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• W2078644413 abstract "We show that Ydr049 (renamed VCP/Cdc48-associated mitochondrial stress-responsive—Vms1), a member of an unstudied pan-eukaryotic protein family, translocates from the cytosol to mitochondria upon mitochondrial stress. Cells lacking Vms1 show progressive mitochondrial failure, hypersensitivity to oxidative stress, and decreased chronological life span. Both yeast and mammalian Vms1 stably interact with Cdc48/VCP/p97, a component of the ubiquitin/proteasome system with a well-defined role in endoplasmic reticulum-associated protein degradation (ERAD), wherein misfolded ER proteins are degraded in the cytosol. We show that oxidative stress triggers mitochondrial localization of Cdc48 and this is dependent on Vms1. When this system is impaired by mutation of Vms1, ubiquitin-dependent mitochondrial protein degradation, mitochondrial respiratory function, and cell viability are compromised. We demonstrate that Vms1 is a required component of an evolutionarily conserved system for mitochondrial protein degradation, which is necessary to maintain mitochondrial, cellular, and organismal viability. We show that Ydr049 (renamed VCP/Cdc48-associated mitochondrial stress-responsive—Vms1), a member of an unstudied pan-eukaryotic protein family, translocates from the cytosol to mitochondria upon mitochondrial stress. Cells lacking Vms1 show progressive mitochondrial failure, hypersensitivity to oxidative stress, and decreased chronological life span. Both yeast and mammalian Vms1 stably interact with Cdc48/VCP/p97, a component of the ubiquitin/proteasome system with a well-defined role in endoplasmic reticulum-associated protein degradation (ERAD), wherein misfolded ER proteins are degraded in the cytosol. We show that oxidative stress triggers mitochondrial localization of Cdc48 and this is dependent on Vms1. When this system is impaired by mutation of Vms1, ubiquitin-dependent mitochondrial protein degradation, mitochondrial respiratory function, and cell viability are compromised. We demonstrate that Vms1 is a required component of an evolutionarily conserved system for mitochondrial protein degradation, which is necessary to maintain mitochondrial, cellular, and organismal viability. Vms1—an evolutionarily conserved protein that translocates to mitochondria under stress Loss of Vms1 causes progressive mitochondrial failure and cell death Vms1 binds to and causes the mitochondrial translocation of Cdc48 and Npl4 for protein degradation Vms1 is required for normal activity of the ubiquitin/proteasome system at mitochondria Mitochondria are dynamic and complex organelles that are essential for many aspects of cellular function including metabolism and cell death. Consistent with these critical roles, mitochondrial dysfunction is associated with most aging-related human diseases, including neurodegenerative disorders, type 2 diabetes, and cancer (Wallace, 2005Wallace D.C. A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine.Annu. Rev. Genet. 2005; 39: 359-407Crossref PubMed Scopus (2344) Google Scholar). The best current inventory of mammalian mitochondrial resident proteins consists of 1098 proteins (Pagliarini et al., 2008Pagliarini D.J. Calvo S.E. Chang B. Sheth S.A. Vafai S.B. Ong S.E. Walford G.A. Sugiana C. Boneh A. Chen W.K. et al.A mitochondrial protein compendium elucidates complex I disease biology.Cell. 2008; 134: 112-123Abstract Full Text Full Text PDF PubMed Scopus (1362) Google Scholar). Surprisingly, nearly 300 of these proteins have completely undefined functions, including many that are highly conserved throughout eukarya, indicating that they perform a fundamental and important function (Meisinger et al., 2008Meisinger C. Sickmann A. Pfanner N. The mitochondrial proteome: from inventory to function.Cell. 2008; 134: 22-24Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, Pagliarini et al., 2008Pagliarini D.J. Calvo S.E. Chang B. Sheth S.A. Vafai S.B. Ong S.E. Walford G.A. Sugiana C. Boneh A. Chen W.K. et al.A mitochondrial protein compendium elucidates complex I disease biology.Cell. 2008; 134: 112-123Abstract Full Text Full Text PDF PubMed Scopus (1362) Google Scholar). The genes that encode the mitochondrial proteome are heavily represented among known human disease genes, with about 20% of predicted human mitochondrial proteins implicated in one or more hereditary diseases (Andreoli et al., 2004Andreoli C. Prokisch H. Hortnagel K. Mueller J.C. Munsterkotter M. Scharfe C. Meitinger T. MitoP2, an integrated database on mitochondrial proteins in yeast and man.Nucleic Acids Res. 2004; 32: D459-D462Crossref PubMed Google Scholar, Elstner et al., 2008Elstner M. Andreoli C. Ahting U. Tetko I. Klopstock T. Meitinger T. Prokisch H. MitoP2: an integrative tool for the analysis of the mitochondrial proteome.Mol. Biotechnol. 2008; 40: 306-315Crossref PubMed Scopus (61) Google Scholar). Presumably, the quarter of the mitochondrial proteome that is uncharacterized contains other proteins with links to human disease that await discovery. Making these connections would be greatly facilitated by an understanding of the biochemical and physiological function of these proteins. Therefore, we initiated studies to determine the genetic and biochemical functions of a subset of these conserved but uncharacterized mitochondrial proteins (Hao and Rutter, 2009Hao H.X. Rutter J. Revealing human disease genes through analysis of the yeast mitochondrial proteome.Cell Cycle. 2009; 8: 4007-4008Crossref PubMed Scopus (6) Google Scholar). As a result of this project, we previously identified the unstudied Yol071 yeast protein, which we named Sdh5, as a critical assembly factor for the succinate dehydrogenase complex/complex II (Hao et al., 2009Hao H.X. Khalimonchuk O. Schraders M. Dephoure N. Bayley J.P. Kunst H. Devilee P. Cremers C.W. Schiffman J.D. Bentz B.G. et al.SDH5, a gene required for flavination of succinate dehydrogenase, is mutated in paraganglioma.Science. 2009; 325: 1139-1142Crossref PubMed Scopus (546) Google Scholar). By virtue of this observation, we identified the human SDH5 ortholog as the causative gene in a familial form of the paraganglioma neuroendocrine tumor syndrome (Hao et al., 2009Hao H.X. Khalimonchuk O. Schraders M. Dephoure N. Bayley J.P. Kunst H. Devilee P. Cremers C.W. Schiffman J.D. Bentz B.G. et al.SDH5, a gene required for flavination of succinate dehydrogenase, is mutated in paraganglioma.Science. 2009; 325: 1139-1142Crossref PubMed Scopus (546) Google Scholar). We describe herein another unstudied conserved mitochondrial protein, Ydr049, which we now designate VCP/Cdc48-associated mitochondrial stress-responsive 1 (Vms1). VMS1 is highly evolutionarily conserved, with one ortholog existing in most eukaryotic species. Initially using yeast, we show that Vms1 protects mitochondrial respiratory function and combats cell death in response to various stress stimuli. Both yeast and human Vms1 copurify with Cdc48/VCP/p97, and we show that Vms1 stably associates with both Cdc48 and its cofactor Npl4, which have well-defined roles in the degradation of endoplasmic reticulum (ER) proteins by the proteasome. We find that Cdc48 recruitment to mitochondria is Vms1 dependent and that this system is required for normal mitochondrial protein degradation under stress conditions. Clear links between mitochondria, which are membrane-confined organelles, and the cytosolic ubiquitin/proteasome system have recently been described (Livnat-Levanon and Glickman, 2010Livnat-Levanon N. Glickman M.H. Ubiquitin-proteasome system and mitochondria—reciprocity.Biochim. Biophys. Acta. 2010; (Published online July 30, 2010)https://doi.org/10.1016/j.bbagrm.2010.07.005Crossref PubMed Scopus (137) Google Scholar). Fzo1, a mitochondrial outer membrane protein, was shown to be ubiquitinated by the Mdm30 cytosolic E3 ubiquitin ligase and degraded by the proteasome (Cohen et al., 2008Cohen M.M. Leboucher G.P. Livnat-Levanon N. Glickman M.H. Weissman A.M. Ubiquitin-proteasome-dependent degradation of a mitofusin, a critical regulator of mitochondrial fusion.Mol. Biol. Cell. 2008; 19: 2457-2464Crossref PubMed Scopus (132) Google Scholar, Fritz et al., 2003Fritz S. Weinbach N. Westermann B. Mdm30 is an F-box protein required for maintenance of fusion-competent mitochondria in yeast.Mol. Biol. Cell. 2003; 14: 2303-2313Crossref PubMed Scopus (113) Google Scholar). Mitochondria in cells lacking MDM30 aggregate in clumps and respire inadequately, leading to shortened life span in response to stress. This is likely a manifestation of a broader system for degradation of mitochondria-associated proteins. The flux of imported proteins and the proximity to oxidative phosphorylation result in significant protein damage and misfolding at mitochondria, necessitating a responsive quality control system. The mitochondria contains an intrinsic system of proteases dedicated to quality control (Tatsuta, 2009Tatsuta T. Protein quality control in mitochondria.J. Biochem. 2009; 146: 455-461Crossref PubMed Scopus (41) Google Scholar), but the cytosolic ubiquitin/proteasome system appears to also play a role. Based on the data presented herein, we propose that Vms1 plays a conserved role in recruiting the ubiquitin/proteasome system for stress-responsive mitochondrial protein degradation. The Vms1 protein was detected by mass spectrometry in highly purified mitochondria (Sickmann et al., 2003Sickmann A. Reinders J. Wagner Y. Joppich C. Zahedi R. Meyer H.E. Schonfisch B. Perschil I. Chacinska A. Guiard B. et al.The proteome of Saccharomyces cerevisiae mitochondria.Proc. Natl. Acad. Sci. USA. 2003; 100: 13207-13212Crossref PubMed Scopus (680) Google Scholar). Surprisingly, however, a functional Vms1-GFP fusion localized primarily to the cytosol in synthetic glucose-containing medium (Figure 1A , top) as well as rich and synthetic media containing glycerol or raffinose (data not shown). In a small fraction of cells grown in synthetic glucose medium, Vms1-GFP partially colocalized with the mitochondrial marker mito-RFP (Figure 1A, top, marked with arrow). Hypothesizing that this small population might be cells that have lost mitochondrial DNA (rho°), we directly tested a rho° strain and found that Vms1 was partially mitochondrial in all cells (Figure 1A). Loss of mitochondrial DNA has a number of effects on mitochondrial physiology, including decreased mitochondrial membrane potential (Petit et al., 1996Petit P. Glab N. Marie D. Kieffer H. Metezeau P. Discrimination of respiratory dysfunction in yeast mutants by confocal microscopy, image, and flow cytometry.Cytometry. 1996; 23: 28-38Crossref PubMed Scopus (21) Google Scholar). The combination of Antimycin A and oligomycin, which blocks the establishment of the mitochondrial membrane potential (Priault et al., 2005Priault M. Salin B. Schaeffer J. Vallette F.M. di Rago J.P. Martinou J.C. Impairing the bioenergetic status and the biogenesis of mitochondria triggers mitophagy in yeast.Cell Death Differ. 2005; 12: 1613-1621Crossref PubMed Scopus (224) Google Scholar), caused near-complete localization of Vms1 to mitochondria (see Figure S1A available online). Treatment with the uncouplers CCCP or FCCP, which directly dissipate the membrane potential, also caused mitochondrial localization of Vms1 (Figure S1A). We also found that oxidative stress, which indirectly impairs mitochondrial function, elicited by hydrogen peroxide or deletion of the mitochondrial superoxide dismutase, Sod2, caused mitochondrial translocation of Vms1 (Figure 1A and data not shown). Hydrogen peroxide also caused Vms1-GFP localization to punctae that do not have mito-RFP, which may be damaged mitochondria that fail to import mito-RFP. Treatment with the TOR protein kinase inhibitor rapamycin, which increases mitochondrial oxidative damage (Kissova et al., 2006Kissova I. Deffieu M. Samokhvalov V. Velours G. Bessoule J.J. Manon S. Camougrand N. Lipid oxidation and autophagy in yeast.Free Radic. Biol. Med. 2006; 41: 1655-1661Crossref PubMed Scopus (57) Google Scholar), also caused robust mitochondrial translocation of Vms1-GFP (Figure 1A). We hypothesize that perturbation of mitochondrial function is the proximal signal that causes Vms1 translocation, with rapamycin and oxidative stress acting indirectly through mitochondrial oxidative damage. To address the functional significance of Vms1 mitochondrial localization, we analyzed the growth of the vms1Δ mutant under conditions that cause Vms1 mitochondrial translocation. While the vms1Δ mutant was indistinguishable from wild-type in normal conditions, it exhibited severe hypersensitivity to hydrogen peroxide, an effect exacerbated when combined with loss of the mitochondrial Sod2 (Figure 1B). In fact, the sod2Δ vms1Δ double mutant exhibited a modest growth defect even on normal medium (Figure 1B, top). Similarly, the vms1Δ mutant failed to grow in the presence of rapamycin, which was completely rescued by plasmid-borne VMS1 and partially rescued by expression of the human VMS1 gene (Figure 1C). This failure to grow is a result of cell death caused by rapamycin treatment (Figure S1B). A high-copy suppressor screen yielded three genes that, when provided in high copy, rescue the vms1Δ mutant rapamycin hypersensitivity: XBP1, GRX3, and ZWF1 (Figures S1C and S1D). Each of the three has a role in response to stress, particularly oxidative stress. Xbp1 is a stress-induced transcription factor. Grx3 is a glutaredoxin and is a component of a major antioxidant system. Loss of Grx3 leads to increased protein oxidation and hypersensitivity to oxidizing agents (Rodriguez-Manzaneque et al., 1999Rodriguez-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Grx5 glutaredoxin plays a central role in protection against protein oxidative damage in Saccharomyces cerevisiae.Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (257) Google Scholar). ZWF1-encoded glucose-6-phosphate dehydrogenase produces NADPH, which is essential for the reduction of oxidized glutathione. The zwf1Δ mutant is hypersensitive to oxidative stress agents, particularly hydrogen peroxide (Nogae and Johnston, 1990Nogae I. Johnston M. Isolation and characterization of the ZWF1 gene of Saccharomyces cerevisiae, encoding glucose-6-phosphate dehydrogenase.Gene. 1990; 96: 161-169Crossref PubMed Scopus (146) Google Scholar, Outten and Culotta, 2003Outten C.E. Culotta V.C. A novel NADH kinase is the mitochondrial source of NADPH in Saccharomyces cerevisiae.EMBO J. 2003; 22: 2015-2024Crossref PubMed Scopus (131) Google Scholar). These data suggest that Vms1 is required for normal tolerance to oxidative stress and this is manifest by hypersensitivity to both hydrogen peroxide and rapamycin. During the course of these experiments, we observed that cultures maintained past log phase exhibited mitochondrial Vms1 in the absence of other stressors. For example, maintenance in culture past log phase (e.g., 1.5 days) caused mitochondrial localization of Vms1-GFP (Figure 2A ). We confirmed the mitochondrial localization of Vms1-HA by biochemical fractionation after mild crosslinking. Vms1 was detectable in both the cytosolic and crude mitochondrial fractions, and a portion of Vms1-HA comigrated exactly with the mitochondrial marker Tom20 in a sucrose gradient (Figure 2B). As the vms1Δ mutant grows poorly under the same conditions that cause mitochondrial translocation of Vms1, we hypothesized that the primary defect of Vms1 loss involves mitochondrial failure. We tested mitochondrial respiration, and the wild-type and vms1Δ strains consumed oxygen equivalently in log phase. After 1.5 days of culture, however, the vms1Δ mutant had a significant impairment in oxygen consumption (Figure 2C). Due to an exposed iron-sulfur cluster, the activity of aconitase is exquisitely sensitive to mitochondrial oxidative stress. As in the respiration assay, there was no difference in aconitase activity in log phase, but the activity of the vms1Δ mutant was significantly reduced relative to wild-type at day 1.5 without a reduction in protein level (Figure 2D and data not shown). We hypothesize that the progressive mitochondrial stress in stationary phase culture necessitates Vms1 for the maintenance of respiration and aconitase activity. For cells in stationary phase, mitochondrial respiration is required for ATP synthesis and cell survival (Werner-Washburne et al., 1993Werner-Washburne M. Braun E. Johnston G.C. Singer R.A. Stationary phase in the yeast Saccharomyces cerevisiae.Microbiol. Rev. 1993; 57: 383-401Crossref PubMed Google Scholar). Therefore, we sought to determine whether the time-dependent loss of mitochondrial function in the vms1Δ mutant might be manifest as a time-dependent defect in colony formation on glycerol medium, wherein ATP generation and growth requires mitochondrial respiration. At day 1.5 of culture, glycerol growth of the vms1Δ strain remained similar to the wild-type level (Figure 2E). This suggests that the mitochondrial impairment observed at day 1.5, as manifest by decreased oxygen consumption and aconitase activity, is reversible at this point. In contrast, the vms1Δ mutant was greatly impaired in glycerol colony formation at day 3.5 (Figure 2E). By day 5.5, colony formation on glycerol was nearly absent (Figure 2E). After 8.5 days of culture, the vms1Δ mutant also showed greatly reduced ability to form colonies on glucose medium (Figure 2E). This is likely due to cell death, as we obtained similar results using exclusion of trypan blue as a measure of viability (data not shown). Two independent vms1Δ isolates in a different strain background (BY4741) exhibited an even more rapid loss of viability than was observed in the W303 background (Figure 2F). Oxidative stress has been proposed to be the key mediator in causing cell death in static culture (Fabrizio et al., 2001Fabrizio P. Pozza F. Pletcher S.D. Gendron C.M. Longo V.D. Regulation of longevity and stress resistance by Sch9 in yeast.Science. 2001; 292: 288-290Crossref PubMed Scopus (677) Google Scholar), and the vms1Δ mutant showed a significant increase in the oxidation of DHE to ethidium (Figure 2G and Figure S2A). In addition to measuring mitochondrial function and glycerol growth in static culture, we also examined the effect of oxidative stress. As with growth to day 1.5, combination with the sod2Δ mutant caused a significant loss of oxygen consumption and aconitase activity in the vms1Δ mutant (Figures S2B and S2C). Similarly, while the vms1Δ mutant exhibited normal glycerol growth in the absence of additional stressors, combination with a deletion of SOD2 caused a complete loss of growth on glycerol medium (Figure 2H). Combined, these data show that the vms1Δ mutant has severely compromised mitochondrial activity under the conditions that cause Vms1 mitochondrial translocation. To determine whether VMS1 orthologs from other eukaryotes function similarly, we examined vms-1 in C. elegans, which contains a single VMS1 ortholog encoded by K06H7.3. Either of two nonoverlapping RNAi constructs targeting vms-1 caused a marked reduction in viability in response to hydrogen peroxide (Figure 3A ). The surviving vms-1-depleted individuals were dramatically more lethargic than controls. This was confirmed in a mutant line carrying a deletion predicted to remove the majority of the VMS-1 protein (data not shown). Under standard growth conditions, vms-1 mutants and RNAi-treated animals had normal morphology and wild-type growth and development. Activation of numerous stress-response genes, including the mitochondrial superoxide dismutase genes sod-2 and sod-3, is dependent on the function of the insulin-regulated FOXO transcription factor DAF-16 (Murphy et al., 2003Murphy C.T. McCarroll S.A. Bargmann C.I. Fraser A. Kamath R.S. Ahringer J. Li H. Kenyon C. Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans.Nature. 2003; 424: 277-283Crossref PubMed Scopus (1574) Google Scholar, Oh et al., 2006Oh S.W. Mukhopadhyay A. Dixit B.L. Raha T. Green M.R. Tissenbaum H.A. Identification of direct DAF-16 targets controlling longevity, metabolism and diapause by chromatin immunoprecipitation.Nat. Genet. 2006; 38: 251-257Crossref PubMed Scopus (1) Google Scholar). As expected, daf-16 mutant animals were also hypersensitive to hydrogen peroxide treatment (Figure 3A). Treatment of daf-16 mutants with vms-1 RNAi caused further hypersensitivity of similar relative magnitude to that observed in wild-type vms-1 RNAi-treated animals (Figure 3A). Knockdown of vms-1 also caused a significant decrease in life span of wild-type animals and a further decrease in the already shortened life span of daf-16 mutants (Figure 3B). These findings suggest that vms-1 functions in parallel with insulin signaling to regulate stress resistance and life span. To determine the tissue and subcellular expression patterns of C. elegans VMS-1, we generated transgenic lines in which full-length VMS-1 fused to a GFP reporter was expressed from the native vms-1 promoter. This reporter was expressed broadly during embryonic development. In larval stages and in adults, expression was noted in intestinal cells, specific neurons in the head and the tail, and in the ventral nerve cord (Figure 3C and Figure S3). In untreated animals, VMS-1::GFP localized to the cytoplasm in intestinal cells and neurons. In head amphid neurons, VMS-1::GFP was uniformly detected in the dendritic processes, where it was specifically excluded from mitochondria, as determined by lack of colocalization with DIC-1::mCherry (Kass et al., 2001Kass J. Jacob T.C. Kim P. Kaplan J.M. The EGL-3 proprotein convertase regulates mechanosensory responses of Caenorhabditis elegans.J. Neurosci. 2001; 21: 9265-9272PubMed Google Scholar) (Figure 3C). Exposure of animals to hydrogen peroxide, however, caused colocalization of VMS-1::GFP with DIC-1::mCherry (Figure 3C), an identical pattern to that seen for other mitochondrial proteins (Hu and Barr, 2005Hu J. Barr M.M. ATP-2 interacts with the PLAT domain of LOV-1 and is involved in Caenorhabditis elegans polycystin signaling.Mol. Biol. Cell. 2005; 16: 458-469Crossref PubMed Scopus (46) Google Scholar). Together, these findings indicate that, as in yeast, C. elegans vms-1 function is dispensable for viability and growth but is required for protection against oxidative stress and for wild-type life span. While the Vms1 protein expressed in E. coli migrated as a monomer in gel filtration chromatography, endogenous Vms1 from crude yeast lysates migrated in a large >500 kDa complex (data not shown). To identify subunits of the putative Vms1 complex, we purified a functional Vms1-TAP fusion and identified associated proteins by mass spectrometry. Cdc48, a hexameric AAA-ATPase with a well-studied role in protein degradation (Jentsch and Rumpf, 2007Jentsch S. Rumpf S. Cdc48 (p97): a “molecular gearbox” in the ubiquitin pathway?.Trends Biochem. Sci. 2007; 32: 6-11Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar), copurified almost stoichiometrically with Vms1-TAP (Figure 4A ). The Vms1-Cdc48 interaction was confirmed by coimmunoprecipitation of epitope-tagged versions of Vms1 and Cdc48 expressed under their endogenous promoters. Immunoprecipitation of Vms1-HA pulled down Cdc48-myc (Figure 4B). This interaction was also observed following 2 hr treatment with rapamycin (Figure S4A), a condition that causes Vms1 mitochondrial translocation. The S565G mutant of Cdc48 was previously reported to cause increased sensitivity to oxidative stress, reduced respiratory activity, and increased cell death (Braun et al., 2006Braun R.J. Zischka H. Madeo F. Eisenberg T. Wissing S. Buttner S. Engelhardt S.M. Buringer D. Ueffing M. Crucial mitochondrial impairment upon CDC48 mutation in apoptotic yeast.J. Biol. Chem. 2006; 281: 25757-25767Crossref PubMed Scopus (70) Google Scholar, Madeo et al., 1997Madeo F. Frohlich E. Frohlich K.U. A yeast mutant showing diagnostic markers of early and late apoptosis.J. Cell Biol. 1997; 139: 729-734Crossref PubMed Scopus (654) Google Scholar). This mutant was expressed at wild-type levels, but interacted much more weakly with Vms1 than did wild-type Cdc48 (Figure 4B). Thus, Vms1 exists in a stable complex with Cdc48, and this interaction is disrupted by a Cdc48 mutation associated with increased oxidative stress sensitivity and cell death. Cell death in Cdc48-S565G mutant strains has been previously shown by increased annexin V and propidium iodide (PI) staining (Madeo et al., 1997Madeo F. Frohlich E. Frohlich K.U. A yeast mutant showing diagnostic markers of early and late apoptosis.J. Cell Biol. 1997; 139: 729-734Crossref PubMed Scopus (654) Google Scholar). Like Cdc48-S565G mutant strains, the vms1Δ mutant strain had a significantly increased fraction of Annexin V and PI-positive cells (Figures S4B and S4C). Cdc48 is a component of the ubiquitin/proteasome system and promotes protein degradation in a variety of cellular contexts, including membrane fusion, cell-cycle regulation, transcription factor activation, and ERAD (Bays and Hampton, 2002Bays N.W. Hampton R.Y. Cdc48-Ufd1-Npl4: stuck in the middle with Ub.Curr. Biol. 2002; 12: R366-R371Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, Ye, 2006Ye Y. Diverse functions with a common regulator: ubiquitin takes command of an AAA ATPase.J. Struct. Biol. 2006; 156: 29-40Crossref PubMed Scopus (170) Google Scholar). To carry out these diverse functions, Cdc48 interacts with different cofactor proteins that target Cdc48 activity to distinct cellular sites and also mediate ubiquitin and proteasome binding (Jentsch and Rumpf, 2007Jentsch S. Rumpf S. Cdc48 (p97): a “molecular gearbox” in the ubiquitin pathway?.Trends Biochem. Sci. 2007; 32: 6-11Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). We found that Vms1 also associated with the Cdc48 cofactor Npl4 (Figure 4C) and this interaction was maintained upon rapamycin treatment (Figure S4A). In the same experiment, however, we could not detect an interaction between Vms1 and Ufd1, another Cdc48 cofactor that has been implicated, along with Npl4, in the activity of Cdc48 in ERAD (Figure 4C). To confirm the lack of interaction with Vms1, we tested the ability of Ufd1 to interact with Vms1 and Npl4 in parallel in the same experiment. While Ufd1 exhibited a strong interaction with Npl4 as expected, it failed to interact with Vms1 (Figure 4D). We therefore speculate that Vms1 and Ufd1 are mutually exclusive components of the Cdc48-Npl4 complex. This idea is corroborated by high-throughput protein-protein interaction analyses that show both Vms1 and Ufd1 as interacting with Cdc48 and Npl4, but no interaction between Vms1 and Ufd1 (Jensen et al., 2009Jensen L.J. Kuhn M. Stark M. Chaffron S. Creevey C. Muller J. Doerks T. Julien P. Roth A. Simonovic M. et al.STRING 8—a global view on proteins and their functional interactions in 630 organisms.Nucleic Acids Res. 2009; 37: D412-D416Crossref PubMed Scopus (1719) Google Scholar). The Vms1 C-terminal region (Figure S4D) was necessary and sufficient for Cdc48 interaction (Figure 4E). For an unknown reason, deletion of the N terminus actually resulted in increased interaction with Cdc48 relative to wild-type Vms1. The Vms1 interaction with Npl4 exhibited the exact same pattern of domain dependence (Figure 4F). We identified a sequence at the extreme C terminus of Vms1 that showed similarity with the VCP (mammalian Cdc48 ortholog) interaction motif, or VIM, found in the human E3 ubiquitin ligase GP78 and SVIP proteins (Yeung et al., 2008Yeung H.O. Kloppsteck P. Niwa H. Isaacson R.L. Matthews S. Zhang X. Freemont P.S. Insights into adaptor binding to the AAA protein p97.Biochem. Soc. Trans. 2008; 36: 62-67Crossref PubMed Scopus (98) Google Scholar). The putative VIM sequence in yeast Vms1 is highly conserved in Vms1 orthologs (Figure 5A ). A mutant lacking this motif (as indicated in Figure 5A) exhibited a wild-type pattern of localization—cytosolic in normal conditions, with mitochondrial translocation in rapamycin and hydrogen peroxide (Figure S5). This mutant, however, completely failed to interact with either Cdc48 or Npl4 (Figures 5B and 5C), suggesting that Vms1 interacts directly with Cdc48 through a C-terminal VIM sequence and the interaction with Npl4 is mediated by Cdc48. Specific loss of Cdc48 and Npl4 binding in this mutant enabled us to test whether the genetic function of Vms1 requires interaction with Cdc48 and Npl4. We assayed the ability of wild-type and VIM mutant VMS1, expressed from the native promoter, to rescue the rapamycin hypersensitivity of the vms1Δ mutant strain. As shown in Figure 5D, wild-type Vms1 rescued fully, but the VIM mutant Vms1 had no effect on growth despite being expressed at levels equivalent to wild-type Vms1 (see Figure 5C). These data suggest that the function of Vms1 required for rapamycin resistance, which we hypothesize relates to protection of mitochondrial function from oxidative damage (see Figure 2), is completely dependent upon interaction with Cdc48. To identify the protein interactions of mammalian Vms1, we conducted a tandem-affinity purification of a Flag/HA-tagged mouse Vms1 expressed in mouse C2C12 cells. The final elution had essentially only two bands (Figure 5E), Vms1-Flag/HA and VCP, the mammalian ortholog of Cdc48. This interaction was confirmed by coimmunoprecipitation experiments. While wild-type Vms1 precipitated endogenous VCP (Figure 5F, lane 6), this interaction was completely lost in a mutant wherein three highly conserved VIM residues (as indicated in Figure 5A) had been mutated to alanine (Figure 5F, lane 9). This experiment was performed quantitatively to enable determination of the amount of VCP that associates with Vms1. While gr" @default.
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• W2078644413 date "2010-11-01" @default.
• W2078644413 modified "2023-10-13" @default.
• W2078644413 title "A Stress-Responsive System for Mitochondrial Protein Degradation" @default.
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