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• W2770297849 abstract "Modification by the ubiquitin-like protein SUMO affects hundreds of cellular substrate proteins and regulates a wide variety of physiological processes. While the SUMO system appears to predominantly target nuclear proteins and, to a lesser extent, cytosolic proteins, hardly anything is known about the SUMOylation of proteins targeted to membrane-enclosed organelles. Here, we identify a large set of structurally and functionally unrelated mitochondrial proteins as substrates of the SUMO pathway in yeast. We show that SUMO modification of mitochondrial proteins does not rely on mitochondrial targeting and, in fact, is strongly enhanced upon import failure, consistent with the modification occurring in the cytosol. Moreover, SUMOylated forms of mitochondrial proteins particularly accumulate in HSP70- and proteasome-deficient cells, suggesting that SUMOylation participates in cellular protein quality control. We therefore propose that SUMO serves as a mark for nonfunctional mitochondrial proteins, which only sporadically arise in unstressed cells but strongly accumulate upon defective mitochondrial import and impaired proteostasis. Overall, our findings provide support for a role of SUMO in the cytosolic response to aberrant proteins. Modification by the ubiquitin-like protein SUMO affects hundreds of cellular substrate proteins and regulates a wide variety of physiological processes. While the SUMO system appears to predominantly target nuclear proteins and, to a lesser extent, cytosolic proteins, hardly anything is known about the SUMOylation of proteins targeted to membrane-enclosed organelles. Here, we identify a large set of structurally and functionally unrelated mitochondrial proteins as substrates of the SUMO pathway in yeast. We show that SUMO modification of mitochondrial proteins does not rely on mitochondrial targeting and, in fact, is strongly enhanced upon import failure, consistent with the modification occurring in the cytosol. Moreover, SUMOylated forms of mitochondrial proteins particularly accumulate in HSP70- and proteasome-deficient cells, suggesting that SUMOylation participates in cellular protein quality control. We therefore propose that SUMO serves as a mark for nonfunctional mitochondrial proteins, which only sporadically arise in unstressed cells but strongly accumulate upon defective mitochondrial import and impaired proteostasis. Overall, our findings provide support for a role of SUMO in the cytosolic response to aberrant proteins. Posttranslational modification by the small ubiquitin-like modifier (SUMO) 4The abbreviations used are: SUMOsmall ubiquitin-like modifierDRP1dynamin-related protein 1HisSUMOHis-tagged SUMOHSP70heat shock protein 70MTSmatrix-targeting sequenceNi-NTAnickel-nitrilotriacetic acidSCsynthetic completeSILACstable isotope labeling with amino acids in cell cultureYPDyeast extract peptone dextrose. is of fundamental importance for the regulation of a wide variety of physiological processes. Consistent with the large number of cellular SUMO substrates and its crucial roles in cell homeostasis, SUMOylation is essential for viability in most eukaryotes. Moreover, SUMO has been widely implicated in cellular responses to stress, including hypoxic, osmotic, genotoxic, and nutrient stress (1Enserink J.M. Sumo and the cellular stress response.Cell Div. 2015; 10 (26101541): 410.1186/s13008-015-0010-1Crossref PubMed Scopus (109) Google Scholar). In particular, SUMOylation is strongly induced under conditions of proteotoxic stress and targets a diverse array of substrate proteins upon protein misfolding caused by heat shock (2Golebiowski F. Matic I. Tatham M.H. Cole C. Yin Y. Nakamura A. Cox J. Barton G.J. Mann M. Hay R.T. System-wide changes to SUMO modifications in response to heat shock.Sci. Signal. 2009; 2 (19471022): ra2410.1126/scisignal.2000282Crossref PubMed Scopus (385) Google Scholar, 3Seifert A. Schofield P. Barton G.J. Hay R.T. Proteotoxic stress reprograms the chromatin landscape of SUMO modification.Sci. Signal. 2015; 8 (26152697): rs710.1126/scisignal.aaa2213Crossref PubMed Scopus (65) Google Scholar4Niskanen E.A. Malinen M. Sutinen P. Toropainen S. Paakinaho V. Vihervaara A. Joutsen J. Kaikkonen M.U. Sistonen L. Palvimo J.J. Global SUMOylation on active chromatin is an acute heat stress response restricting transcription.Genome Biol. 2015; 16 (26259101): 15310.1186/s13059-015-0717-yCrossref PubMed Scopus (63) Google Scholar) or proteasome inhibition (5Castorálová M. Bezinová D. Svéda M. Lipov J. Ruml T. Knejzlík Z. SUMO-2/3 conjugates accumulating under heat shock or MG132 treatment result largely from new protein synthesis.Biochim. Biophys. Acta. 2012; 1823 (22306003): 911-91910.1016/j.bbamcr.2012.01.010Crossref PubMed Scopus (28) Google Scholar, 6Tatham M.H. Matic I. Mann M. Hay R.T. Comparative proteomic analysis identifies a role for SUMO in protein quality control.Sci. Signal. 2011; 4 (21693764): rs410.1126/scisignal.2001484Crossref PubMed Scopus (140) Google Scholar7Schimmel J. Larsen K.M. Matic I. van Hagen M. Cox J. Mann M. Andersen J.S. Vertegaal A.C. The ubiquitin-proteasome system is a key component of the SUMO-2/3 cycle.Mol. Cell Proteomics. 2008; 7 (18565875): 2107-212210.1074/mcp.M800025-MCP200Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). small ubiquitin-like modifier dynamin-related protein 1 His-tagged SUMO heat shock protein 70 matrix-targeting sequence nickel-nitrilotriacetic acid synthetic complete stable isotope labeling with amino acids in cell culture yeast extract peptone dextrose. Most SUMO substrates identified to date are nuclear proteins (8Hendriks I.A. Vertegaal A.C. A comprehensive compilation of SUMO proteomics.Nat. Rev. Mol. Cell Biol. 2016; 17 (27435506): 581-59510.1038/nrm.2016.81Crossref PubMed Scopus (289) Google Scholar) and also most SUMO conjugating and deconjugating enzymes show a primarily nuclear localization (9Hickey C.M. Wilson N.R. Hochstrasser M. Function and regulation of SUMO proteases.Nat. Rev. Mol. Cell Biol. 2012; 13 (23175280): 755-76610.1038/nrm3478Crossref PubMed Scopus (432) Google Scholar10Johnson E.S. Protein modification by SUMO.Annu. Rev. Biochem. 2004; 73 (15189146): 355-38210.1146/annurev.biochem.73.011303.074118Crossref PubMed Scopus (1384) Google Scholar, 11Seeler J.S. Dejean A. Nuclear and unclear functions of SUMO.Nat. Rev. Mol. Cell Biol. 2003; 4 (14506472): 690-69910.1038/nrm1200Crossref PubMed Scopus (578) Google Scholar12Flotho A. Melchior F. Sumoylation: A regulatory protein modification in health and disease.Annu. Rev. Biochem. 2013; 82 (23746258): 357-38510.1146/annurev-biochem-061909-093311Crossref PubMed Scopus (744) Google Scholar). However, SUMOylation is not restricted to the nucleus and several cytosolic SUMO targets have been identified as well (13Geiss-Friedlander R. Melchior F. Concepts in sumoylation: A decade on.Nat. Rev. Mol. Cell Biol. 2007; 8 (18000527): 947-95610.1038/nrm2293Crossref PubMed Scopus (1359) Google Scholar). Well-studied examples of SUMO substrates outside the nucleus are the septins in budding yeast, which become SUMOylated by the cytosolic pool of the SUMO E3 ligase Siz1 during mitosis (14Makhnevych T. Ptak C. Lusk C.P. Aitchison J.D. Wozniak R.W. The role of karyopherins in the regulated sumoylation of septins.J. Cell Biol. 2007; 177 (17403926): 39-4910.1083/jcb.200608066Crossref PubMed Scopus (59) Google Scholar15Johnson E.S. Blobel G. Cell cycle-regulated attachment of the ubiquitin-related protein SUMO to the yeast septins.J. Cell Biol. 1999; 147 (10579719): 981-99410.1083/jcb.147.5.981Crossref PubMed Scopus (327) Google Scholar, 16Johnson E.S. Gupta A.A. An E3-like factor that promotes SUMO conjugation to the yeast septins.Cell. 2001; 106 (11572779): 735-74410.1016/S0092-8674(01)00491-3Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar, 17Takahashi Y. Iwase M. Konishi M. Tanaka M. Toh-e A. Kikuchi Y. Smt3, a SUMO-1 homolog, is conjugated to Cdc3, a component of septin rings at the mother-bud neck in budding yeast.Biochem. Biophys. Res. Commun. 1999; 259 (10364461): 582-58710.1006/bbrc.1999.0821Crossref PubMed Scopus (85) Google Scholar, 18Takahashi Y. Kahyo T. Toh-e A. Yasuda H. Kikuchi Y. Yeast Ull1/Siz1 is a novel SUMO1/Smt3 ligase for septin components and functions as an adaptor between conjugating enzyme and substrates.J. Biol. Chem. 2001; 276 (11577116): 48973-4897710.1074/jbc.M109295200Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar19Takahashi Y. Toh-e A. Kikuchi Y. A novel factor required for the SUMO1/Smt3 conjugation of yeast septins.Gene. 2001; 275 (11587849): 223-23110.1016/S0378-1119(01)00662-XCrossref PubMed Scopus (107) Google Scholar) and deSUMOylated by the SUMO-specific isopeptidase Ulp1 during cytokinesis (14Makhnevych T. Ptak C. Lusk C.P. Aitchison J.D. Wozniak R.W. The role of karyopherins in the regulated sumoylation of septins.J. Cell Biol. 2007; 177 (17403926): 39-4910.1083/jcb.200608066Crossref PubMed Scopus (59) Google Scholar, 15Johnson E.S. Blobel G. Cell cycle-regulated attachment of the ubiquitin-related protein SUMO to the yeast septins.J. Cell Biol. 1999; 147 (10579719): 981-99410.1083/jcb.147.5.981Crossref PubMed Scopus (327) Google Scholar, 20Takahashi Y. Mizoi J. Toh-e A. Kikuchi Y. Yeast Ulp1, an Smt3-specific protease, associates with nucleoporins.J. Biochem. 2000; 128 (11056382): 723-72510.1093/oxfordjournals.jbchem.a022807Crossref PubMed Scopus (70) Google Scholar). Interestingly, SUMO substrates in the cytosol also include proteins that are located at the interfaces of the plasma membrane and cellular organelles such as the nucleus, the endoplasmic reticulum, and mitochondria (13Geiss-Friedlander R. Melchior F. Concepts in sumoylation: A decade on.Nat. Rev. Mol. Cell Biol. 2007; 8 (18000527): 947-95610.1038/nrm2293Crossref PubMed Scopus (1359) Google Scholar). This group of substrates includes the GTPase DRP1 in mammals, which currently is the only well-characterized SUMO substrate that localizes to mitochondria. DRP1 associates with the cytosolic side of the outer mitochondrial membrane. SUMOylation of DRP1 was found to be mediated by the mitochondrial anchored SUMO E3 ligase MAPL (21Braschi E. Zunino R. McBride H.M. MAPL is a new mitochondrial SUMO E3 ligase that regulates mitochondrial fission.EMBO Rep. 2009; 10 (19407830): 748-75410.1038/embor.2009.86Crossref PubMed Scopus (259) Google Scholar) and to promote mitochondrial fission under normal growth conditions (22Harder Z. Zunino R. McBride H. Sumo1 conjugates mitochondrial substrates and participates in mitochondrial fission.Curr. Biol. 2004; 14 (14972687): 340-34510.1016/j.cub.2004.02.004Abstract Full Text Full Text PDF PubMed Google Scholar) as well as during apoptosis (23Wasiak S. Zunino R. McBride H.M. Bax/Bak promote sumoylation of DRP1 and its stable association with mitochondria during apoptotic cell death.J. Cell Biol. 2007; 177 (17470634): 439-45010.1083/jcb.200610042Crossref PubMed Scopus (427) Google Scholar). Ubiquitin has also been identified as regulator of mitochondrial homeostasis and has been linked to mitochondrial protein quality control (24Escobar-Henriques M. Langer T. Dynamic survey of mitochondria by ubiquitin.EMBO Rep. 2014; 15 (24569520): 231-24310.1002/embr.201338225Crossref PubMed Scopus (45) Google Scholar, 25Bragoszewski P. Turek M. Chacinska A. Control of mitochondrial biogenesis and function by the ubiquitin-proteasome system.Open Biol. 2017; 7 (28446709)17000710.1098/rsob.170007Crossref PubMed Scopus (119) Google Scholar). Notably, the ubiquitin-proteasome system was shown to mediate the degradation of nonimported mitochondrial proteins under physiological conditions (26Bragoszewski P. Gornicka A. Sztolsztener M.E. Chacinska A. The ubiquitin-proteasome system regulates mitochondrial intermembrane space proteins.Mol. Cell Biol. 2013; 33 (23508107): 2136-214810.1128/MCB.01579-12Crossref PubMed Scopus (100) Google Scholar) and acutely upon import failure (26Bragoszewski P. Gornicka A. Sztolsztener M.E. Chacinska A. The ubiquitin-proteasome system regulates mitochondrial intermembrane space proteins.Mol. Cell Biol. 2013; 33 (23508107): 2136-214810.1128/MCB.01579-12Crossref PubMed Scopus (100) Google Scholar27Habelhah H. Laine A. Erdjument-Bromage H. Tempst P. Gershwin M.E. Bowtell D.D. Ronai Z. Regulation of 2-oxoglutarate (α-ketoglutarate) dehydrogenase stability by the RING finger ubiquitin ligase Siah.J. Biol. Chem. 2004; 279 (15466852): 53782-5378810.1074/jbc.M410315200Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 28Radke S. Chander H. Schäfer P. Meiss G. Krüger R. Schulz J.B. Germain D. Mitochondrial protein quality control by the proteasome involves ubiquitination and the protease Omi.J. Biol. Chem. 2008; 283 (18362145): 12681-1268510.1074/jbc.C800036200Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 29Pearce D.A. Sherman F. Differential ubiquitin-dependent degradation of the yeast apo-cytochrome c isozymes.J. Biol. Chem. 1997; 272 (9395529): 31829-3183610.1074/jbc.272.50.31829Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar30Itakura E. Zavodszky E. Shao S. Wohlever M.L. Keenan R.J. Hegde R.S. Ubiquitins chaperone and triage mitochondrial membrane proteins for degradation.Mol. Cell. 2016; 63 (27345149): 21-3310.1016/j.molcel.2016.05.020Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). In this scenario, ubiquitin is conjugated to proteins that normally localize to and function within the inner mitochondrial subcompartments (26Bragoszewski P. Gornicka A. Sztolsztener M.E. Chacinska A. The ubiquitin-proteasome system regulates mitochondrial intermembrane space proteins.Mol. Cell Biol. 2013; 33 (23508107): 2136-214810.1128/MCB.01579-12Crossref PubMed Scopus (100) Google Scholar, 27Habelhah H. Laine A. Erdjument-Bromage H. Tempst P. Gershwin M.E. Bowtell D.D. Ronai Z. Regulation of 2-oxoglutarate (α-ketoglutarate) dehydrogenase stability by the RING finger ubiquitin ligase Siah.J. Biol. Chem. 2004; 279 (15466852): 53782-5378810.1074/jbc.M410315200Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). By contrast, SUMO modification of proteins from inner mitochondrial subcompartments has not been analyzed to date, even though previous large-scale “SUMOylome” studies have suggested a small number of putative SUMO substrates from these compartments (31Denison C. Rudner A.D. Gerber S.A. Bakalarski C.E. Moazed D. Gygi S.P. A proteomic strategy for gaining insights into protein sumoylation in yeast.Mol. Cell Proteomics. 2005; 4 (15542864): 246-25410.1074/mcp.M400154-MCP200Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar32Wohlschlegel J.A. Johnson E.S. Reed S.I. Yates 3rd., J.R. Global analysis of protein sumoylation in Saccharomyces cerevisiae.J. Biol. Chem. 2004; 279 (15326169): 45662-4566810.1074/jbc.M409203200Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 33Panse V.G. Hardeland U. Werner T. Kuster B. Hurt E. A proteome-wide approach identifies sumoylated substrate proteins in yeast.J. Biol. Chem. 2004; 279 (15292183): 41346-4135110.1074/jbc.M407950200Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 34Zhou W. Ryan J.J. Zhou H. Global analyses of sumoylated proteins in Saccharomyces cerevisiae. Induction of protein sumoylation by cellular stresses.J. Biol. Chem. 2004; 279 (15166219): 32262-3226810.1074/jbc.M404173200Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 35Hannich J.T. Lewis A. Kroetz M.B. Li S.J. Heide H. Emili A. Hochstrasser M. Defining the SUMO-modified proteome by multiple approaches in Saccharomyces cerevisiae.J. Biol. Chem. 2005; 280 (15590687): 4102-411010.1074/jbc.M413209200Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar36Wykoff D.D. O'Shea E.K. Identification of sumoylated proteins by systematic immunoprecipitation of the budding yeast proteome.Mol. Cell Proteomics. 2005; 4 (15596868): 73-8310.1074/mcp.M400166-MCP200Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Starting from a mass spectrometry (MS) approach, we provide here evidence that a substantial fraction of the mitochondrial proteome is targeted by the SUMO modification system. We corroborate our MS data by individually confirming the SUMO modification of several mitochondrial matrix proteins in vivo. In agreement with the presence of SUMO enzymes in the cytosol but not in the mitochondrial matrix, we find that the SUMOylation of mitochondrial proteins does not rely on mitochondrial import. By contrast, our data rather indicate that SUMOylation of mitochondrial proteins is strongly enhanced upon import failure. Moreover, whereas SUMO modification of these substrates occurs only sporadically in unstressed cells, it is particularly induced when canonical components of the proteostasis network, such as the HSP70 system or the proteasome, are defective. Finally, we propose a model in which the SUMO modification pathway targets nonfunctional mitochondrial proteins as an element of cellular protein quality control. Following our long-standing interest in the SUMO system, we screened for novel SUMO substrates using a strategy that involves the purification of His-tagged SUMO (HisSUMO) conjugates from yeast cells and analysis of the enriched proteins using quantitative mass spectrometry (37Psakhye I. Jentsch S. Identification of substrates of protein-group SUMOylation.Methods Mol. Biol. 2016; 1475 (27631809): 219-23110.1007/978-1-4939-6358-4_16Crossref PubMed Scopus (12) Google Scholar, 38Psakhye I. Jentsch S. Protein group modification and synergy in the SUMO pathway as exemplified in DNA repair.Cell. 2012; 151 (23122649): 807-82010.1016/j.cell.2012.10.021Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). Previous large-scale studies in budding yeast had suggested a small number of mitochondrial proteins as potential SUMO substrates (31Denison C. Rudner A.D. Gerber S.A. Bakalarski C.E. Moazed D. Gygi S.P. A proteomic strategy for gaining insights into protein sumoylation in yeast.Mol. Cell Proteomics. 2005; 4 (15542864): 246-25410.1074/mcp.M400154-MCP200Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar32Wohlschlegel J.A. Johnson E.S. Reed S.I. Yates 3rd., J.R. Global analysis of protein sumoylation in Saccharomyces cerevisiae.J. Biol. Chem. 2004; 279 (15326169): 45662-4566810.1074/jbc.M409203200Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 33Panse V.G. Hardeland U. Werner T. Kuster B. Hurt E. A proteome-wide approach identifies sumoylated substrate proteins in yeast.J. Biol. Chem. 2004; 279 (15292183): 41346-4135110.1074/jbc.M407950200Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 34Zhou W. Ryan J.J. Zhou H. Global analyses of sumoylated proteins in Saccharomyces cerevisiae. Induction of protein sumoylation by cellular stresses.J. Biol. Chem. 2004; 279 (15166219): 32262-3226810.1074/jbc.M404173200Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 35Hannich J.T. Lewis A. Kroetz M.B. Li S.J. Heide H. Emili A. Hochstrasser M. Defining the SUMO-modified proteome by multiple approaches in Saccharomyces cerevisiae.J. Biol. Chem. 2005; 280 (15590687): 4102-411010.1074/jbc.M413209200Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar36Wykoff D.D. O'Shea E.K. Identification of sumoylated proteins by systematic immunoprecipitation of the budding yeast proteome.Mol. Cell Proteomics. 2005; 4 (15596868): 73-8310.1074/mcp.M400166-MCP200Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Notably, we consistently identified peptides of several different mitochondrial proteins in our HisSUMO purifications. By compiling the results of multiple MS experiments, our approach revealed a set of 89 inner mitochondrial proteins as potential SUMO substrates (Table S1). For 61 of these proteins we also identified a total of 81 SUMOylation sites, further suggesting that these proteins are modified in vivo (Table S1). Among the 89 potential mitochondrial SUMO substrates, we found components of all inner mitochondrial subcompartments (intermembrane space, inner membrane, and matrix) (Fig. 1A). A comparison with the localization of known mitochondrial proteins listed in the Yeast Deletion and Proteomics of Mitochondria (YDPM) database (39Prokisch H. Scharfe C. Camp 2nd, D.G. Xiao W. David L. Andreoli C. Monroe M.E. Moore R.J. Gritsenko M.A. Kozany C. Hixson K.K. Mottaz H.M. Zischka H. Ueffing M. Herman Z.S. Davis R.W. Meitinger T. Oefner P.J. Smith R.D. Steinmetz L.M. Integrative analysis of the mitochondrial proteome in yeast.PLoS Biol. 2004; 2 (15208715): e16010.1371/journal.pbio.0020160Crossref PubMed Scopus (165) Google Scholar) suggested that the number of SUMO substrates from each subcompartment largely scaled with the overall number of proteins in each subcompartment. Seemingly, therefore, submitochondrial localization is not a determinant for SUMO modification. Importantly, only a small fraction of substrates (six proteins) were annotated as having a dual localization (mitochondrial and cytosolic). We therefore conclude that a large number of proteins that function in mitochondria are modified by SUMO in vivo. To ascertain the MS results, we analyzed the SUMOylation of several structurally and functionally unrelated mitochondrial proteins individually. Using denaturing Ni-NTA pulldowns and subsequent Western blot analysis we were able to confirm that these mitochondrial matrix proteins are indeed modified by SUMO. These confirmed SUMO substrates include Ilv6 (Fig. 1B), a protein involved in branched-chain amino acid biosynthesis (40Cullin C. Baudin-Baillieu A. Guillemet E. Ozier-Kalogeropoulos O. Functional analysis of YCL09C: Evidence for a role as the regulatory subunit of acetolactate synthase.Yeast. 1996; 12 (8972574): 1511-151810.1002/(SICI)1097-0061(199612)12:15%3C1511::AID-YEA41%3E3.0.CO%3B2-BCrossref PubMed Scopus (29) Google Scholar, 41Pang S.S. Duggleby R.G. Expression, purification, characterization, and reconstitution of the large and small subunits of yeast acetohydroxyacid synthase.Biochemistry. 1999; 38 (10213630): 5222-523110.1021/bi983013mCrossref PubMed Scopus (88) Google Scholar), Adh3 (Fig. 1C), a mitochondrial alcohol dehydrogenase isoform (42Sugar J. Schimpfessel L. Rozen E. Crokaert R. The mitochondrial alcohol dehydrogenase of the yeast “Saccharomyces cerevisiae.”.Arch. Int. Physiol. Biochim. 1970; 78 (4101911): 1009-1010PubMed Google Scholar, 43Lutstorf U. Megnet R. Multiple forms of alcohol dehydrogenase in Saccharomyces cerevisiae. I. Physiological control of ADH-2 and properties of ADH-2 and ADH-4.Arch. Biochem. Biophys. 1968; 126 (5686604): 933-94410.1016/0003-9861(68)90487-6Crossref PubMed Scopus (175) Google Scholar), and Mrpl23 (Fig. 1D), a mitochondrial ribosomal protein (44Kitakawa M. Graack H.R. Grohmann L. Goldschmidt-Reisin S. Herfurth E. Wittmann-Liebold B. Nishimura T. Isono K. Identification and characterization of the genes for mitochondrial ribosomal proteins of Saccharomyces cerevisiae.Eur. J. Biochem. 1997; 245 (9151978): 449-45610.1111/j.1432-1033.1997.t01-2-00449.xCrossref PubMed Scopus (35) Google Scholar). We next aimed to identify the SUMO acceptor sites on mitochondrial SUMO substrates. To this end, we systematically replaced individual lysine residues on Ilv6, Adh3, and Mrpl23 to arginine. For Ilv6, this identified lysine 260 as major SUMO acceptor site (Fig. S1, A and B), but additional removal of three adjacent lysine residues (Lys-218, Lys-284, and Lys-296) in a stepwise manner further reduced SUMOylation and a mutant variant lacking all four lysine residues (Ilv63HA-K218R, K260R, K284R, K296R termed Ilv63HA-4KR) did not show any detectable SUMOylation (Fig. S1, A and B). For Adh3, we found lysine 305 as major SUMO acceptor site (Fig. S1, C and D) and for Mrpl23, replacement of the two most C-terminal lysine residues by arginine reduced the levels of SUMO conjugates to less than 50% (Fig. S1, E and F). We next asked whether SUMOylation of mitochondrial proteins requires specific SUMO E3 ligases. Analysis of Ilv6 SUMOylation in cells lacking the known SUMO E3 ligases Siz1 (siz1Δ) or Siz2 (siz2Δ) indicated that the SUMO modification of Ilv6 is catalyzed by Siz1 and to a minor extent by Siz2 (Fig. 2A). Accordingly, Ilv6 SUMOylation was undetectable by Western blotting in samples from cells lacking both Siz1 and Siz2 (siz1Δ siz2Δ) (Fig. 2A). Moreover, we found strikingly similar roles for Siz1 and Siz2 in the SUMO modification of Adh3 (Fig. 2B) and Mrpl23 (Fig. 2C). Thus, all tested SUMO substrates require the same SUMO E3 ligases of the conserved Siz/PIAS (protein inhibitor of activated STAT) family for modification. The vast majority of mitochondrial proteins are synthesized on cytosolic ribosomes and subsequently imported into mitochondria (45Neupert W. Herrmann J.M. Translocation of proteins into mitochondria.Annu. Rev. Biochem. 2007; 76 (17263664): 723-74910.1146/annurev.biochem.76.052705.163409Crossref PubMed Scopus (1101) Google Scholar, 46Chacinska A. Koehler C.M. Milenkovic D. Lithgow T. Pfanner N. Importing mitochondrial proteins: Machineries and mechanisms.Cell. 2009; 138 (19703392): 628-64410.1016/j.cell.2009.08.005Abstract Full Text Full Text PDF PubMed Scopus (987) Google Scholar). We therefore asked whether SUMOylation of mitochondrial proteins is linked to the import process or requires the presence of a mitochondrial targeting signal. Classical mitochondrial targeting signals are N-terminal presequences, which in most cases are proteolytically removed from mitochondrial precursor proteins during import. Presequences frequently target proteins into the mitochondrial matrix and therefore are also referred to as matrix-targeting sequences (MTS) (45Neupert W. Herrmann J.M. Translocation of proteins into mitochondria.Annu. Rev. Biochem. 2007; 76 (17263664): 723-74910.1146/annurev.biochem.76.052705.163409Crossref PubMed Scopus (1101) Google Scholar). Accordingly, we generated an Ilv6 mutant variant (MTSΔ-Ilv63HA), which lacks the N-terminal MTS (amino acids 1–24) required for mitochondrial import (Fig. 3A). Microscopic analysis of corresponding GFP fusion proteins demonstrated that removal of the 24 N-terminal amino acids of Ilv6 is indeed sufficient to prevent mitochondrial import, thereby causing a presumably cytosolic localization of the mutant protein variant (Fig. S2, A and B). Importantly, this mutant was efficiently SUMOylated (Fig. 3B) and the modification was again dependent on the SUMO E3 ligases Siz1 and Siz2 (Fig. S2C) and occurred at a similar set of SUMO acceptor sites as for wild-type Ilv6 (Fig. S2D). Therefore, SUMO modification of Ilv6 does not rely on mitochondrial import and does not require the presence of an MTS. In fact, MTS deletion even strongly enhanced the SUMOylation of Ilv6 (Fig. 3B), indicating that SUMO modification is induced by import failure. We also analyzed an import-incompetent variant of Adh3 (MTSΔ-Adh33HA) (Fig. 3C). Again, SUMO modification of the import-incompetent variant was enhanced compared with the wild-type protein (Fig. 3D). Furthermore, SUMOylation of import-incompetent Adh3 also required the SUMO E3 ligases Siz1 and Siz2 (Fig. S2E) and the predominantly targeted lysine 305 (Fig. S2F), similar to wild-type Adh3. We therefore conclude that import-incompetent mutant variants of mitochondrial proteins are SUMOylated with requirements as wild-type substrates, but that deletion of targeting sequences strongly enhances the modification. This may suggest that SUMOylation of wild-type proteins specifically occurs upon sporadic mistargeting in unstressed cells and under conditions where mitochondrial protein import is impaired. Indeed, we observed an accumulation of SUMOylated Ilv6 precursors (p) in strains defective in mitochondrial import (Fig. 3E), which harbor a temperature-sensitive mutant variant of mitochondrial HSP70 (ssc1-3) (47Gambill B.D. Voos W. Kang P.J. Miao B. Langer T. Craig E.A. Pfanner N. A dual role for mitochondrial heat shock protein 70 in membrane translocation of preproteins.J. Cell Biol. 1993; 123 (8408191): 109-11710.1083/jcb.123.1.109Crossref PubMed Scopus (221) Google Scholar). Furthermore, we specifically observed the accumulation of SUMOylated Ilv6 precursors in the ssc1-3 mutant, whereas in wild-type cells Ilv6-SUMO conjugates appeared to be proteolytically processed (Fig. 3E; note the shift of the precursor (p) compared with the mature form (m)). This indicates that the major pool of Ilv6-SUMO conjugates in unstressed wild-type cells possesses a proteolytically processed N terminus. It is therefore conceivable that these protein species have at some point initiated mitochondrial import, but that they become modified by SUMO in the cytosol. Based on our observation that the SUMOylation of mitochondrial proteins can occur in the cytosol, we speculated that the modification might be affected by factors which bind nonimported mitochondrial precursor proteins. Several factors are involved in posttranslational protein import into mitochondria (48Mihara K. Omura T. Cytosolic factors in mitochondrial protein import.Experientia. 1996; 52 (8988247): 1063-106810.1007/BF01952103Crossref PubMed Scopus (28) Google Scholar, 49Mori M. Terada K. Mitochondrial protein import in animals.Biochim. Biophys. Acta. 1998; 1403 (9622585): 12-2710.1016/S0167-4889(98)00021-4Crossref PubMed Scopus (50) Google Scholar50Mihara K. Omura T. Cytoplasmic chaperones in precursor targeting to mitochondria: The role of MSF and hsp 70.Trends Cell Biol. 1996; 6 (15157486): 104-10810.1016/0962-8924(96)81000-2Abstract Full Text PDF PubMed Scopus (100) Google Scholar), of which the SSA family HSP70 proteins (Ssa1–4) are of particular importance in budding yeast (51Deshaies R.J. Koch B.D. Werner-Washbu" @default.
• W2770297849 created "2017-12-04" @default.
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• W2770297849 date "2018-01-01" @default.
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• W2770297849 title "Failed mitochondrial import and impaired proteostasis trigger SUMOylation of mitochondrial proteins" @default.
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• W2770297849 doi "https://doi.org/10.1074/jbc.m117.817833" @default.
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