This would elirninate the need for selective growth in minimal media and the mutant cells could be grown in rich medium containing non- repressive carbon sources such as galactose. Further mutational analysis of the Qcr7p-~7amino-terminus (in which residues 2 to 8 are deleted) has been performed by substituting residues 9 to 15 with a poly-alanine segment (So-Young Lee, personal communication). In this experiment the Qcr7 protein is not detectable in the rnitochondria and the assembly phenotype of the mutant strain is comparable to that in YSM-qcr7a. REFERENCES REFERENCES

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