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Previous Article | Next Article 
Molecular and Cellular Biology, July 2007, p. 4664-4673, Vol. 27, No. 13
0270-7306/07/$08.00+0 doi:10.1128/MCB.01955-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
and
Thomas D. Fox*
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853-2703
Received 17 October 2006/ Returned for modification 22 January 2007/ Accepted 16 April 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Insertion of Cox2 into the inner membrane has been thought to occur cotranslationally (26) and results in the translocation of hydrophilic N- and C-terminal domains to the intermembrane space (IMS) anchored by two transmembrane domains. Nuclearly encoded Oxa1, the founding member of the conserved Oxa1/YidC/Alb3 family of membrane proteins (59), is required for translocation of both N- and C-terminal domains of Cox2 (21, 23). Following translocation of the precursor of yeast Cox2 to the IMS, an N-terminal leader peptide (15 residues) is cleaved by the Imp1/Imp2/Som1 protease complex (27, 38) in a reaction dependent upon the specific chaperone Cox20 (25). Two nuclear genes, COX18 and MSS2, appear to be specifically required for Cox2 C-tail export (6, 46). Cox18 is an integral membrane protein related to Oxa1 (41, 46, 48) that is also functionally conserved among animals and fungi (14). Mss2 is a peripheral membrane protein facing the matrix (6). A third integral inner membrane protein, Pnt1, appears to facilitate Cox2 C-tail domain export but is not essential in S. cerevisiae (22). Cox18, Mss2, and Pnt1 interact genetically and coimmunoprecipitate (46), suggesting that they function together.
The translocated C-tail domain of Cox2 is 144 residues in length and negatively charged. The machinery that recognizes and exports this domain from the matrix can also translocate a soluble passenger protein, Arg8m, when Arg8m is translationally fused to the Cox2 C terminus (21). However, when Arg8m was fused just downstream of the second transmembrane domain, replacing the C-tail domain, it remained in the matrix, suggesting that the Cox2 C-tail domain might contain signals necessary for its own translocation (21).
In this study, we have further investigated translocation of the N- and C-terminal domains of Cox2 through analysis of variants that lack the C-tail domain or portions of it. We constructed mutant cox2 genes encoding epitope-tagged variant proteins, inserted them into yeast mitochondrial DNA (mtDNA), and examined the behavior of their products. These studies revealed that the C-tail domain is not required for insertion of the transmembrane domains of Cox2 but does contain sequences that direct its own translocation across the mitochondrial membrane. Furthermore, we found that Mss2 interacts with newly synthesized full-length Cox2 but not a truncated Cox2 variant entirely lacking this domain. Our data suggest that the C-tail domain is recognized posttranslationally by a specialized translocation apparatus.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Yeast strains.
S. cerevisiae strains used in this study are listed in Table 1. All strains are congenic to D273-10B (ATCC 25657) except CM1, HLF5-22, SCS32, and NAB33, which are congenic to DBY947 (36). Genes encoding truncated, epitope-tagged variants of Cox2 were constructed in the vector pTZ18u (Bio-Rad), transformed by microprojectile bombardment into mitochondria of [rho°] strain NAB33, and inserted into [rho+] mtDNA by mating the transformants with the cox2 mutant NB40-3C followed by selection of recombinant cytoductants HLF3-60 {[rho+] cox2(1-109)::3xHA} and HLF4-13 {[rho+] cox2(1-211)::3xHA}. YME1 and OXA1 were deleted using deletion constructs pPT45 (52) and pNB60 (3), respectively. COX18 was disrupted using construct pG34/ST12 (48). MSS2 was disrupted with an mss2::LEU2 disruption cassette amplified by PCR from strain SB12 (6). An imp1
::kanMX4 mutation was generated as described previously (55, 56). A MYC tag was added to the C terminus of MSS2 and COX18 using plasmid pMPY-3x-MYC (47). Strain HLF5-22 was created by homologous mitochondrial recombination after mating the synthetic [rho–] strain CM1 (cox2[C225H H229C]) (49) with NB41 (5). All strains were verified by functional complementation tests when appropriate and/or by PCR analysis and sequencing.
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Translation in isolated mitochondria with [35S]methionine (ICN Biochemicals) was performed essentially as described elsewhere (58). Following translation for 10 min at 30°C, the mitochondria were washed with 0.6 M sorbitol, 20 mM HEPES pH 7.4 and resuspended to 10 mg/ml before further processing. For immunoprecipitations of mitochondrial translation products, the labeled mitochondria were chased with 25 mM unlabeled methionine for 5 min, washed, resuspended in lysis buffer containing 1% digitonin, 100 mM NaCl, and 20 mM Tris pH 7.4 for 10 min, and spun at 40,000 x g for 45 min. The clarified supernatant was incubated with anti-HA (Roche, Inc.) or anti-MYC (Santa Cruz Biotechnology, Inc.) antibody-conjugated beads.
In vivo pulse-labeling of cycloheximide-treated cells with [35S]methionine was performed as previously described (2). As indicated, labeled strains were chased with the addition of unlabeled methionine to 10 mM for 10 min. After labeling, cells were chilled on ice in the presence of 10 mM unlabeled methionine, and mitochondria were prepared using glass bead disruption (11). Radiolabeled mitochondrial proteins were electrophoretically separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels as indicated, dried, and exposed to film.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Translocation of newly synthesized Cox2(1-109)-HA N-tail across the inner membrane is dependent on OXA1 but not COX18 or MSS2.
To determine whether the N-tail of the truncated Cox2(1-109)-HA is translocated following synthesis, processing of the peptide in the IMS by the Imp1 protease was evaluated following pulse-labeling of mitochondrial translation products in vivo. Cycloheximide-treated log-phase cells were incubated with [35S]methionine, followed by analysis of labeled mitochondrial proteins. Unprocessed pre-Cox2(1-109)-HA peptide synthesized in cells lacking Imp1 was slightly longer than the processed peptide in the wild type (Fig. 2). As expected, the leader peptide was not processed by Imp1 in an oxa1 mutant, showing that N-tail export of the truncated protein required Oxa1. Neither mss2 nor cox18 mutations affected Imp1 processing of pre-Cox2(1-109)-HA, confirming that Mss2 and Cox18 are not required for N-tail export. Unprocessed pre-Cox2(1-109)-HA contains 40% more labeled methionines (five versus three), explaining the increased labeling of pre-Cox2(1-109)-HA in the imp1 background. Reduced signal intensity in the oxa1 background is most likely due to decreased stability of Cox2(1-109)-HA (data not shown).
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Insertion of both transmembrane domains in the absence of the C-tail. The tagged C terminus of full-length Cox2-HA is translocated across the inner membrane and is accessible to exogenously added proteases in mitoplasts (46). To ask whether the C-terminal epitope of the Cox2(1-109)-HA truncation was also translocated across the inner membrane, we examined the topology of accumulated, steady-state protein in mitochondria from unlabeled growing cells. Mitoplasts were prepared and exposed to exogenously added protease to digest epitope residues that had been exported to the IMS. Western analysis of the mitoplasts containing Cox2(1-109)-HA revealed that the HA tag was accessible to added proteases (Fig. 4), demonstrating that the C terminus of the accumulated truncated protein was exposed on the IMS side of the inner membrane. This behavior, identical to that of the full-length protein (reference 46 and data not shown), suggests that the C-tail domain of Cox2 is not required for the insertion of both the first and second transmembrane domains nor for translocation of the small hydrophilic epitope.
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, cox18, and mss2 strains harboring Cox2(1-109)-HA (Fig. 4). In the absence of Oxa1, the Cox2(1-109)-HA C-terminal epitope remained on the matrix side of the inner membrane, protected from exogenous protease. As expected, the protein was not shortened by protease treatment, indicating that the N terminus of Cox2(1-109)-HA also remained on the matrix side.
Since Cox18 and Mss2 have roles in translocation of the C-tail domain of Cox2, we expected that cox18 and mss2 mutations might not affect translocation of the N or C termini of Cox2(1-109)-HA, allowing export of the epitope. Consistent with this idea, most of the epitope on accumulated Cox2(1-109)-HA in mitoplasts disappeared after protease treatment (Fig. 4), indicating a substantial level of export in both cox18 and mss2 mutants. However, mitoplasts from a cox18 strain also accumulated a significant amount of peptide whose N and C termini remained protected. Mitoplasts from the mss2 mutant accumulated Cox2(1-109)-HA whose N terminus was translocated across the membrane (as judged by the shortening of the immunoreactive peptide) but whose C terminus remained protected. The accumulation of some untranslocated N-tail of Cox2(1-109)-HA in cox18 and mss2 mitochondria despite the apparently normal N-tail export of newly synthesized protein in these mutants suggests that there is a subtle slowing of N-tail translocation in these mutants.
Sequences within the Cox2 C-tail domain affect C-tail translocation. To ask whether the C-tail domain might contain sequence or structural information necessary to direct its own membrane translocation, we created another cox2 truncation mutant, cox2(1-211)::3xHA. This altered allele encodes a C-terminally tagged truncated form of Cox2 lacking the last 40 residues of the 144-residue C-tail domain. The stability of newly synthesized Cox2(1-211)-HA was evaluated by [35S]methionine pulse-labeling of mitochondrial translation products in cycloheximide-treated cells followed by an unlabeled chase. Like Cox2(1-109)-HA, newly synthesized Cox2(1-211)-HA was degraded during the chase, in contrast to the wild-type protein (Fig. 5A). Its steady-state level was also reduced relative to full-length Cox2-HA (not shown).
We evaluated Cox2(1-211)-HA N-tail translocation by asking whether the N terminus was processed by the Imp1 protease after synthesis in cycloheximide-treated cells or in isolated mitochondria (Fig. 5B). In both cases, newly synthesized Cox2(1-211)-HA was processed by Imp1. An unidentified high-molecular-weight mitochondrial translation product, apparently also processed by Imp1, appeared in the cox2(1-211)::3xHA mitochondria (Fig. 5A and B). It is likely to be an aggregated form of Cox2(1-211)-HA. No corresponding band appears in Western blots of cox2(1-211)::3xHA mitochondria probed with anti-HA or anti-Cox2 antibodies, suggesting that this species is unstable.
We evaluated the location of accumulated Cox2(1-211)-HA C-tail in mitochondria by Western analysis to detect the HA epitope (Fig. 6A). Addition of exogenous protease to mitoplasts shortened the protein but left the epitope intact, indicating that the N-tail was exposed on the IMS side of the inner membrane but the C-tail remained protected in the matrix. Thus, removal of the last 40 amino acids of the C-tail prevented its translocation.
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Interaction of Mss2 with newly synthesized Cox2 requires the C-tail. Mss2 is a hydrophilic protein located on the inner surface of the inner membrane and could therefore interact directly with newly synthesized Cox2 to carry out its role in the export process (6). To test this idea, we asked whether immunoprecipitation of Mss2 would coprecipitate newly synthesized Cox2 labeled in isolated mitochondria. Radiolabeling of mitochondrial translation products was carried out in mitochondria purified from MSS2 or MSS2::HA strains. The mitochondria were then solubilized in digitonin and immunoprecipitated using anti-HA antibody coupled to beads (Fig. 7A). Mature Cox2 was selectively immunoprecipitated with Mss2-HA but not Mss2, suggesting that Mss2 interacts physically with newly synthesized Cox2 molecules whose N termini have been exported and processed. In addition, we found that Cox2-HA was selectively coimmunoprecipitated with Mss2 bearing a MYC epitope (Mss2-MYC) (Fig. 7B). Finally, we asked whether Cox18, which has been shown to coimmunoprecipitate with Mss2 (46), would also pull down newly synthesized Cox2. Precipitation of Cox18-MYC from solubilized mitochondria with anti-MYC antibody selectively enriched newly synthesized Cox2-HA (Fig. 7C).
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DISCUSSION |
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Oxa1 appears to have two functional domains (41). Membrane protein insertion is carried out by the N-terminal polytypic integral membrane segment of Oxa1, which is homologous to bacterial YidC as well as yeast Cox18. The C-terminal domain of Oxa1 is exposed on the inner surface of the mitochondrial membrane and interacts with mitochondrial ribosomes, apparently to promote cotranslational targeting of nascent proteins (28, 50). Both Cox18, which is functionally conserved among eukaryotes (14), and YidC lack this C-terminal domain. Fusion of this domain of Oxa1 to the C terminus of bacterial YidC generates a chimeric protein that can partially compensate for the absence of normal Oxa1 in yeast, while unmodified YidC can partially compensate for the absence of Cox18 (41). These observations suggest that direct interaction with the ribosome presents the N terminus of pre-Cox2 to the translocase domain of Oxa1 but that other functions may be required to present the Cox2 C-terminal domain to Cox18.
Mss2 is exposed on the inner surface of the inner membrane and thus could function to recognize the Cox2 C-tail prior to translocation by Cox18 (6). Consistent with this idea, Mss2 has a tetratricopeptide motif (6) similar to one in the mitochondrial import receptor Tom70, which binds cytoplasmic chaperone-associated translocation substrates (9, 60). We observed here that newly synthesized full-length Cox2 could be coimmunoprecipitated with Mss2 from solubilized mitochondria, consistent with the idea that Mss2 recognizes the Cox2 C-tail prior to export. The coprecipitated Cox2 was processed at its N terminus, demonstrating that N-tail export can occur while Cox2 is still bound, directly or indirectly, to Mss2 on the inner surface of the inner membrane. Thus, C-tail translocation may be a posttranslational event that can occur subsequent to cotranslational N-tail export and N-terminal processing. Mss2-Cox2 coprecipitation was not observed when the entire C-tail domain was completely deleted, consistent with the possibility that Mss2 might bind to the C-tail. If so, Mss2 could hold the C-tail domain of Cox2 in an unfolded conformation, or in an intermediate folded conformation (51), required for translocation after translation termination. (The N-tail domain of truncated Cox2 lacking the C-tail was not processed during synthesis in isolated mitochondria, making it formally possible that interaction with Mss2 depends on N-terminal processing rather than the absence of the C-tail per se.)
To explore the features of Cox2 that are required for membrane insertion and translocation, we studied the behavior of truncated Cox2 variants bearing an epitope tag at their C termini, encoded in mtDNA. The N-tail of a protein lacking the entire C-tail domain, Cox2(1-109)-HA, was efficiently translocated across the inner membrane during pulse-labeling of whole cells in a reaction that was dependent upon Oxa1 but not Cox18 or Mss2. Interestingly, N-tail export of the Cox2(1-109)-HA N-tail did not occur during pulse-labeling of isolated mitochondria, demonstrating that the isolated organelles are deficient in some feature of this process relative to intact cells.
Unlabeled Cox2(1-109)-HA accumulated (albeit in greatly diminished amounts relative to wild-type Cox2) with both its N terminus and epitope-tagged C terminus on the outside of the inner membrane. Thus, insertion of the two transmembrane domains of Cox2, and export of the C-terminal epitope, can occur in the absence of the C-tail. Both the N and C termini of Cox2(1-109)-HA accumulated in the matrix in an oxa1 mutant. Since previous work indicated that Cox18 and Mss2 are required only for C-tail translocation, we expected export of the Cox2(1-109)-HA N-tail to be unaffected in cox18 and mss2 mutants. However, both mutations caused accumulation of detectable levels of Cox2(1-109)-HA, whose N-tail was in the matrix (cox18 more so than mss2). This could indicate either a direct but minor role for Cox18 and Mss2 in N-tail export or a general inefficiency of a larger translocation complex lacking either protein, in either case leading to accumulation of species not detected during pulse-labeling. Similarly, while most of the C-terminal epitope of Cox2(1-109)-HA was exported in both mutants, demonstrating that Cox18 and Mss2 are not required for insertion of the second transmembrane domain, both mutations clearly reduced export of the C terminus relative to wild type. In this context it is important to note that the addition of the 33-residue hydrophilic epitope tag to the C terminus of the second transmembrane domain at residue 109 may reduce the efficiency of its insertion. This insertion is completely blocked by the presence of a 402-residue hydrophilic passenger protein moiety (Arg8m) fused at the same position (21).
We also examined features of the 144-residue Cox2 C-tail domain that could contribute to its recognition as a translocation substrate. A truncated variant of Cox2 lacking the 40 C-terminal residues of this domain [Cox2(1-211)-HA] accumulated in mitochondria with its N-tail exported to the IMS and its C terminus remaining in the matrix. Thus, it is clear that sequence and/or structural features of the C-tail domain itself are recognized by its translocation apparatus independently of N-tail translocation. Recognition of these features in the C-tail can also lead to translocation of a fused passenger protein (21). Despite the fact that the truncated C-tail of Cox2(1-211)-HA is not translocated, this variant protein nevertheless did coimmunoprecipitate with Mss2. Thus, while the Cox2-Mss2 interaction is highly likely to be necessary for C-tail export, recognition of the C-tail as a translocation substrate must involve other steps as well.
Three of the four copper-ligating residues of the cytochrome c oxidase CuA site are absent from the truncated protein Cox2(1-211)-HA. While copper insertion is thought to occur in the IMS after export (7), the mitochondrial matrix has an accessible nonproteinaceous copper pool (10) that could provide metal ions to the C-tail domain. However, we found that point mutations altering the copper-binding residues C225 and H229 (corresponding to C200 and H204 from the bovine Cox2 sequence) (49) are not required for efficient C-tail translocation. Furthermore, Sco1, a mitochondrial protein required for copper delivery to Cox2 (37, 42), is not required for C-tail translocation (45). Thus, a fully folded C-tail domain containing copper ions is not required for C-tail translocation.
Both the exported 22-residue N-tail and 144-residue C-tail domains of Cox2 are negatively charged, with predicted pI values of 3.8 and 4.4, respectively. Thus, these translocation systems adhere to the "positive-inside rule" (15, 20, 43). However, while charge may play a role in driving the Cox2 C-tail across the membrane in a potential-dependent fashion, neither acidity nor the charged residues immediately flanking the transmembrane domains are sufficient to direct this process, since the acidic C-tail domain of the truncated variant Cox2(1-211)-HA (pI 4.0) is not translocated.
The molecular mechanism(s) of bacterial YidC has been studied extensively but remains to be precisely defined (reviewed in reference 32). YidC is at least partially associated with the bacterial Sec translocase, although it is not required for most Sec-dependent reactions. YidC is necessary for the assembly of the Escherichia coli respiratory chain (54). Plasma membrane insertion of the E. coli protein CyoA, a subunit of the bacterial cytochrome bo oxidase that is similar to mitochondrial Cox2, also depends upon distinct mechanisms for insertion of its N- and C-terminal domains into the membrane (8, 12, 53). In this case, the Oxa1/Cox18 homolog YidC is required for translocation of the N-terminal domain, while the C-terminal domain is translocated by the Sec translocase. While cells depleted for YidC do not translocate either domain of the wild-type protein, this is due to the fact that prior YidC-dependent translocation of the N-terminal sequences and insertion of the transmembrane domains are a prerequisite for Sec-dependent translocation of the C-terminal domain (8). Interestingly, both translocation reactions are independent of membrane potential when carried out in vitro in proteoliposomes (12). Thus, there are both similarities and clear differences between the yeast mitochondrial and bacterial systems: in both cases the early events are catalyzed by Oxa1/YidC, while in mitochondria the Cox2-specific functions of Cox18/YidC and Mss2 have taken the place of the Sec translocon.
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ACKNOWLEDGMENTS |
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This study was supported by a postdoctoral fellowship to H.L.F. (GM65664) and a research grant to T.D.F. (GM29362) from the U.S. National Institutes of Health.
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FOOTNOTES |
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Published ahead of print on 23 April 2007.
Present address: Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.
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REFERENCES |
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2. Bonnefoy, N., N. Bsat, and T. D. Fox. 2001. Mitochondrial translation of Saccharomyces cerevisiae COX2 mRNA is controlled by the nucleotide sequence specifying the pre-Cox2p leader peptide. Mol. Cell. Biol. 21:2359-2372.
3. Bonnefoy, N., F. Chalvet, P. Hamel, P. P. Slonimski, and G. Dujardin. 1994. OXA1, a Saccharomyces cerevisiae nuclear gene whose sequence is conserved from prokaryotes to eukaryotes controls cytochrome oxidase biogenesis. J. Mol. Biol. 239:201-212.[CrossRef][Medline]
4. Bonnefoy, N., and T. D. Fox. 2001. Genetic transformation of Saccharomyces cerevisiae mitochondria. Methods Cell Biol. 65:381-396.[Medline]
5. Bonnefoy, N., and T. D. Fox. 2000. In vivo analysis of mutated initiation codons in the mitochondrial COX2 gene of Saccharomyces cerevisiae fused to the reporter gene ARG8m reveals lack of downstream reinitiation. Mol. Gen. Genet. 262:1036-1046.[CrossRef][Medline]
6. Broadley, S. A., C. M. Demlow, and T. D. Fox. 2001. Peripheral mitochondrial inner membrane protein, Mss2p, required for export of the mitochondrially coded Cox2p C tail in Saccharomyces cerevisiae. Mol. Cell. Biol. 21:7663-7672.
7. Carr, H. S., and D. R. Winge. 2003. Assembly of cytochrome c oxidase within the mitochondrion. Acc. Chem. Res. 36:309-316.[CrossRef][Medline]
8. Celebi, N., L. Yi, S. J. Facey, A. Kuhn, and R. E. Dalbey. 2006. Membrane biogenesis of subunit II of cytochrome bo oxidase: contrasting requirements for insertion of N-terminal and C-terminal domains. J. Mol. Biol. 357:1428-1436.[CrossRef][Medline]
9. Chan, N. C., V. A. Likic, R. F. Waller, T. D. Mulhern, and T. Lithgow. 2006. The C-terminal TPR domain of Tom70 defines a family of mitochondrial protein import receptors found only in animals and fungi. J. Mol. Biol. 358:1010-1022.[CrossRef][Medline]
10. Cobine, P. A., L. D. Ojeda, K. M. Rigby, and D. R. Winge. 2004. Yeast contain a non-proteinaceous pool of copper in the mitochondrial matrix. J. Biol. Chem. 279:14447-14455.
11. Diekert, K., A. I. de Kroon, G. Kispal, and R. Lill. 2001. Isolation and subfractionation of mitochondria from the yeast Saccharomyces cerevisiae. Methods Cell Biol. 65:37-51.[Medline]
12. du Plessis, D. J. F., N. Nouwen, and A. J. M. Driessen. 2006. Subunit a of cytochrome o oxidase requires both YidC and SecYEG for membrane insertion. J. Biol. Chem. 281:12248-12252.
13. Fontanesi, F., I. C. Soto, D. Horn, and A. Barrientos. 2006. Assembly of mitochondrial cytochrome c oxidase, a complicated and highly regulated cellular process. Am. J. Physiol. Cell Physiol. 291:C1129-C1147.
14. Gaisne, M., and N. Bonnefoy. 2006. The COX18 gene, involved in mitochondrial biogenesis, is functionally conserved and tightly regulated in humans and fission yeast. FEMS Yeast Res. 6:869-882.[CrossRef][Medline]
15. Gavel, Y., and G. von Heijne. 1992. The distribution of charged amino acids in mitochondrial inner-membrane proteins suggests different modes of membrane integration for nuclearly and mitochondrially encoded proteins. Eur. J. Biochem. 205:1207-1215.[Medline]
16. Glick, B. S., and L. A. Pon. 1995. Isolation of highly purified mitochondria from Saccharomyces cerevisiae. Methods Enzymol. 260:213-223.[Medline]
17. Glick, B. S., and G. Von Heijne. 1996. Saccharomyces cerevisiae mitochondria lack a bacterial-type sec machinery. Protein Sci. 5:2651-2652.[Abstract]
18. Green-Willms, N. S., C. A. Butler, H. M. Dunstan, and T. D. Fox. 2001. Pet111p, an inner membrane-bound translational activator that limits expression of the Saccharomyces cerevisiae mitochondrial gene COX2. J. Biol. Chem. 276:6392-6397.
19. Guthrie, C., and G. R. Fink. 1991. Guide to yeast genetics and molecular biology. Methods Enzymol. 194:1-863.[Medline]
20. Harley, C. A., J. A. Holt, R. Turner, and D. J. Tipper. 1998. Transmembrane protein insertion orientation in yeast depends on the charge difference across transmembrane segments, their total hydrophobicity, and its distribution. J. Biol. Chem. 273:24963-24971.
21. He, S., and T. D. Fox. 1997. Membrane translocation of mitochondrially coded Cox2p: distinct requirements for export of N and C termini and dependence on the conserved protein Oxa1p. Mol. Biol. Cell 8:1449-1460.[Abstract]
22. He, S., and T. D. Fox. 1999. Mutations affecting a yeast mitochondrial inner membrane protein, Pnt1p, block export of a mitochondrially synthesized fusion protein from the matrix. Mol. Cell. Biol. 19:6598-6607.
23. Hell, K., J. Herrmann, E. Pratje, W. Neupert, and R. A. Stuart. 1997. Oxa1p mediates the export of the N- and C-termini of pCoxII from the mitochondrial matrix to the intermembrane space. FEBS Lett. 418:367-370.[CrossRef][Medline]
24. Hell, K., W. Neupert, and R. A. Stuart. 2001. Oxa1p acts as a general membrane insertion machinery for proteins encoded by mitochondrial DNA. EMBO J. 20:1281-1288.[CrossRef][Medline]
25. Hell, K., A. Tzagoloff, W. Neupert, and R. A. Stuart. 2000. Identification of Cox20p, a novel protein involved in the maturation and assembly of cytochrome oxidase subunit 2. J. Biol. Chem. 275:4571-4578.
26. Herrmann, J. M., and S. Funes. 2005. Biogenesis of cytochrome oxidase-sophisticated assembly lines in the mitochondrial inner membrane. Gene 354:43-52.[CrossRef][Medline]
27. Jan, P. S., K. Esser, E. Pratje, and G. Michaelis. 2000. Som1, a third component of the yeast mitochondrial inner membrane peptidase complex that contains Imp1 and Imp2. Mol. Gen. Genet. 263:483-491.[CrossRef][Medline]
28. Jia, L., M. Dienhart, M. Schramp, M. McCauley, K. Hell, and R. A. Stuart. 2003. Yeast Oxa1 interacts with mitochondrial ribosomes: the importance of the C-terminal region of Oxa1. EMBO J. 22:6438-6447.[CrossRef][Medline]
29. Kermorgant, M., N. Bonnefoy, and G. Dujardin. 1997. Oxa1p, which is required for cytochrome c oxidase and ATP synthase complex formation, is embedded in the mitochondrial inner membrane. Curr. Genet. 31:302-307.[CrossRef][Medline]
30. Leonhard, K., B. Guiard, G. Pellecchia, A. Tzagoloff, W. Neupert, and T. Langer. 2000. Membrane protein degradation by AAA proteases in mitochondria: extraction of substrates from either membrane surface. Mol. Cell 5:629-638.[CrossRef][Medline]
31. Leonhard, K., J. M. Herrmann, R. A. Stuart, G. Mannhaupt, W. Neupert, and T. Langer. 1996. AAA proteases with catalytic sites on opposite membrane surfaces comprise a proteolytic system for the ATP-dependent degradation of inner membrane proteins in mitochondria. EMBO J. 15:4218-4229.[Medline]
32. Luirink, J., G. von Heijne, E. N. Houben, and J. W. Gier. 2005. Biogenesis of inner membrane proteins in Escherichia coli. Annu. Rev. Microbiol. 59:329-355.[CrossRef][Medline]
33. Mulero, J. J., and T. D. Fox. 1993. PET111 acts in the 5'-leader of the Saccharomyces cerevisiae mitochondrial COX2 mRNA to promote its translation. Genetics 133:509-516.[Abstract]
34. Naithani, S., S. A. Saracco, C. A. Butler, and T. D. Fox. 2003. Interactions among COX1, COX2, and COX3 mRNA-specific translational activator proteins on the inner surface of the mitochondrial inner membrane of Saccharomyces cerevisiae. Mol. Biol. Cell 14:324-333.
35. Nakai, T., T. Yasuhara, Y. Fujiki, and A. Ohashi. 1995. Multiple genes, including a member of the AAA family, are essential for degradation of unassembled subunit 2 of cytochrome c oxidase in yeast mitochondria. Mol. Cell. Biol. 15:4441-4452.[Abstract]
36. Neff, N. F., J. H. Thomas, P. Grisafi, and D. Botstein. 1983. Isolation of the beta-tubulin gene from yeast and demonstration of its essential function in vivo. Cell 33:211-219.[CrossRef][Medline]
37. Nittis, T., G. N. George, and D. R. Winge. 2001. Yeast Sco1, a protein essential for cytochrome c oxidase function is a Cu(I)-binding protein. J. Biol. Chem. 276:42520-42526.
38. Nunnari, J., T. D. Fox, and P. Walter. 1993. A mitochondrial protease with two catalytic subunits of nonoverlapping specificities. Science 262:1997-2004.
39. Pearce, D. A., and F. Sherman. 1995. Degradation of cytochrome oxidase subunits in mutants of yeast lacking cytochrome c and suppression of the degradation by mutation of YME1. J. Biol. Chem. 270:20879-20882.
40. Perez-Martinez, X., S. A. Broadley, and T. D. Fox. 2003. Mss51p promotes mitochondrial Cox1p synthesis and interacts with newly synthesized Cox1p. EMBO J. 22:5951-5961.[CrossRef][Medline]
41. Preuss, M., M. Ott, S. Funes, J. Luirink, and J. M. Herrmann. 2005. Evolution of mitochondrial Oxa proteins from bacterial YidC. Inherited and acquired functions of a conserved protein insertion machinery. J. Biol. Chem. 280:13004-13011.
42. Rentzsch, A., G. Krummeck-Weiss, A. Hofer, A. Bartuschka, K. Ostermann, and G. Rodel. 1999. Mitochondrial copper metabolism in yeast: mutational analysis of Sco1p involved in the biogenesis of cytochrome c oxidase. Curr. Genet. 35:103-108.[CrossRef][Medline]
43. Rojo, E. E., B. Guiard, W. Neupert, and R. A. Stuart. 1999. N-terminal tail export from the mitochondrial matrix. Adherence to the prokaryotic "positive-inside" rule of membrane protein topology. J. Biol. Chem. 274:19617-19622.
44. Sanchirico, M. E., T. D. Fox, and T. L. Mason. 1998. Accumulation of mitochondrially synthesized Saccharomyces cerevisiae Cox2p and Cox3p depends on targeting information in untranslated portions of their mRNAs. EMBO J. 17:5796-5804.[CrossRef][Medline]
45. Saracco, S. A. 2003. Analysis of the export of Saccharomyces cerevisiae Cox2p from the mitochondrial matrix. Ph.D. thesis. Cornell University, Ithaca, NY.
46. Saracco, S. A., and T. D. Fox. 2002. 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 13:1122-1131.
47. Schneider, B. L., W. Seufert, B. Steiner, Q. H. Yang, and A. B. Futcher. 1995. Use of polymerase chain reaction epitope tagging for protein tagging in Saccharomyces cerevisiae. Yeast 11:1265-1274.[CrossRef][Medline]
48. Souza, R. L., N. S. Green-Willms, T. D. Fox, A. Tzagoloff, and F. G. Nobrega. 2000. Cloning and characterization of COX18, a Saccharomyces cerevisiae PET gene required for the assembly of cytochrome oxidase. J. Biol. Chem. 275:14898-15902.
49. Speno, H., M. R. Taheri, D. Sieburth, and C. T. Martin. 1995. Identification of essential amino acids within the proposed CuA binding site in subunit II of Cytochrome c oxidase. J. Biol. Chem. 270:25363-25369.
50. Szyrach, G., M. Ott, N. Bonnefoy, W. Neupert, and J. M. Herrmann. 2003. Ribosome binding to the Oxa1 complex facilitates co-translational protein insertion in mitochondria. EMBO J. 22:6448-6457.[CrossRef][Medline]
51. Teter, S. A., and D. J. Klionsky. 1999. How to get a folded protein across a membrane. Trends Cell Biol. 9:428-431.[CrossRef][Medline]
52. Thorsness, P. E., K. H. White, and T. D. Fox. 1993. Inactivation of YME1, a member of the ftsH-SEC18-PAS1-CDC48 family of putative ATPase-encoding genes, causes increased escape of DNA from mitochondria in Saccharomyces cerevisiae. Mol. Cell. Biol. 13:5418-5426.
53. van Bloois, E., G. J. Haan, J. W. de Gier, B. Oudega, and J. Luirink. 2006. Distinct requirements for translocation of the N-tail and C-tail of the Escherichia coli inner membrane protein CyoA. J. Biol. Chem. 281:10002-10009.
54. Van Der Laan, M., M. L. Urbanus, C. M. Ten Hagen-Jongman, N. Nouwen, B. Oudega, N. Harms, A. J. Driessen, and J. Luirink. 2003. A conserved function of YidC in the biogenesis of respiratory chain complexes. Proc. Natl. Acad. Sci. USA 100:5801-5806.
55. Wach, A. 1996. PCR-synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae. Yeast 12:259-265.[CrossRef][Medline]
56. Wach, A., A. Brachat, R. Pohlmann, and P. Philippsen. 1994. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10:1793-1808.[CrossRef][Medline]
57. Weber, E. R., T. Hanekamp, and P. E. Thorsness. 1996. Biochemical and functional analysis of the YME1 gene product, an ATP and zinc-dependent mitochondrial protease from S. cerevisiae. Mol. Biol. Cell 7:307-317.[Abstract]
58. Westermann, B., J. M. Herrmann, and W. Neupert. 2001. Analysis of mitochondrial translation products in vivo and in organello in yeast. Methods Cell Biol. 65:429-438.[Medline]
59. Yi, L., and R. E. Dalbey. 2005. Oxa1/Alb3/YidC system for insertion of membrane proteins in mitochondria, chloroplasts and bacteria (review). Mol. Membr. Biol. 22:101-111.[Medline]
60. Young, J. C., N. J. Hoogenraad, and F. U. Hartl. 2003. Molecular chaperones Hsp90 and Hsp70 deliver preproteins to the mitochondrial import receptor Tom70. Cell 112:41-50.[CrossRef][Medline]
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