THE ROLE OF COX20 IN COX2 MATURATION AND

CYTOCHROME C OXIDASE ASSEMBLY

by

Elliott Carter Ferris

A thesis submitted to the faculty of The University of Utah

in partial fulfillment of the requirement for the degree of

Master of Science

Department of Biochemistry

University of Utah

December 2011

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Provided by The University of Utah: J. Willard Marriott Digital Library

Copyright © Elliott Carter Ferris 2011

All Rights Reserved

T h e U n i v e r s i t y o f U t a h G r a d u a t e S c h o o l

STATEMENT OF THESIS APPROVAL

The thesis of Elliott Carter Ferris

has been approved by the following supervisory committee members:

Dennis R. Winge , Chair 10/27/11

Date Approved

Chris Hill , Member 10/27/11

Date Approved

Martin Horvath , Member 10/28/11

Date Approved

and by Chris Hill , Chair of

the Department of Biochemistry

and by Charles A. Wight, Dean of The Graduate School.

ABSTRACT

Proper assembly of Cytochrome c Oxidase (CcO) is vital to mitochondrial

respiration. The metallation and incorporation of CcO subunit 2 (Cox2) are important

assembly steps that remain poorly understood. The Cox2 assembly factor Cox20 may

provide a unique window on CcO assembly. Cox20 is known to bind Cox2 before the

latter is incorporated into the larger CcO assembly in S. cerevisiae. Conservation of

Cox20 in higher organisms and the lethality of its deletion in Drosophila melanogaster

may suggest Cox20 plays a conserved role in CcO assembly.

In an attempt to understand the nature of the hypothesized conserved role of

Cox20, conserved residues within Cox20 were mutated and the resulting mutants studied.

Mutation of a conserved cysteine pair impacted mitochondrial respiration, but did not

block CcO assembly. The mutant Cox20 C87A forms a mixed disulfide species.

Identifying the partner molecule may shed light on the role of Cox20.

The present study finds Cox20, a 23 kD protein, in high molecular weight

complexes that are unlikely to be homo-oligomeric. Identifying any other proteins in

these complexes may shed light on the role Cox20 plays in CcO assembly. To this end,

Cox20 was purified and analyzed by mass spectrometry (MS) to identify any

accompanying proteins. Small quantities of the known CcO assembly factors Mss2,

Coa1, and Mss51 were identified.

TABLE OF CONTENTS

ABSTRACT ............................................................................................................. iii

Chapter

1 COX2 AND COX20 IN CCO ASSEMBLY ................................................... 1

Intoduction ..................................................................................................... 1 Cytochrome c Oxidase Assembly ................................................................... 2 Cox2 Maturation............................................................................................. 4 Cox20............................................................................................................. 4

2 THE ROLE OF COX20 in CCO ASSEMBLY ..............................................10

A Conserved Role for Cox20.........................................................................10 Characterization of the Role of Cox20 in CcO Assembly...............................11 Mutational Analysis of Cox20 .......................................................................11 Identify Components of the Cox2-Cox20 Pre-Assembly Intermediate............13 Progression of the Cox2-Cox20 Interaction....................................................15 Conclusions ...................................................................................................16

REFERENCES..............................................................................................25

CHAPTER 1

COX2 AND COX20 IN CYTOCHORME

C OXIDASE ASSEMBLY

Introduction

Mitochondria are host to important metabolic reactions including oxidative

phosphorylation (OXPHOS), which generates the bulk of cellular ATP. OXPHOS relies

on the respiratory chain, a series of membrane embedded protein complexes, to couple

electron transfer to the movement of H+ ions across the mitochondrial inner membrane

(IM). The resulting electro-chemical gradient is harnessed by another membrane

embedded complex, ATP synthase, to generate ATP. Mitochondrial diseases are marked

by defects in OXPHOS and occur in 1 in 5000 humans.1 These diseases affect tissues

with high energy demands such as nerves and muscles. Mitochondria contain a separate

genome that encodes key mitochondrial proteins, but the bulk of mitochondrial proteins

are nuclear encoded proteins that are imported from the cytosol. Nuclear encoded

assembly factors are required for the complicated assembly of respiratory complexes,

coordinating the incorporation of mitochondrial and nuclear encoded components as well

as cofactors. Mitochondrial disease thus may arise from mutations in mitochondrial or

nuclear encoded components of respiratory complexes, or nuclear encoded respiratory

complex assembly factors. Autosomally inherited mitochondrial diseases are often

2

caused by defects in the assembly of respiratory complexes. The study of the

complicated assembly of respiratory complexes is thus vital to understanding these

mitochondrial diseases.

Cytochme c Oxidase Assembly

The terminal enzyme in the mitochondrial respiratory chain is cytochrome c

oxidase (CcO or Complex IV). CcO catalyzes the transfer of electrons from the electron

carrier cytochrome c to oxygen and uses the resulting energy to move protons across the

IM. This enzyme is comprised of eleven subunits in Saccharomyces cerevisiae and

thirteen in humans. Three core subunits, Cox1-3, are encoded in the mitochondrial

genome while the remaining nuclear encoded subunits are imported into mitochondria.

Cox1-3, play an active role in catalysis and require a number of cofactors. The maturation

of the CcO holoenzyme thus requires insertion of three copper atoms and two modified

heme cofactors. In the assembled enzyme, the binuclear copper A (CuA) site in the

soluble domain of subunit 2 (Cox2) conveys electrons from the electron carrier

cytochrome c in the inner membrane space (IMS) to a catalytic center within the

membrane-integrated portion of subunit 1 (Cox1). This site relies on the heme moieties

heme a and heme a3 and a second copper site (CuB) to catalyze the final step of the

electron transport chain, the reduction of oxygen to water on the matrix side of the

membrane. The dual genetic origin of the CcO subunits and the potential toxicity of the

required cofactors demand a highly regulated assembly process involving a number of

nuclear encoded assembly factors.2, 3

Defects in complex assembly are a common cause of electron transport chain

related disease in humans.3 The human encephalomyelopathy, Leigh’s syndrome, for

3

example, is defined by compromised mitochondrial respiration affecting the central

nervous system. Autosomally inherited mutations responsible for such disorders often

affect factors important for the assembly of respiratory complexes as opposed to subunits

present in the mature enzyme. Mutations in the CcO assembly factors SURF1 (Shy1 in

S. cerevisiae), SCO1, and SCO2, for example, cause severe disorders including Leigh’s

syndrome.4, 5 Study of Complex IV is thus now largely focused on the enigmatic

assembly of the enzyme.

CcO assembly likely occurs in a sequential manner.3 Cox1 is translated on

mitochondrial ribosomes and inserted into the mitochondrial inner membrane (IM) as it is

translated. The first definable Cox1 containing intermediate includes the assembly

factors Mss51 and Cox14.6 Mss51 is lost as Cox1 progresses to Shy1 (SURF1)

containing intermediates.5 The maturation of Cox1 within Shy1 containing intermediates

requires the insertion of heme cofactors, the maturation of the CuB site, and the

incorporation of additional subunits.

Cox2 is presumably incorporated into a Cox1 containing intermediate after the

heme moieties and the CuB site are introduced (in the crystal structure of bovine CcO, it

appears that Cox2 would block access to the CuB and heme sites).7 Cox2 itself contains a

binuclear copper site, but whether this site is formed before or after incorporation into the

larger Cox1 assembly is unclear. The importance of Cox2 maturation to human health is

underscored by the disease caused in humans by mutations in the proteins SCO1 and

SCO2; these copper chaperones function in maturing the CuA site.4, 8

4

Cox2 Maturation

Cox2 is also translated on mitochondrial ribosomes recruited to the mitochondrial

inner membrane.9 The N-terminus of the nascent polypeptide is inserted into the inner

membrane as it exits the ribosomal tunnel by the ribosome-associated translocase Oxa1

(Figure 1A).10 Once the N-terminus is inserted into the membrane, Cox2 is bound by the

membrane integrated chaperone Cox20.11 In S. cerevisiae, Cox20 is required for the

proteolytic processing of an N-terminal leader peptide of Cox2 by the inner membrane

peptidase complex Imp1/Imp2 (Figure 1B). The larger C-terminal domain is translocated

across the IM by the Oxa1 paralog Cox18 and the assembly factor Mss2 (Figure 1C).12

Cox20 remains associated with Cox2 after proteolytic processing (Figure 1D). Cox2 is

ultimately loaded with two copper atoms, presumably by the copper chaperone Sco1, and

incorporated into the Cox1 subassembly. The soluble IMS copper chaperon Cox17

metallates the IM integrated chaperone Sco1, which in turn metallates Cox2.13 Whether

CuA site maturation takes place before or after the Cox1 assembly incorporates Cox2 is

unclear.

Cox20

Cox20 is a nuclear encoded protein containing two transmembrane domains and

adopts an N-out C-out topology in the IM. Cox20 is conserved in higher eukaryotes

(Figure 2).8 A conserved cysteine pair flanks the two transmembrane helices. Hell et al.

previously explored the role of Cox20 in CcO assembly.11 Their work indicates that

Cox20 is necessary for the proteolytic processing of Cox2 by Imp1/Imp2, a step specific

to yeast, and that Cox20 and Cox2 are associated after proteolytic processing.11

5

Another study suggests that Cox20 may play a role in the degradation of excess

Cox2. The IMS oriented i-AAA protease Yme1 degrades Cox2.14 Mutations

compromising a putative substrate recognition motif in Yme1 render its degradation of

Cox2 dependent on Cox20.10 Cox20 thus may accompany Cox2 through maturation steps

as well as degradation.

Several lines of evidence suggest the existence of a role for Cox20 in Cox2

maturation beyond proteolytic processing of Cox2. In S. cerevisiae, a further role is

suggested by the persistence of an association between Cox2 and Cox20 after proteolytic

processing. Furthermore, Cox20 is conserved in species lacking the proteolytic

processing step observed in yeast. Indeed, Cox20 is conserved from yeast to mammals

while the leader peptide-processing step is specific to certain fungi. Additional evidence

for a conserved role for Cox20 in Cox2 maturation comes from the observation that

degradation of Cox2 by a mutant Yme1 is dependent on Cox20, suggesting Cox20 may

accompany Cox2 through maturation as well as degradation.10 While Cox20 seems likely

to play a conserved role in CcO assembly, the nature of this role is unclear.

The placement of the conserved cysteine pair of Cox20 on the IMS side of the

inner membrane may suggest the formation of a disulfide bond. The probable proximity

of these cysteines to the soluble domain of Cox2 coupled with the propensity of cysteine

pairs to traffic reducing equivalents and copper ions raises the possibility that these

residues assist in the maturation of the CuA site.

Previous efforts to elucidate Cox2 maturation have failed to answer basic

questions about Cox2 maturation. Many of these studies have focused on understanding

6

the role of the copper chaperone Sco1 in maturing the CuA site. Studies focused on

Cox20, which is known to associate with Cox2, may prove more revealing

7

Figure 1. Cox2 maturation. A) The N-terminus of Cox2 is cotranslationally inserted into the inner membrane by the translocase Oxa1.10 B) The assembly factor Cox20 associates with Cox2 allowing, in S. cerevisiae, proteolytic processing by the Imp1/Imp2 protease complex. C) Cox18 translocates the C-terminal domain of Cox2 across the mitochondrial inner membrane. In yeast, this process depends on the assembly factor Mss2. D) Cox20 remains associated with Cox2. Cox2 must subsequently be metallated, presumably by the copper chaperone Sco1, and incorporated into the larger CcO assembly. The mechanisms of Cox2 metallation and incorporation into CcO remain unclear.

8

IMS

A

Nacent Cox2 polvpeptide

I. " ~ I ~ o

Mitochondrial Ribosome

B

Cox2 mRNA

Matrix

C

---+ 111111 ~---+ 1. 2

Cox20 \

mature Cox2 precursor

D

S,," I @

Cox2

...: ~/ 'l£~_ ",- 8 ~_

IMS - ,.,,., 1<"," ,';>' -,.,-~ /~1H::1.'Q

'V' '''J u-r ~ Matrix Cox20 -r~' Cytochrome c ~ He,,", 8 3 S CUB U Oxidase

eo., Cox I sub-assembly

9

Figure 2. Cox20 sequence alignment and topology. Cox20 contains two transmembrane segments, indicated in blue, flanked by a conserved cysteine pair, indicated in red.

10

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11

CHAPTER 2

THE ROLE OF COX20 IN CCO ASSEMBLY

Perspective

Mutations causing defects in CcO assembly cause serious human diseases like

Leigh’s syndrome. Elucidating CcO assembly will be vital to understanding the

mechanisms behind these diseases. The incorporation of the CcO subunit Cox2 into a

larger subassembly and its metallation by the copper chaperone Sco1 (SCO1 in humans)

are poorly understood. The importance of these steps to human health is underscored by

the severity of diseases caused by mutations in SCO1.15 The assembly factor Cox20 may

provide a new avenue to a better understanding of these steps. Cox20 has previously

been found to associate with Cox2 before it is incorporated into the larger CcO

assembly.11 In S. cerevisiae, Cox20 is required for a proteolytic processing step important

in Cox2 maturation, but there is evidence to suggest a wider role for Cox20 in CcO

assembly.

A Conserved Role for Cox20

The association between Cox2 and Cox20 persists after proteolytic processing of

Cox2.11 This may indicate a role for Cox20 in CcO assembly beyond proteolytic

12

processing. This possibility is also suggested by the conservation of Cox20 in species

lacking the proteolytic processing step observed in S. cerevisiae. Indeed, Cox20 is

conserved from yeast to mammals while the leader peptide-processing step is specific to

yeast.8 Further evidence for a larger role for Cox20 in Cox2 maturation comes from a

study suggesting a role for Cox20 in the degradation of Cox2. The IMS oriented i-AAA

protease Yme1 degrades Cox2.14 Mutations compromising a putative substrate

recognition motif in Yme1 render its degradation of Cox2 dependent on Cox20.10 Cox20

thus may accompany Cox2 through maturation steps as well as mediate its degradation.

While Cox20 seems likely to play a conserved role in CcO assembly, the nature of this

role is unclear.

Cox20 is conserved in higher eukaryotes (Figure 2).8 A conserved cysteine pair

flanks two transmembrane helices. The placement of this cysteine pair in on the IMS

side of the inner membrane may suggest the formation of a disulfide bond. Their

probable proximity to the soluble domain of Cox2 coupled with the propensity of

cysteine pairs to traffik reducing equivalents and copper ions raises the possibility that

these residues assist in the maturation of the CuA site.

Characterization of the Role of Cox20 in CcO Assembly

Mutational Analysis of Cox20

Cox20 is required for the N-terminal processing of Cox2 in S. cerevisiae.11 While

this processing step is not required in higher eukaryotes, Cox20 is nonetheless conserved.

Additionally, Cox2 remains associated with Cox20 after N-terminal proteolysis

suggesting a role for Cox20 in CcO assembly beyond Cox2 processing. Elucidating this

13

conserved role would be facilitated by identifying Cox20 mutations that compromise

CcO assembly in S. cerevisiea without affecting the proteolytic processing of Cox2.

Results

With the goal of identifying Cox20 mutations that impact the hypothesized

conserved function of the protein, I considered Cox20 residues conserved in higher

eukaryotes for mutational analysis. Conservation across eukaryotic species is limited to a

pair of transmembrane helices and surrounding residues (Figure 2). A conserved cysteine

pair is additionally interesting because of its predicted location in the IMS side of the

inner membrane and its potential for redox chemistry and copper trafficking. These

cysteines are partially reduced in wild-type yeast and thus could conceivably participate

in trafficking reducing equivalents or copper ions, activities that might aid in the

maturation of the CuA site (Figure 3).

Mutation of either cysteine residue to serine results in diminished CcO activity

(Figure 4A, C). In vivo labeling experiments demonstrate that mutation of cysteine 87 to

serine allows proteolytic processing of Cox2 while this activity is not observed in cox20∆

strains (Figure 4B). This raises the possibility that diminished CcO activity observed in

the Cox20 C87S mutant is due to an assembly defect unrelated to impaired Cox2

processing. An apparent intermolecular disulfide species observed in Cox20 C87S

suggests redox activity in this cysteine pair (Figure 5). Mutating cysteine 135 or

mutating both cysteines affects CcO activity rates.

14

Future Directions

Identifying the unknown disulfide partner of Cox20 C87S may shed light on the

activity of Cox20. To identify this protein I have purified the adduct species (Figure 5B).

Ectopically expressed Cox20-13myc C87S was purified by immunoprecipitation.

Alternatively, a two-step purification of Cox20-MBP C87S may afford higher purity.

MS analysis will identify any partner peptide.

Identify Components of the Cox2-Cox20 Pre-Assembly Intermediate

Perspective

Later steps in the maturation of the CcO subunit Cox2 are poorly understood.

After its insertion into the IM but before its incorporation into CcO, Cox2 exists in

complex with the chaperone Cox20.11 Identifying any additional components of the

Cox2-Cox20 pre-assembly complex may shed light on steps required for the

incorporation of Cox2 into CcO.

Results

Cox20 is detected mainly in large complexes by sucrose gradient

ultracentrifugation and clear native PAGE (CN-PAGE) analysis (Figure 6A-B). The

composition of these complexes, however, is unknown. Homo-oligimerization of Cox20

is unlikely to account for the size of these complexes; purification of a Cox20-TMBP

fusion protein from a diploid strain coding for both Cox20-MBP and Cox20-13Myc

proteins fails to capture the preponderance of the 13Myc species (Figure 6C). CN-PAGE

analysis of mitochondria purified from the same diploid strain show distinct migration

15

rates for Cox20-MBP and Cox20-13Myc species, suggesting that the major Cox20

species is not a homo-oligomer (Figure 6B).

In order to purify the Cox2-Cox20 intermediate, I have modified endogenous

COX20 by homologous recombination, to code for a protein with Escherichia coli

maltose binding protein (MBP) and a polyhistidine tag fused to its C-terminus. This

strain is respiratory competent, unlike the cox20∆ mutant strain, suggesting a functional

Cox20 fusion protein (Figure 7). I have purified this fusion protein using nickel and

amylose affinity chromatography. Mass spectrometry analysis (MS) of purified proteins

confirms a Cox20-Cox2 interaction and suggests that Cox20 may interact with small

quantities of the CcO assembly factors Mss51, Mss2, and Coa1. Further work is needed

to verify whether these factors indeed associate with Cox20.

Future Directions

While subtractive logic suggests that Cox20 species identified by CN-PAGE and

ultracentrifugation contain other factors, these factors have yet to be identified. Future

efforts should focus on purifying sufficient quantities of large Cox20 containing complex

for identification of unknown factors by MS. Cox20-MBP could be purified, for

example, by amylose affinity chromatography, and the resulting eluate resolved by CN-

PAGE before MS analysis. It is also possible that the unidentified factors in large Cox20

containing complexes are not proteinaceous. Nuclease treatment experiments could

determine whether Cox20 containing complexes contain a nucleic acid component.

16

Progression of the Cox2-Cox20 Interaction

Overview

Cox2 associates with Cox20 prior to its incorporation into a Cox1 containing

intermediate. A clearer characterization of the progression of this interaction will

contribute to our understanding of Cox2 maturation. To better define the formation and

progression of the Cox2/Cox20 intermediate, I have purified Cox20-TMBP from yeast

with deletions designed to stall CcO assembly at various steps.

Results

While steady-state levels of Cox2 are reduced in ∆Cox4 cells, most of the existing

Cox2 associates with Cox20 (Figure 8). The existence of a Cox20-Cox2 complex in

sco1∆ cells suggests that Sco1 is not necessary for the formation of the Cox20-Cox2

complex. In the absence of Cox18, The C-terminus of Cox2 cannot be inserted into the

membrane and is quickly degraded. The presence of a Cox20-Cox2 complex under these

conditions indicates that it is the N-terminal transmembrane domain of Cox2 that

interacts with Cox20.

Future Directions

The stalled Cox20-Cox2 complexes in cox18∆, sco1∆, and, cox4∆ provide an

opportunity to address outstanding questions about Cox2 maturation. The intermediate in

which Cox2 is metallated, for example, is unknown. Identifying any additional

components of Cox20-Cox2 in the various stalled states would also elucidate the

progression of Cox2 maturation.

17

Conclusions

Studying the conserved role of the CcO assembly factor Cox20 may provide a new

insights into assembly steps involving Cox2. Clear understanding of the conserved role

of Cox20 is obscured in S. cerevisiae by a proteolytic processing step in the maturation of

Cox2 that requires Cox20—this step is not observed in other model organisms.

Identifying mutations in Cox20 that disrupt CcO assembly without affecting Cox2

processing would facilitate the study of Cox20. Alternatively, it may prove advantageous

to study the role of Cox20 in an organism lacking the proteolytic processing demanded

for Cox2 maturation in S. cerevisiae.

18

Figure 3. Redox state of Cox20 cysteine residies. Precipitated protein from purified cox20∆ mitochondria expressing Cox20-13myc were treated with the alkylating agent PEG-maleimide. Cox20-13 myc was visualized by immunoblotting. Cox20-13myc is apparent in both a reduced and oxidized form. Alkylation of reduced cysteines results in decreased electrophoretic mobility.

19

Figure 4. Mutational analysis of Cox20. A) Mutating the conserved residue C87 of Cox20 compromises growth on respiratory media at 37˚C. B) Cox20∆ yeast carrying low-copy expression vectors for Cox20-13Myc were treated with cycloheximide to inhibit cytosolic translation while mitochondrial translation proceeded for 20 min in the presence of [35S]methionine to selectively label mitochondrial translation products. Samples taken before and after 60 min of treatment with unlabeled methionine were analyzed by autoradiography. Cox20 C87S mutants are able to proteolytically process Cox2 while Cox20 C135S cells were severely impaired in processing. C) CcO activity analysis of isolated mitochondria. Yeast were grown at 30˚C and shifted to 37˚C 4 hours before harvest.

20

, [. , .

" ~ ~ •

~ > ~ •

«

! H J I Y I

I :1 C

, u

21

Figure 5. Stability of Cox20 mutants and purification of unknown intrermolecular disulfide species. A) Mitochondria isolated from cox20∆ cells transformed with COX20-13MYC vectors were solubilized in 1% digitonin and analyzed by western blot. B) To identify the disulfide binding partner of Cox20, Cox20-13myc was purified from 1% dodecyl maltoside (DDM) solubilized mitochondria by immuno-precipitation.

22

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« . < 0"

" ~~ 0 . ' ,

u

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4

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0

23

Figure 6. Cox20 is detected in large complexes. A) Sucrose gradient ultracentrifugation reveals Cox20-TMBP containing complexes in a high molecular-weight range. A potential Cox20-Cox2 complex appears in the range of 300-400 kDa. B) Analysis of mitochondria purified from a diploid COX20::TMBP, COX20::13-Myc strain by CN-PAGE show large Cox20 complexes. The visualized complexes don’t appear to be homo-oligomeric; MBP-tagged and 13Myc-tagged proteins travel at different rates. C) Cox20-TMBP purified from a diploid COX20::TMBP, COX20::13-Myc strain is accompanied by only a small fraction of Cox20-13myc, suggesting that only a fraction of Cox20 exists in a homo-oligomeric species.

24

H

~~ M < .. ~ ~ S S " ~

£ .. L U ~ g :E ~ 0 0 0 0 ~ ~ " I . I ""OIl • I

punoqUn I I I

..." I I I

• I I I I ' I

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«

25

Figure 7. Cox20 fusion proteins support respiratory growth. Yeast with endogenously tagged Cox20 respire on media containing the non-fermentable carbon source glycerol/lactate indicating that the fusion protein is functional in CcO assembly.

Figure 8. Cox20-TMBP purification. Mitochondria isolated from wild-type, cox4∆, sco1∆ and cox18∆ mutant yeast with endogenously MBP-Histidine tagged Cox20 were solubilized with digitonin and the fusion protein Cox20-MBP was purified by amylose or Ni affinity chromatography. Accompanying Cox2 was detected by immunoblotting.

26

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2. Barrientos, A.; Gouget, K.; Horn, D.; Soto, I. C.; Fontanesi, F., Suppression mechanisms of COX assembly defects in yeast and human: insights into the COX assembly process. Biochim Biophys Acta 2009, 1793, (1), 97-107.

3. Fontanesi, F.; Soto, I. C.; Horn, D.; Barrientos, A., Assembly of mitochondrial cytochrome c-oxidase, a complicated and highly regulated cellular process. Am J Physiol Cell Physiol 2006, 291, (6), C1129-47.

4. Cobine, P. A.; Pierrel, F.; Leary, S. C.; Sasarman, F.; Horng, Y. C.; Shoubridge, E. A.; Winge, D. R., The P174L mutation in human Sco1 severely compromises Cox17-dependent metallation but does not impair copper binding. J Biol Chem 2006, 281, (18), 12270-6.

5. Mick, D. U.; Wagner, K.; van der Laan, M.; Frazier, A. E.; Perschil, I.; Pawlas, M.; Meyer, H. E.; Warscheid, B.; Rehling, P., Shy1 couples Cox1 translational regulation to cytochrome c oxidase assembly. EMBO J 2007, 26, (20), 4347-58.

6. Pierrel, F.; Khalimonchuk, O.; Cobine, P. A.; Bestwick, M.; Winge, D. R., Coa2 is an assembly factor for yeast cytochrome c oxidase biogenesis that facilitates the maturation of Cox1. Mol Cell Biol 2008, 28, (16), 4927-39.

7. Gennis, R.; Ferguson-Miller, S., Structure of cytochrome c oxidase, energy generator of aerobic life. Science 1995, 269, (5227), 1063-4.

8. Cobine, P. A.; Pierrel, F.; Winge, D. R., Copper trafficking to the mitochondrion and assembly of copper metalloenzymes. Biochim Biophys Acta 2006, 1763, (7), 759-72.

9. Sanchirico, M. E.; Fox, T. D.; Mason, T. L., Accumulation of mitochondrially synthesized Saccharomyces cerevisiae Cox2p and Cox3p depends on targeting information in untranslated portions of their mRNAs. EMBO J 1998, 17, (19), 5796-804.

27

10. Kohler, R.; Boehringer, D.; Greber, B.; Bingel-Erlenmeyer, R.; Collinson, I.; Schaffitzel, C.; Ban, N., YidC and Oxa1 form dimeric insertion pores on the translating ribosome. Mol Cell 2009, 34, (3), 344-53.

11. Hell, K.; Tzagoloff, A.; Neupert, W.; Stuart, R. A., Identification of Cox20p, a novel protein involved in the maturation and assembly of cytochrome oxidase subunit 2. J Biol Chem 2000, 275, (7), 4571-8.

12. Saracco, S. A.; Fox, T. D., Cox18p is required for export of the mitochondrially encoded Saccharomyces cerevisiae Cox2p C-tail and interacts with Pnt1p and Mss2p in the inner membrane. Mol Biol Cell 2002, 13, (4), 1122-31.

13. Horng, Y. C.; Cobine, P. A.; Maxfield, A. B.; Carr, H. S.; Winge, D. R., Specific copper transfer from the Cox17 metallochaperone to both Sco1 and Cox11 in the assembly of yeast cytochrome C oxidase. J Biol Chem 2004, 279, (34), 35334-40.

14. Van Dyck, L.; Langer, T., ATP-dependent proteases controlling mitochondrial function in the yeast Saccharomyces cerevisiae. Cell Mol Life Sci 1999, 56, (9-10), 825-42.

15. Leary, S. C.; Kaufman, B. A.; Pellecchia, G.; Guercin, G. H.; Mattman, A.; Jaksch, M.; Shoubridge, E. A., Human SCO1 and SCO2 have independent, cooperative functions in copper delivery to cytochrome c oxidase. Hum Mol Genet 2004, 13, (17), 1839-48.