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Vol. 19, Issue 12, 5387-5397, December 2008
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Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205
Submitted February 1, 2008;
Revised September 12, 2008;
Accepted September 29, 2008
Monitoring Editor: Peter Walter
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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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). Proteomic studies have shown that mitochondria contain >1000 proteins (Sickmann et al., 2003; Jensen et al., 2004; Prokisch et al., 2004; Zahedi et al., 2006), with the majority encoded on nuclear genes. These nuclear-encoded genes are translated in the cytosol and then sorted to the outer membrane (OM), intermembrane space (IMS), inner membrane (IM), or mitochondrial matrix (Koehler, 2004; Rehling et al., 2004). In contrast, only a small fraction of mitochondrial proteins are encoded in the mitochondrial genome (mtDNA) (Attardi and Schatz, 1988). Most of these mtDNA-encoded proteins are subunits of IM complexes required for oxidative phosphorylation.
Although mtDNA is required for the viability of most eukaryotes, the yeast Saccharomyces cerevisiae can live in the absence of its mitochondrial genome (Tzagoloff, 1982; Chen and Clark-Walker, 2000). These "rho0" or "cytoplasmic petite" mutants lack essential subunits of the electron transport chain and ATP synthase complexes, generating cellular ATP only by glycolysis. Several proteins have been identified that are needed for yeast growth in the absence of mtDNA, even when cells are grown on fermentable medium. Yeast cells lacking these proteins are called "petite-negative" mutants (Chen and Clark-Walker, 2000). Without mtDNA, the electron transport chain is not functional; consequently, the generation of an inner membrane potential (
) requires both the F1 portion of the ATP synthase and the ATP/ADP antiporter (Dupont et al., 1985; Giraud and Velours, 1997; Chen and Clark-Walker, 1999). The exchange via the antiporter of cytosolic ATP–4 for ADP–3 produced by the matrix-localized F1-ATPase is thought to generate the electrical gradient in petite cells. Because is vital for essential mitochondrial functions besides oxidative phosphorylation, cells lacking subunits of the F1-ATPase or the ATP/ADP antiporter are petite-negative.
The i-AAA complex, a mitochondrial protease located in the IM, is also crucial for mtDNA-independent growth (Thorsness et al., 1993). The i-AAA protease is thought to consist of a complex of Yme1p subunits. Yme1p is a member of the AAA+ superfamily (ATPases associated with various cellular activities), which are typically organized into large, hexameric ring structures and use the energy from ATP hydrolysis to unfold substrates and translocate them into the narrow axial channel of the complex for degradation (Baker and Sauer, 2006). Yme1p is anchored in the IM with its proteolytic domain facing the IMS (Leonhard et al., 1996). Although a few substrates for the i-AAA protease are known (Leonhard et al., 1996, 1999, 2000; Augustin et al., 2005), it is not understood why Yme1p-dependent turnover is needed for yeast viability in the absence of mtDNA. Recently, in a genome-wide screen for new petite-negative mutants, we identified Mgr1p (Dunn et al., 2006). Mgr1p associates with Yme1p and is required for full activity of the i-AAA protease, but its role in mitochondrial quality control is unclear. In a survey of yeast knockouts lacking uncharacterized mitochondrial proteins, we now identify a new protein that we call Mgr3p. We find that Mgr3p and Mgr1p function together in an adaptor complex that seems to help target substrates to the i-AAA protease.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Brachmann et al., 1998) and protein tagging (Longtine et al., 1998) are listed in Supplemental Table S7. As indicated, when diploid strains were the recipient of PCR-mediated disruption or tagging, sporulation of the diploid and isolation of meiotic segregants were used to generate haploid constructs. Yeast media and molecular genetic techniques were as described previously (Adams et al., 1997).
Plasmid Construction
The 2µ-MGR3 plasmid pM527 was created by PCR amplification of the MGR3 gene by using primers 1867 and 1868 (Supplemental Table S7), followed by product digestion with XhoI and NotI and ligation into XhoI/NotI-cut pRS426 (Sikorski and Hieter, 1989).
Screening Knockouts for mtDNA Dependence
Wild-type cells and 42 yeast knockout strains (Supplemental Table S1) were grown overnight in YEPD medium containing 25 µg/ml ethidium bromide (EtBr) and then tested for growth on YEPD medium without EtBr. One mutant, yer038w-a, grew moderately better in EtBr-containing medium than the other strains tested. Interestingly, this gene disruption also removes YER039C, an overlapping gene of unknown function, but this strain was not analyzed further. Four knockouts, yor286w, ykr016w, yer182w, and ymr115w, showed reduced growth after EtBr treatment in comparison with wild type.
Mitochondrial Fractionation
Mitochondria were isolated from yeast cells as described previously (Daum et al., 1982), except that SEH buffer (250 mM sucrose, 1 mM EDTA, and 20 mM HEPES-KOH, pH7.4) with a 1:1000 dilution of protease inhibitor cocktail (P8340; Sigma-Aldrich, St. Louis, MO) was used. Osmotic shock of mitochondria and protease treatment of mitochondria and mitoplasts were as described previously (Kerscher et al., 2000), except that 200 µg of mitochondria or mitoplasts at 0.2 mg/ml were used. The separation of OM and IM was as described previously (Pon et al., 1989). Treatment of mitochondria with 0.1 M Na2CO3 was as described previously (Kerscher et al., 2000), except that samples were centrifuged at 100,000 x g for 60 min.
Analysis of i-AAA Complex
Blue native (BN) electrophoresis was performed as described previously (Dunn et al., 2006). For precipitation of Mgr3p-His6, 500 µg of mitochondria was solubilized in 500 µl of extraction buffer (150 mM potassium acetate, 30 mM HEPES-KOH, 10 mM imidazole, 1% digitonin, and 1x protease inhibitor cocktail) on ice for 15 min. Then, 500 µl of extraction buffer lacking detergent was added to samples, and insoluble material was removed by centrifugation (13,000 x g for 10 min). Fifty to 100 µl of nickel-nitrilotriacetic acid (Ni-NTA) agarose (QIAGEN, VALENCIA, CA) was added to the cleared lysate, and samples were rotated for 1 h at 4°C. After removing 100 µl (10% load), Ni-NTA beads were collected by centrifugation. After three washes in buffer containing 0.1% digitonin, 2x SDS-sample buffer with 400 mM imidazole was added to beads and to the 10% load sample before polyacrylamide gel electrophoresis (PAGE) analysis. Precipitation of myc-tagged proteins from digitonin-solubilized mitochondria was as described previously (Dunn et al., 2006).
Substrate Degradation Assays
Degradation of Yta10p(161)DHFRMUT and Nde1p-HA was assayed as described in Dunn et al. (2006).
Substrate Binding Assays
35S-labeled Yta10(161)-DHFRMUT was imported into isolated mitochondria in the presence of ATP (but lacking an ATP regeneration system) at 25°C for 12 min. Import reactions were stopped on ice, and mitochondria were reisolated by centrifugation at 13,000 x g for 10 min through a sucrose cushion. Mitochondria were then resuspended at 1 mg/ml in 400 µl of import buffer lacking ATP and NADH and incubated for 30 min at 31°C. To precipitate Mgr1p-myc along with bound substrate, mitochondria were pelleted by centrifugation (13,000 x g for 10 min) and resuspended in 500 µl in lysis buffer (1% digitonin, 50 mM NaCl, 30 mM HEPES-KOH, pH 7.4, and 1:1000 dilution of protease inhibitor cocktail) on ice for 15 min. After centrifugation at 13,000 x g for 10 min to remove insoluble material, another 500 µl of lysis buffer lacking detergent was added to the samples, followed by 75 µl of a 50% slurry of anti-myc antibodies coupled to agarose beads. After mixing at 4°C for 4 h, 100 µl were removed (10% total), and beads were washed three times with lysis buffer containing 0.1% digitonin and collected by centrifugation. Samples were analyzed by SDS-PAGE and phosphorimaging. Anti-myc antibodies were coupled to beads using a Seize primary immunoprecipitation kit (Pierce Chemical, Rockford, IL). Alternatively, antibodies were cross-linked to protein G-Sepharose beads (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) as described previously (Harlow and Lane, 1999), with the following modifications: Protein G-Sepharose beads (1 ml; settled) were washed and equilibrated in binding buffer (20 mM sodium phosphate, pH 7.0) before mixing with 1 ml of anti-myc ascites fluid at 4°C overnight. After pelleting, myc-beads were resuspended in 1.5 ml of binding buffer containing 14 mM disuccinimidyl suberate (DSS; Pierce Chemical) and incubated for 1 h at room temperature. After washing the myc-beads in binding buffer, the DSS was quenched with 2 ml of 0.1 M ethanolamine for 1 h at room temperature. Beads were washed with 10 ml of 0.1 M glycine, pH 2.8, and then four times with 10 ml of binding buffer.
To precipitate Mgr3p-His6 or Yme1p-His6 along with bound substrate, mitochondria were solubilized on ice in 500 µl of lysis buffer containing 10 mM imidazole. After 15 min, 500 µl of lysis buffer lacking detergent was added to samples and centrifuged at 13,000 x g for 10 min. Ni-NTA beads (
50–100 µl) were added to the mitochondrial extract and mixed at 4°C for 1 h. After removal of 100 µl of mix (10% total), beads were washed three times with wash buffer containing 10 mM imidazole, and proteins from the beads and loading control were extracted with 2x SDS sample buffer containing 0.6 M β-mercaptoethanol and 400 mM imidazole.
Immunoblotting
Proteins were analyzed by SDS-PAGE and immunoblotting to Immobilon filters (Millipore, Billerica, MA) by using standard techniques (Current Protocols Online; http://www.mrw2.interscience.wiley.com/cponline). The myc-tagged proteins were visualized using mouse ascites fluid prepared using 9E10 cells (Covance, Denver, PA; Evan et al., 1985). His6-tagged proteins were detected using rabbit polyclonal antiserum SC-803, purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mitochondrial proteins were identified using anti-serum to Tim23p (Emtage and Jensen, 1993), Mas2p (Jensen and Yaffe, 1988), Tom70p (Hase et al., 1984), Yme1p (Thorsness et al., 1993), OM45 (Yaffe et al., 1989), or F1β (a gift from A. Davis, Tufts University School of Medicine, Boston, MA). Immune complexes were visualized using horseradish peroxidase-conjugated secondary antibody (GE Healthcare) followed by chemiluminescence (West Pico; Pierce Chemical). Immune blots were imaged using a Versadoc imaging system and Quantity One software (Bio-Rad, Hercules, CA) or by exposure to x-ray film.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Zahedi et al., 2006) and that were uncharacterized at the time we began our study. Forty-two mutant strains from the haploid yeast knockout collection (Giaever et al., 2002) were grown overnight in medium containing EtBr to force mtDNA loss (Slonimski et al., 1968; Goldring et al., 1970) and tested for viability (Supplemental Table S1). Although the growth of 38 of the knockout strains was not inhibited by the treatment, as compared to wild-type, four of the mutants grew slowly or not at all after treatment with EtBr. Because the growth of the ymr115w knockout was the most severely affected by EtBr treatment, we chose this strain for further analysis and named the gene Mitochondrial Genome Required (MGR3). We note that an earlier name for MGR3 was FMP24 (Sickmann et al., 2003). mgr3 is inviable when loss of mtDNA is induced by EtBr, similar to yme1 and mgr1, mutants that are deficient in i-AAA protease activity, and atp2, which lacks F1-ATPase function (Figure 1A). Furthermore, we confirmed that the lethality of mgr3 after EtBr treatment is due to the loss of mtDNA and is not due to other effects of EtBr treatment. In particular, mgr3 strains lacking mtDNA were inviable even in the absence of EtBr when they were forced to lose a plasmid carrying wild-type MGR3 (Figure 1B). We do note that the mtDNA dependence of mgr3 is background specific: mgr3 generated in an unrelated strain (Thorsness and Fox, 1993) is not petite negative and continues to grow after EtBr treatment (Figure 1C). This strain dependence was also seen for mgr1 mutants (Dunn et al., 2006).
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knockouts are not petite negative (Dunn, unpublished data). Orthologues of Mgr3p exist in other yeast, such as Ashbya gossypii (AFR197W; 52% identical), Kluyveromyces lactis (KLLA0B12419g; 52% identical), and Candida albicans (CaO19.11835; 32% identical), but their function in these different organisms is not known. We do not find significant homology between the amino acid sequence of Mgr3p and any nonfungal proteins.
Mgr3p Is a Mitochondrial Inner Membrane Protein
Mgr3 protein was previously found in the proteome of highly purified yeast mitochondria (Sickmann et al., 2003) and also localized to mitochondria during a genome-wide survey of green fluorescent protein fusion protein localization (Huh et al., 2003). To confirm this location, we fused the myc epitope to the carboxy terminus of Mgr3p and monitored the fully functional Mgr3p-myc protein during subcellular fractionation. Cells expressing Mgr3p-myc were homogenized and separated into a mitochondrial fraction and a postmitochondrial supernatant. We found by immunoblotting that Mgr3p-myc cofractionated with the mitochondrial protein Tim23p (Emtage and Jensen, 1993) and not with hexokinase, a cytosolic marker (Kerscher et al., 2000) (Figure 2A). When mitochondria were treated with alkali, both Mgr3p-myc and Tim23p, an integral IM protein (Emtage and Jensen, 1993), were found in the pellet fraction (Figure 2B). In contrast, Mas2p, a soluble matrix marker (Jensen and Yaffe, 1988), was released by the carbonate treatment. Our results indicate that Mgr3p is an integral membrane protein, consistent with the prediction of a transmembrane segment near its amino terminus. When mitochondria were sonicated and the resulting membrane vesicles separated on a sucrose gradient, Mgr3p-myc cofractionated with the IM marker, the β-subunit of the F1-ATPase (F1β), but not with the OM marker, OM45 (Yaffe et al., 1989) (Figure 2C). Thus, Mgr3p is a mitochondrial inner membrane protein.
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), was digested by the protease treatment. However, when the OM was ruptured by osmotic shock (OS) to form mitoplasts, the immunoreactive amino terminus of Tim23p (Ryan et al., 1998) and the myc tag of Mgr3p-myc were both digested, whereas matrix-localized Mas2p was protected. Because no protease-protected Mgr3p-myc fragments were detected after protease digestion of mitoplasts, we conclude that Mgr3p is inserted into the mitochondrial IM with a small amino-terminal segment exposed to the matrix and a large carboxy-terminal domain facing the IMS.
Mgr3p Associates with the i-AAA Protease
We find that Mgr3p coprecipitates from mitochondrial extracts with Yme1p and the i-AAA-associated Mgr1 protein. For these studies, we used a Mgr1p-myc fusion protein (Dunn et al., 2006) and also engineered six histidine residues at the carboxy terminus of Mgr3p, forming a fully functional Mgr3p-His6 construct. Mitochondria were isolated from cells expressing both Mgr3p-His6 and Mgr1p-myc, solubilized in digitonin-containing buffer, and the Mgr3p-His6 protein precipitated with Ni-NTA beads. Both Yme1p and Mgr1p-myc coprecipitated with Mgr3p (Figure 3A, lane 4). Although nearly all of the Mgr3p-His6 protein was found in the pellet fraction, we note that only 10% of Yme1p and Mgr1p-myc precipitated along with Mgr3p. Therefore, the binding of Mgr3p to Mgr1p and Yme1p may be weak or dynamic. Nonetheless, the Mgr3p–Mgr1p–Yme1p interaction is specific, because we did not see any coprecipitation when mitochondria from cells lacking the Mgr3p-His6 fusion protein were used (Figure 3A, lane 2). In addition, similar amounts of Yme1p were found to coprecipitate with an Mgr3p-myc fusion protein (Dunn, unpublished data).
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). Most Mgr1p-myc comigrated with the larger of two resolvable Yme1p-containing complexes (Figure 3B, lanes 1 and 2, white arrow), both larger than 667 kDa, in agreement with previous findings (Dunn et al., 2006). Supporting the association of Mgr3p with the i-AAA protease complex, Mgr3p-His6 was found to comigrate with Mgr1p-myc and Yme1p during BN-PAGE (Figure 3B, lane 3, white arrow). Further evidence that Mgr3p interacts with the i-AAA complex comes from examining the migration of the catalytic Yme1p subunit after deletion of MGR3. Mitochondria were isolated from wild-type cells, as well as from mgr1, mgr3, and mgr1 mgr3 mutants. As shown in Figure 3B, Yme1p in mitochondrial extracts from wild-type cells migrates in two complexes (Figure 3C, lanes 1 and 5; white and black arrows). Because the larger band disappeared and the smaller band increased in intensity in mitochondria lacking Mgr1p (Figure 3C, lane 2), Mgr3p (Figure 3C, lane 3), or both Mgr1p and Mgr3p (Figure 3C, lane 4), we conclude that the upper band consists of a supercomplex containing Mgr3p, Mgr1p, and the i-AAA protease. Supporting this view, most of Mgr1p (Figure 4B, lane 1, white arrow) migrates in a single, large band that is dependent upon the presence of Mgr3p and Yme1p (Figure 4B, lanes 2–4). Similar results were obtained when the mobility of Mgr3p was examined in the presence or absence of Mgr1p and Yme1p (Supplemental Figure S3).
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450 kDa (Figure 4B, lane 2, black arrow) (Dunn et al., 2006). However, the 450-kDa band containing Mgr1p-myc disappeared when mitochondria were isolated from yme1 mgr3 (Figure 4B, lane 3) cells, indicating that the 450-kDa subcomplex contains both Mgr1p and Mgr3p. Mgr3p-Mgr1p subcomplex assembly is also demonstrated by experiments tracking the Mgr3 protein. In mitochondria from cells expressing Mgr1p and Yme1p (Supplemental Figure S3, lane 1), Mgr3p-His6 migrated similarly to Mgr1p and Yme1p in a band above the 667-kDa marker. However, in the absence of Yme1p (Supplemental Figure S3, lane 2), Mgr3p-His6 was found in the 450-kDa form. In mitochondria lacking Mgr1p, Mgr3p-His6 was found in an even smaller, 200-kDa band (Supplemental Figure S3, lane 3) that is unaffected by further deletion of Yme1p (Supplemental Figure S3, lane 4). We conclude that Mgr3p and Mgr1p normally interact with i-AAA, but Mgr1p and Mgr3p remain stably bound in the absence of Yme1p.
Mgr1p Tethers the Mgr3p-Mgr1p Subcomplex to the i-AAA Protease
Although Mgr1p can bind to Yme1p in mitochondria lacking Mgr3p, the Mgr3p-Yme1p interaction is Mgr1p-dependent. When Mgr1p-myc was precipitated from digitonin-treated mitochondria, the amount of Yme1p coprecipitated was reduced by an average of 64% (n = 5) in mitochondria lacking Mgr3p (Figure 5A, lane 4) in comparison with mitochondria containing Mgr3p (Figure 5A, lane 2). This Mgr1p–Yme1p interaction in the absence of Mgr3p was not apparent using BN-PAGE analysis (Figure 4B, lane 4); perhaps the weaker interaction between Mgr1p and Yme1p in the absence of Mgr3p cannot survive BN-PAGE conditions.
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cells (Figure 5B, lane 4). Thus, our results suggest that Mgr3p and Mgr1p interact with the Yme1p-containing i-AAA complex via the Mgr1 protein.
Protein Degradation Is Defective in Mitochondria Lacking Mgr3p
Like mgr1 mutants (Dunn et al., 2006), mitochondria lacking Mgr3p are defective in the turnover of two known i-AAA substrates. First, we examined the proteolysis of Yta10(161)-DHFRMUT, a fusion between the amino-terminal region of the IM protein Yta10p and a loosely folded form of dihydrofolate reductase (DHFRMUT). After import into isolated mitochondria, Yta10(161)-DHFRMUT is rapidly degraded by the i-AAA protease (Leonhard et al., 1999). We incubated Yta10(161)-DHFRMUT with mitochondria isolated from wild-type, mgr1, mgr3, and yme1 cells, and we then monitored Yta10(161)-DHFRMUT levels over time. After import into mitochondria, Yta10(161)-DHFRMUT is processed by the matrix-localized processing protease and occurs as a lower molecular weight doublet (Dunn et al., 2006). Because the significance of these two forms is not known, we quantified both bands together in our analyses (Figure 6A). In wild-type mitochondria, Yta10(161)-DHFRMUT is quickly degraded, with a half-life of 15 min. In contrast, in mitochondria from the mgr3, mgr1 or yme1 mutant, proteolysis is greatly inhibited. Little or no turnover of Yta10(161)-DHFRMUT was seen in mitochondria lacking Mgr3p, Mgr1p or Yme1p during the 60-min incubation.
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). We engineered the Nde1p-HA construct into wild-type, mgr1, mgr3, mgr1mgr3, and yme1 cells. We then added cycloheximide to cultures of the five strains to stop protein synthesis and monitored Nde1p-HA levels at different time points after translation arrest (Figure 6B). In wild-type cells, Nde1p-HA was rapidly degraded with a half-life of 25 min. As described previously (Augustin et al., 2005; Dunn et al., 2006), Nde1p-HA was completely stabilized in yme1 cells, and cells lacking Mgr1p were inhibited in turnover but not as severely as yme1 cells. Loss of Mgr3p also significantly reduced the rate at which the i-AAA complex degraded Nde1p-HA; Nde1p-HA exhibited a half-life of 36 min in the mgr3 mutant, compared with 25 min in wild-type cells. Our studies with intact cells and isolated mitochondria indicate that Mgr3p, like Mgr1p, plays a critical, but not essential, role in the proteolysis of i-AAA substrates. We also found that there was no additive effect of combining mgr1 and mgr3 deletions (Figure 6B). These data are in agreement with the more severe phenotypes seen in yme1 strains compared with mgr3, mgr1, and mgr1mgr3 mutants. In particular, although yme1 is temperature-sensitive on nonfermentable medium and cold-sensitive on rich glucose medium, the mgr1 or mgr3 single mutants and the mgr1mgr3 double mutant are not cold-sensitive or temperature sensitive (Supplemental Figure S4).
In preliminary studies, we found that the amount of full-length Cox2p, a substrate of the i-AAA complex (Nakai et al., 1995), is normal in mgr1, mgr3, and yme1 mutants under conditions where the all members of the cytochrome oxidase complex are present. However, we discovered an accumulation of Cox2p breakdown products in these mutants compared with wild type (Dunn, unpublished data), suggesting a possible role for Mgr1p and Mgr3p in the degradation pathway of Cox2p.
Maximal Association of an Unfolded Substrate with Yme1p Requires Mgr3p and Mgr1p
To further probe the role of Mgr3p and Mgr1p, we examined the association of the folding-defective Yta10(161)-DHFRMUT protein to Yme1p in mgr1 and mgr3 mutants. For these studies, we imported 35S-labeled Yta10(161)-DHFRMUT into mitochondria in the presence of ATP. Then, to inhibit Yme1p-dependent degradation, mitochondria were washed and resuspended in buffer lacking ATP. After further incubation, mitochondria were solubilized in digitonin-containing buffer and binding of Yta10(161)-DHFRMUT to a fully functional Yme1p-His6 fusion protein was monitored using coprecipitation analyses. Consistent with a previous report (Leonhard et al., 1999), we found that in mitochondria containing both Mgr1p and Mgr3p, the imported Yta10(161)-DHFRMUT protein pelleted along with Yme1p-His6 (Figure 7). Although the amount of Yta10(161)-DHFRMUT that associated with Yme1p-His6 only averaged 4% of the total amount imported, this interaction is significant. In particular, when mitochondria lacked the Yme1p-His6 fusion protein, Yta10(161)-DHFRMUT did not pellet with the Ni-NTA beads. Moreover, when we precipitated Atp21p-His6, an abundant IM protein carrying the hexahistidine tag, only background levels of Yta10(161)-DHFRMUT were found in the pellet (Supplemental Figure S5A).
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or mgr3 mutants were used, <1% of the imported Yta10(161)-DHFRMUT protein coprecipitated with Yme1p-His6 (Figure 7). Although binding of Yta10(161)-DHFRMUT to Yme1p-His6 was lower without Mgr1p or Mgr3p, the amount of Yta10(161)-DHFRMUT in the pellet was clearly greater than background levels. Thus, we conclude that Mgr1p and Mgr3p play an important, but not absolute, role in substrate-Yme1p interaction.
Mgr1p and Mgr3p Bind Substrate in the Absence of Yme1p
We then investigated substrate binding to the Mgr1 and Mgr3 proteins. Using antibodies against the myc epitope, we found that 6% of imported Yta10(161)-DHFRMUT coprecipitated with Mgr1p-myc from solubilized mitochondria (Figure 8A). This binding is specific, because Yta10(161)-DHFRMUT was not found in the pellet fraction when mitochondria lacked myc-tagged Mgr1p (Figure 8A) or when an abundant Atp21p-myc fusion protein was precipitated from mitochondrial extracts (Supplemental Figure S5B). Surprisingly, similar amounts of Yta10(161)-DHFRMUT coprecipitated with Mgr1p-myc in mitochondria lacking Yme1p, Mgr3p, or both Yme1p and Mgr3p (Figure 8A). Similar to our results with Mgr1p, Mgr3p can bind substrate in the absence of both Yme1p and Mgr1p. Approximately 4% of imported substrate coprecipitated with Mgr3p-His6. Comparable amounts of Yta10(161)-DHFRMUT coprecipitated with Mgr3p-His6 in mitochondria expressing Yme1p and Mgr1p or in mitochondria lacking Yme1p, Mgr1p, or both Yme1p and Mgr1p (Figure 8B). Our results suggest that both Mgr1p and Mgr3p are required for the maximal binding of at least one substrate to Yme1p. Furthermore, because both Mgr1p and Mgr3p associate with Yta10(161)-DHFRMUT in the absence of Yme1p, our studies raise the possibility that Mgr1p and Mgr3p function in targeting substrates to the i-AAA protease.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Zahedi et al., 2006), we uncovered MGR3, a new gene required for cell growth without mtDNA. We found that the Mgr3 protein associates with both Mgr1p and Yme1p in the mitochondrial IM, and we discovered that mitochondria lacking Mgr3p were defective in Yme1p-mediated protein turnover, as demonstrated previously for the mgr1 mutant (Dunn et al., 2006). We conclude that Mgr3p is required for full activity of the i-AAA protease. Because MGR3 was missed in our earlier screen for petite-negative yeast mutants (Dunn et al., 2006), our prior screen clearly was not exhaustive and additional petite-negative mutants may await discovery. Interestingly, findings from a genome-wide chemical screen (Hillenmeyer et al., 2008) also support a common function for Mgr1p and Mgr3p. In this study, mgr1 and mgr3 mutants behaved more similarly to one another than to nearly 5000 other yeast knockout strains in their response to hundreds of different drug treatments. We note that mgr3 was recently reported to have a weak synthetic fitness interaction with gem1 (Frederick et al., 2008), which lacks a protein required for wild-type mitochondrial shape and inheritance (Frederick et al., 2004). This synthetic growth defect in gem1 mgr3 double mutants, however, may be due to the combination of the increased rate of mtDNA loss in gem1 mutants and the petite-negative phenotype of mgr3. In support of this view, three of the five strongest synthetic fitness interactions with gem1 are with mutations that also cause a petite-negative phenotype: fmp13 (Supplemental Table S1), tom70 (Dunn and Jensen, 2003), and phb2 (Dunn et al., 2006).
Our blue native gels results suggest that there are at least two different complexes containing the i-AAA subunit Yme1p. The largest resolvable form, which we call the "i-AAA supercomplex," contains Yme1p, Mgr1p, and Mgr3p, whereas the smaller, i-AAA core complex lacks Mgr1p and Mgr3p. Both complexes migrated well above our largest molecular mass standard of 667 kDa, preventing us from accurately determining their sizes. However, because previous gel filtration analyses found only a single 850-kDa complex (Leonhard et al., 1996) consisting solely of Yme1p subunits (Langer, personal communication), we presume that the higher mobility Yme1p band found in mitochondria lacking Mgr3p or Mgr1p after BN-PAGE analysis represents this 850-kDa form. Structural studies indicate that members of the AAA family frequently form hexameric ATPase ring structures (Baker and Sauer, 2006). If this is true for the i-AAA protease, then it is likely that the 850-kDa complex is composed of at least one hexamer, composed of 82-kDa Yme1p subunits. However, because these studies are done in the presence of detergent, it is difficult to determine precise complex sizes. Discerning the organization of the i-AAA supercomplex is even more problematic. In the absence of the i-AAA core complex, Mgr3p and Mgr1p form a subcomplex of 450-kDa. Therefore, it is likely that the largest resolvable species on our blue native gels represents a connection between the 850-kDa Yme1p multimer and the 450-kDa Mgr3p–Mgr1p-containing subcomplex. Because the Mgr3p–Yme1p interaction requires the Mgr1 protein, we speculate that the Mgr3p–Mgr1p subcomplex is tethered to the Yme1p-containing complex via the Mgr1 subunit. Although our pull-down studies confirm Mgr3p–Mgr1p–Yme1p interactions, they also suggest that the interplay between the three proteins may be dynamic. For example, only 10% of Mgr3p, Mgr1p, and Yme1p coprecipitate with each other. Finally, our immunoblots commonly show that some amount of Mgr1p, Mgr3p, and Yme1p is incapable of entering the blue native separating gel, suggesting that even larger, so far irresolvable, i-AAA supercomplexes exist. The exact composition and arrangement of i-AAA clearly awaits further study.
Two observations suggest that Mgr3p and Mgr1p play a direct role in the binding of substrates to the i-AAA protease. First, efficient binding of the loosely folded Yta10(161)-DHFRMUT protein to Yme1p requires both Mgr3p and Mgr1p. In the absence of either protein, approximately fivefold less Yta10(161)-DHFRMUT coprecipitates with Yme1p. Second, Mgr3p and Mgr1p can interact with substrate independently of other known subunits. A similar amount of Yta10(161)-DHFRMUT pellets with Mgr1p-myc from mitochondrial extracts containing Yme1p and Mgr3p as from lysates from mitochondria lacking one or both proteins. Likewise, Yme1p and Mgr1p are not necessary for the association of Yta10(161)-DHFRMUT with Mgr3p-His6. Because Mgr3p and Mgr1p interact with substrate independently, it will be interesting to determine if the two proteins function in a concerted or sequential manner. Surprisingly, equivalent levels of substrate can be coprecipitated with Mgr1p in the presence or in the absence of both Yme1p and Mgr3p; yet, in mitochondria lacking Mgr3p, substrate coprecipitation with Yme1p is substantially reduced despite a detectable Mgr1p–Yme1p interaction in coprecipitation assays. We therefore believe that the Mgr1 protein associated with Yme1p in the absence of Mgr3p is likely to be in an abnormal, perhaps misfolded conformation that leaves it unable to bind substrate and that the substrate/Mgr1p interaction seen in mgr3 mitochondria is due to substrate interaction with a pool of binding-competent, non–Yme1p-bound Mgr1p.
We note that the phenotypes of cells lacking Mgr1p, Mgr3p, or both proteins are much less severe than the yme1 mutant. For example, although the yme1 mutant has a temperature-sensitive growth defect on nonfermentable medium and is cold sensitive on glucose-containing medium, the mgr1, mgr3, and mgr1mgr3 cells show no growth defects. Moreover, mgr1 and mgr3 show more variation in their phenotypes in different strain backgrounds than does the yme1 knockout. These data are consistent with at least two scenarios: the Mgr1p–Mgr3p subcomplex could play a minor role in the degradation of all endogenous i-AAA substrates, or the Mgr1p–Mgr3p subcomplex could play an critical role in the degradation of a subset of i-AAA targets. The analyses of different endogenous substrates for their dependence upon the Mgr1p–Mgr3p subcomplex is needed to distinguish between these (and other) possibilities.
Our results suggest that Mgr3p and Mgr1p function like some bacterial adaptor proteins, such as SspB, ClpS, and others that recruit substrates to specific AAA proteases (Baker and Sauer, 2006). The SspB adaptor promotes efficient proteolysis by binding to both the exposed ssrA-tag of target proteins and to the ClpXP protease, stimulating degradation at low concentrations of substrate (Levchenko et al., 2000, 2003, 2005; Wah et al., 2002). Like Mgr3p and Mgr1p, SspB is not essential for ClpXP proteolysis, but SspB increases turnover rates by severalfold (Levchenko et al., 2000; Flynn et al., 2004). Likewise, the ClpS adaptor enhances the degradation of N-end rule substrates by the ClpAP protease at low substrate concentrations (Erbse et al., 2006; Wang et al., 2007). However, ClpS also inhibits degradation of ssrA-tagged proteins (Dougan et al., 2002). It is unclear whether Mgr1p or Mgr3p could, like ClpS, promote the degradation of some substrates while preventing the degradation of others. Finally, we note the additional possibility that Mgr3p and Mgr1p promote Yme1p nucleotide hydrolysis upon binding to substrate, similar to yeast Vta1p, which interacts with the Vps4p AAA protein at the endosomal membrane to promote its assembly and stimulate its ATPase activity (Azmi et al., 2006; Lottridge et al., 2006).
Recent evidence suggests that two distinct regions of the Yme1 protein are capable of binding to substrates. It was previously shown that an N-terminal, juxtamembrane portion of Yme1p lacking the AAA and protease domains, Yme1(1-313)p, is capable of binding to unfolded substrate at the mitochondrial inner membrane (Leonhard et al., 2000). Additionally, recent in vivo and in vitro work demonstrated the ability of a helical region at the very C terminus of Yme1p, Yme1(650-709)p, to bind substrate in the absence of both the AAA and protease domain (Graef et al., 2007). Interestingly, our preliminary data (Dunn, unpublished data) suggest that Mgr1p is able to bind to the Yme1(1-313) truncation, raising the possibility that Mgr1p or Mgr3p might directly mediate substrate binding to this surface. Future experiments should address how Mgr3p or Mgr1p contribute or control substrate binding to these two separate domains of Yme1p.
Although we have uncovered new components contributing to i-AAA activity, it remains puzzling why protein turnover at the IM is required for mtDNA-dependent yeast cell growth. In cells lacking mtDNA, the electron transport chain and the F0 portion of the ATP synthase are nonfunctional, and the F1 portion of the ATP synthase becomes critical for generating the IM potential (Chen and Clark-Walker, 2000). In this case, now results from the electrogenic exchange of cytosolic ATP–4 for ADP–3 produced by the matrix-localized F1-ATPase (Giraud and Velours, 1997; Chen and Clark-Walker, 2000). In petite mutants, i-AAA activity is required for efficient F1-ATPase function. Mitochondria lacking Yme1p have reduced ATPase activity and are deficient in the ATP-driven generation of (Kominsky et al., 2002), whereas constructs that express forms of the F1-ATPase with increased activity restore mtDNA independence to the yme1 mutant (Kominsky et al., 2002; Dunn et al., 2006). It has been suggested that the i-AAA protease degrades one or more inhibitors of the F1-ATPase. However, this scenario is complicated by the fact that the catalytic domains of i-AAA and the F1-ATPase are on opposite sides of the IM. Alternatively, some evidence suggests that peptides released from the mitochondria as a result of proteolysis could act in mitochondria-to-nucleus communication (Arnold et al., 2006; Haynes et al., 2007). Perhaps a putative signaling pathway, activated by i-AAA proteolysis products, plays a critical role in petite cells. The identification of additional substrates for the i-AAA protease complex will help help unravel the connection between mitochondrial protein quality control and mtDNA-independent cell growth.
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ACKNOWLEDGMENTS |
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Footnotes |
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* Present address: Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University College of Physicians and Surgeons, 701 West 168th St., Room 720, New York, NY 10032. 
Address correspondence to: Robert Jensen (rjensen{at}jhmi.edu).
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Arnold, I., Wagner-Ecker, M., Ansorge, W., and Langer, T. (2006). Evidence for a novel mitochondria-to-nucleus signalling pathway in respiring cells lacking i-AAA protease and the ABC-transporter Mdl1. Gene 367, 74–88.[CrossRef][Medline]
Attardi, G., and Schatz, G. (1988). Biogenesis of mitochondria. Annu. Rev. Cell Biol 4, 289–333.[CrossRef][Medline]
Augustin, S., Nolden, M., Muller, S., Hardt, O., Arnold, I., and Langer, T. (2005). Characterization of peptides released from mitochondria: evidence for constant proteolysis and peptide efflux. J. Biol. Chem 280, 2691–2699.
Azmi, I., Davies, B., Dimaano, C., Payne, J., Eckert, D., Babst, M., and Katzmann, D. J. (2006). Recycling of ESCRTs by the AAA-ATPase Vps4 is regulated by a conserved VSL region in Vta1. J. Cell Biol 172, 705–717.
Baker, T. A., and Sauer, R. T. (2006). ATP-dependent proteases of bacteria: recognition logic and operating principles. Trends Biochem. Sci 31, 647–653.[CrossRef][Medline]
Brachmann, C. B., Davies, A., Cost, G. J., Caputo, E., Li, J., Hieter, P., and Boeke, J. D. (1998). Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115–132.[CrossRef][Medline]
Chen, X. J., and Clark-Walker, G. D. (1999). Alpha and beta subunits of F1-ATPase are required for survival of petite mutants in Saccharomyces cerevisiae. Mol. Gen. Genet 262, 898–908.[CrossRef][Medline]
Chen, X. J., and Clark-Walker, G. D. (2000). The petite mutation in yeasts: 50 years on. Int. Rev. Cytol 194, 197–238.[Medline]
Daum, G., Bohni, P. C., and Schatz, G. (1982). Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. J. Biol. Chem 257, 13028–13033.
Dougan, D. A., Reid, B. G., Horwich, A. L., and Bukau, B. (2002). ClpS, a substrate modulator of the ClpAP machine. Mol. Cell 9, 673–683.[CrossRef][Medline]
Dunn, C. D., and Jensen, R. E. (2003). Suppression of a defect in mitochondrial protein import identifies cytosolic proteins required for viability of yeast cells lacking mitochondrial DNA. Genetics 165, 35–45.
Dunn, C. D., Lee, M. S., Spencer, F. A., and Jensen, R. E. (2006). A genomewide screen for petite-negative yeast strains yields a new subunit of the i-AAA protease complex. Mol. Biol. Cell 17, 213–226.
Dupont, C. H., Mazat, J. P., and Guerin, B. (1985). The role of adenine nucleotide translocation in the energization of the inner membrane of mitochondria isolated from rho + and rho degree strains of Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun 132, 1116–1123.[CrossRef][Medline]
Emtage, J. L., and Jensen, R. E. (1993). MAS6 encodes an essential inner membrane component of the yeast mitochondrial protein import pathway. J. Cell Biol 122, 1003–1012.
Erbse, A., Schmidt, R., Bornemann, T., Schneider-Mergener, J., Mogk, A., Zahn, R., Dougan, D. A., and Bukau, B. (2006). ClpS is an essential component of the N-end rule pathway in Escherichia coli. Nature 439, 753–756.[CrossRef][Medline]
Evan, G. I., Lewis, G. K., Ramsay, G., and Bishop, J. M. (1985). Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol. Cell Biol 5, 3610–3616.
Flynn, J. M., Levchenko, I., Sauer, R. T., and Baker, T. A. (2004). Modulating substrate choice: the SspB adaptor delivers a regulator of the extracytoplasmic-stress response to the AAA+ protease ClpXP for degradation. Genes Dev 18, 2292–2301.
Frederick, R. L., McCaffery, J. M., Cunningham, K. W., Okamoto, K., and Shaw, J. M. (2004). Yeast Miro GTPase, Gem1p, regulates mitochondrial morphology via a novel pathway. J. Cell Biol 167, 87–98.
Frederick, R. L., Okamoto, K., and Shaw, J. M. (2008). Multiple pathways influence mitochondrial inheritance in budding yeast. Genetics 178, 825–837.
Giaever, G. et al. (2002). Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387–391.[CrossRef][Medline]
Giraud, M. F., and Velours, J. (1997). The absence of the mitochondrial ATP synthase delta subunit promotes a slow growth phenotype of rho– yeast cells by a lack of assembly of the catalytic sector F1. Eur. J. Biochem 245, 813–818.[Medline]
Goldring, E. S., Grossman, L. I., Krupnick, D., Cryer, D. R., and Marmur, J. (1970). The petite mutation in yeast. Loss of mitochondrial deoxyribonucleic acid during induction of petites with ethidium bromide. J. Mol. Biol 52, 323–335.[CrossRef][Medline]
Graef, M., Seewald, G., and Langer, T. (2007). Substrate recognition by AAA+ ATPases: distinct substrate binding modes in ATP-dependent protease Yme1 of the mitochondrial intermembrane space. Mol. Cell Biol 27, 2476–2485.
Harlow, E., and Lane, D. (1999). Using Antibodies: A Laboratory Manual, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Hase, T., Muller, U., Riezman, H., and Schatz, G. (1984). A 70-kd protein of the yeast mitochondrial outer membrane is targeted and anchored via its extreme amino terminus. EMBO J 3, 3157–3164.[Medline]
Haynes, C. M., Petrova, K., Benedetti, C., Yang, Y., and Ron, D. (2007). ClpP mediates activation of a mitochondrial unfolded protein response in C. elegans. Dev. Cell 13, 467–480.[CrossRef][Medline]
Hillenmeyer, M. E. et al. (2008). The chemical genomic portrait of yeast: uncovering a phenotype for all genes. Science 320, 362–365.
Huh, W. K., Falvo, J. V., Gerke, L. C., Carroll, A. S., Howson, R. W., Weissman, J. S., and O'Shea, E. K. (2003). Global analysis of protein localization in budding yeast. Nature 425, 686–691.[CrossRef][Medline]
Jensen, R. E., Dunn, C. D., Youngman, M. J., and Sesaki, H. (2004). Mitochondrial building blocks. Trends Cell Biol 14, 215–218.[CrossRef][Medline]
Jensen, R. E., and Yaffe, M. P. (1988). Import of proteins into yeast mitochondria: the nuclear MAS2 gene encodes a component of the processing protease that is homologous to the MAS1-encoded subunit. EMBO J 7, 3863–3871.[Medline]
Kerscher, O., Sepuri, N. B., and Jensen, R. E. (2000). Tim18p is a new component of the Tim54p-Tim22p translocon in the mitochondrial inner membrane. Mol. Biol. Cell 11, 103–116.
Koehler, C. M. (2004). New developments in mitochondrial assembly. Annu. Rev. Cell Dev. Biol 20, 309–335.[CrossRef][Medline]
Kominsky, D. J., Brownson, M. P., Updike, D. L., and Thorsness, P. E. (2002). Genetic and biochemical basis for viability of yeast lacking mitochondrial genomes. Genetics 162, 1595–1604.
Leonhard, K., Guiard, B., Pellecchia, G., Tzagoloff, A., Neupert, W., and Langer, T. (2000). Membrane protein degradation by AAA proteases in mitochondria: extraction of substrates from either membrane surface. Mol. Cell 5, 629–638.[CrossRef][Medline]
Leonhard, K., Herrmann, J. M., Stuart, R. A., Mannhaupt, G., Neupert, W., and Langer, T. (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]
Leonhard, K., Stiegler, A., Neupert, W., and Langer, T. (1999). Chaperone-like activity of the AAA domain of the yeast Yme1 AAA protease. Nature 398, 348–351.
Levchenko, I., Grant, R. A., Flynn, J. M., Sauer, R. T., and Baker, T. A. (2005). Versatile modes of peptide recognition by the AAA+ adaptor protein SspB. Nat. Struct. Mol. Biol 12, 520–525.[CrossRef][Medline]
Levchenko, I., Grant, R. A., Wah, D. A., Sauer, R. T., and Baker, T. A. (2003). Structure of a delivery protein for an AAA+ protease in complex with a peptide degradation tag. Mol. Cell 12, 365–372.[CrossRef][Medline]
Levchenko, I., Seidel, M., Sauer, R. T., and Baker, T. A. (2000). A specificity-enhancing factor for the ClpXP degradation machine. Science 289, 2354–2356.
Longtine, M. S., McKenzie, A., 3rd, Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P., and Pringle, J. R. (1998). Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961.[CrossRef][Medline]
Lottridge, J. M., Flannery, A. R., Vincelli, J. L., and Stevens, T. H. (2006). Vta1p and Vps46p regulate the membrane association and ATPase activity of Vps4p at the yeast multivesicular body. Proc. Natl. Acad. Sci. USA 103, 6202–6207.
Nakai, T., Yasuhara, T., Fujiki, Y., and Ohashi, A. (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]
Pon, L., Moll, T., Vestweber, D., Marshallsay, B., and Schatz, G. (1989). Protein import into mitochondria: ATP-dependent protein translocation activity in a submitochondrial fraction enriched in membrane contact sites and specific proteins. J. Cell Biol 109, 2603–2616.
Prokisch, H. et al. (2004). Integrative analysis of the mitochondrial proteome in yeast. PLoS Biol 2, e160.[CrossRef][Medline]
Rehling, P., Brandner, K., and Pfanner, N. (2004). Mitochondrial import and the twin-pore translocase. Nat. Rev. Mol. Cell Biol 5, 519–530.[CrossRef][Medline]
Ryan, K. R., Leung, R. S., and Jensen, R. E. (1998). Characterization of the mitochondrial inner membrane translocase complex: the Tim23p hydrophobic domain interacts with Tim17p but not with other Tim23p molecules. Mol. Cell Biol 18, 178–187.
Schagger, H., and von Jagow, G. (1991). Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal. Biochem 199, 223–231.[CrossRef][Medline]
Sickmann, A. et al. (2003). The proteome of Saccharomyces cerevisiae mitochondria. Proc. Natl. Acad. Sci. USA 100, 13207–13212.
Sikorski, R. S., and Hieter, P. (1989). A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19–27.
Slonimski, P. P., Perrodin, G., and Croft, J. H. (1968). Ethidium bromide induced mutation of yeast mitochondria: complete transformation of cells into respiratory deficient non-chromosomal "petites". Biochem. Biophys. Res. Commun 30, 232–239.[CrossRef][Medline]
Thorsness, P. E., and Fox, T. D. (1993). Nuclear mutations in Saccharomyces cerevisiae that affect the escape of DNA from mitochondria to the nucleus. Genetics 134, 21–28.[Abstract]
Thorsness, P. E., White, K. H., and Fox, T. D. (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.
Tzagoloff, A. (1982). Mitochondria, New York: Plenum Press.
Wah, D. A., Levchenko, I., Baker, T. A., and Sauer, R. T. (2002). Characterization of a specificity factor for an AAA+ ATPase: assembly of SspB dimers with ssrA-tagged proteins and the ClpX hexamer. Chem. Biol 9, 1237–1245.[CrossRef][Medline]
Wang, K. H., Sauer, R. T., and Baker, T. A. (2007). ClpS modulates but is not essential for bacterial N-end rule degradation. Genes Dev 21, 403–408.
Winston, F., Dollard, C., and Ricupero-Hovasse, S. L. (1995). Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast 11, 53–55.[CrossRef][Medline]
Yaffe, M. P., Jensen, R. E., and Guido, E. C. (1989). The major 45-kDa protein of the yeast mitochondrial outer membrane is not essential for cell growth or mitochondrial function. J. Biol. Chem 264, 21091–21096.
Zahedi, R. P., Sickmann, A., Boehm, A. M., Winkler, C., Zufall, N., Schonfisch, B., Guiard, B., Pfanner, N., and Meisinger, C. (2006). Proteomic analysis of the yeast mitochondrial outer membrane reveals accumulation of a subclass of preproteins. Mol. Biol. Cell 17, 1436–1450.
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