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• W3082078584 abstract "Studies in the yeast Saccharomyces cerevisiae have helped define mechanisms underlying the activity of the ubiquitin–proteasome system (UPS), uncover the proteasome assembly pathway, and link the UPS to the maintenance of cellular homeostasis. However, the spectrum of UPS substrates is incompletely defined, even though multiple techniques—including MS—have been used. Therefore, we developed a substrate trapping proteomics workflow to identify previously unknown UPS substrates. We first generated a yeast strain with an epitope tagged proteasome subunit to which a proteasome inhibitor could be applied. Parallel experiments utilized inhibitor insensitive strains or strains lacking the tagged subunit. After affinity isolation, enriched proteins were resolved, in-gel digested, and analyzed by high resolution liquid chromatography-tandem MS. A total of 149 proteasome partners were identified, including all 33 proteasome subunits. When we next compared data between inhibitor sensitive and resistant cells, 27 proteasome partners were significantly enriched. Among these proteins were known UPS substrates and proteins that escort ubiquitinated substrates to the proteasome. We also detected Erg25 as a high-confidence partner. Erg25 is a methyl oxidase that converts dimethylzymosterol to zymosterol, a precursor of the plasma membrane sterol, ergosterol. Because Erg25 is a resident of the endoplasmic reticulum (ER) and had not previously been directly characterized as a UPS substrate, we asked whether Erg25 is a target of the ER associated degradation (ERAD) pathway, which most commonly mediates proteasome-dependent destruction of aberrant proteins. As anticipated, Erg25 was ubiquitinated and associated with stalled proteasomes. Further, Erg25 degradation depended on ERAD-associated ubiquitin ligases and was regulated by sterol synthesis. These data expand the cohort of lipid biosynthetic enzymes targeted for ERAD, highlight the role of the UPS in maintaining ER function, and provide a novel tool to uncover other UPS substrates via manipulations of our engineered strain. Studies in the yeast Saccharomyces cerevisiae have helped define mechanisms underlying the activity of the ubiquitin–proteasome system (UPS), uncover the proteasome assembly pathway, and link the UPS to the maintenance of cellular homeostasis. However, the spectrum of UPS substrates is incompletely defined, even though multiple techniques—including MS—have been used. Therefore, we developed a substrate trapping proteomics workflow to identify previously unknown UPS substrates. We first generated a yeast strain with an epitope tagged proteasome subunit to which a proteasome inhibitor could be applied. Parallel experiments utilized inhibitor insensitive strains or strains lacking the tagged subunit. After affinity isolation, enriched proteins were resolved, in-gel digested, and analyzed by high resolution liquid chromatography-tandem MS. A total of 149 proteasome partners were identified, including all 33 proteasome subunits. When we next compared data between inhibitor sensitive and resistant cells, 27 proteasome partners were significantly enriched. Among these proteins were known UPS substrates and proteins that escort ubiquitinated substrates to the proteasome. We also detected Erg25 as a high-confidence partner. Erg25 is a methyl oxidase that converts dimethylzymosterol to zymosterol, a precursor of the plasma membrane sterol, ergosterol. Because Erg25 is a resident of the endoplasmic reticulum (ER) and had not previously been directly characterized as a UPS substrate, we asked whether Erg25 is a target of the ER associated degradation (ERAD) pathway, which most commonly mediates proteasome-dependent destruction of aberrant proteins. As anticipated, Erg25 was ubiquitinated and associated with stalled proteasomes. Further, Erg25 degradation depended on ERAD-associated ubiquitin ligases and was regulated by sterol synthesis. These data expand the cohort of lipid biosynthetic enzymes targeted for ERAD, highlight the role of the UPS in maintaining ER function, and provide a novel tool to uncover other UPS substrates via manipulations of our engineered strain. The 26S proteasome, a multi-catalytic cytosolic protease, serves as the major proteolytic factory in eukaryotes. The 26S particle is formed by two species, a 20S core particle, which houses pairs of trypsin, chymotrypsin, and caspase-like enzymes, and two 19S “caps” (or PA700 particles) that abut each end of the core particle (1Bard J.A.M. Goodall E.A. Greene E.R. Jonsson E. Dong K.C. Martin A. Structure and Function of the 26S Proteasome.Annu. Rev. Biochem. 2018; 87: 697-724Crossref PubMed Scopus (243) Google Scholar, 2Voges D. Zwickl P. Baumeister W. The 26S proteasome: a molecular machine designed for controlled proteolysis.Annu. Rev. Biochem. 1999; 68: 1015-1068Crossref PubMed Scopus (1561) Google Scholar). Based on its abundance and robust activity, the targeting and destruction of substrates to this protease are tightly controlled because the majority of proteasome substrates are covalently modified by an isopeptide ubiquitin (Ub) linkage. Ub is added onto a target via a cascade of Ub activating, conjugating, and ligating enzymes, and covalent Ub appendages are formed between the C terminus of Ub and most commonly the ε amino group on a Lys in a protein substrate or on a previously attached Ub (3Varshavsky A. Discovery of cellular regulation by protein degradation.J. Biol. Chem. 2008; 283: 34469-34489Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Although numerous varieties of poly-Ub “chains” have been described—due to the presence of seven internal Lys residues in Ub as well as the N terminus—the most common poly-Ub linkage recognized by the proteasome is formed by the sequential addition of the C terminus of Ub onto a Lys in Ub at position 48 (4Yau R. Rape M. The increasing complexity of the ubiquitin code.Nat. Cell Biol. 2016; 18: 579-586Crossref PubMed Scopus (476) Google Scholar, 5Stolz A. Dikic I. Heterotypic Ubiquitin Chains: Seeing is Believing.Trends Cell Biol. 2018; 28: 1-3Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 6Kulathu Y. Komander D. Atypical ubiquitylation - the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages.Nat. Rev. Mol. Cell Biol. 2012; 13: 508-523Crossref PubMed Scopus (450) Google Scholar). Early results indicated that a poly-Ub chain of at least four Ub substituents is required for proteasome recognition (7Thrower J.S. Hoffman L. Rechsteiner M. Pickart C.M. Recognition of the polyubiquitin proteolytic signal.EMBO J. 2000; 19: 94-102Crossref PubMed Scopus (1270) Google Scholar). As a result of fine-tuned mechanisms underlying the recognition and Ub-tagging of substrates, the Ub-proteasome system (UPS) plays a critical role in myriad cellular processes, which include the cell cycle, gene expression, immune system function, signal transduction, and cell fate (8Finley D. Ulrich H.D. Sommer T. Kaiser P. The ubiquitin-proteasome system of Saccharomyces cerevisiae.Genetics. 2012; 192: 319-360Crossref PubMed Scopus (244) Google Scholar). The UPS also plays a critical role in protein “quality control,” which mediates the destruction of damaged, mutated, unassembled, and improperly folded proteins in the cell. Perhaps not surprisingly, the UPS is linked to an ever-growing number of human diseases. One prominent protein quality control pathway is known as endoplasmic reticulum (ER) associated degradation, or ERAD. Approximately one-third of all proteins in eukaryotes are synthesized at the ER, including all membrane and the majority of secreted proteins (9Ghaemmaghami S. Huh W.K. Bower K. Howson R.W. Belle A. Dephoure N. O'Shea E.K. Weissman J.S. Global analysis of protein expression in yeast.Nature. 2003; 425: 737-741Crossref PubMed Scopus (2917) Google Scholar). The maintenance of ER homeostasis requires the high fidelity folding, post-translational modification, and assembly of proteins in the ER (10Braakman I. Bulleid N.J. Protein folding and modification in the mammalian endoplasmic reticulum.Annu. Rev. Biochem. 2011; 80: 71-99Crossref PubMed Scopus (420) Google Scholar, 11Brodsky J.L. Skach W.R. Protein folding and quality control in the endoplasmic reticulum: Recent lessons from yeast and mammalian cell systems.Curr. Opin. Cell Biol. 2011; 23: 464-475Crossref PubMed Scopus (164) Google Scholar, 12Smith M.H. Ploegh H.L. Weissman J.S. Road to ruin: targeting proteins for degradation in the endoplasmic reticulum.Science. 2011; 334: 1086-1090Crossref PubMed Scopus (441) Google Scholar, 13Berner N. Reutter K.R. Wolf D.H. Protein quality control of the endoplasmic reticulum and ubiquitin-proteasome-triggered degradation of aberrant proteins: yeast pioneers the path.Annu. Rev. Biochem. 2018; 87: 751-782Crossref PubMed Scopus (63) Google Scholar). During ERAD, proteins are first selected by molecular chaperones and chaperone-like proteins that reside in or on the cytosolic face of the ER. After recognition, the proteins are polyubiquitinated. However, because the Ub machinery is absent from the ER lumen, many ERAD substrates—such as soluble proteins that reside completely within the ER—must be extracted or “retrotranslocated” prior to Ub tagging (14Zattas D. Hochstrasser M. Ubiquitin-dependent protein degradation at the yeast endoplasmic reticulum and nuclear envelope.Crit. Rev. Biochem. Mol. Biol. 2015; 50: 1-17Crossref PubMed Scopus (54) Google Scholar, 15Christianson J.C. Ye Y. Cleaning up in the endoplasmic reticulum: ubiquitin in charge.Nat. Struct. Mol. Biol. 2014; 21: 325-335Crossref PubMed Scopus (241) Google Scholar). In contrast, integral membrane proteins in the ER contain domains facing the cytosol that can be polyubiquitinated before retrotranslocation and proteasome-mediated destruction. Based on its central role in maintaining ER and secretory protein homeostasis, the ERAD pathway has also been linked to numerous human diseases (16Needham P.G. Guerriero C.J. Brodsky J.L. Chaperoning endoplasmic reticulum-associated degradation (ERAD) and protein conformational diseases.Cold Spring Harb. Perspect. Biol. 2019; 11: a033928Crossref PubMed Scopus (42) Google Scholar). Initially, ERAD substrates were identified by the analysis of mutated secreted proteins, primarily in yeast, along with disease-causing proteins in human cells (17McCracken A.A. Brodsky J.L. Assembly of ER-associated protein degradation in vitro: dependence on cytosol, calnexin, and ATP.J. Cell Biol. 1996; 132: 291-298Crossref PubMed Scopus (339) Google Scholar, 18Hiller M.M. Finger A. Schweiger M. Wolf D.H. ER degradation of a misfolded luminal protein by the cytosolic ubiquitin-proteasome pathway.Science. 1996; 273: 1725-1728Crossref PubMed Scopus (604) Google Scholar, 19Hampton R.Y. Gardner R.G. Rine J. Role of 26S proteasome and HRD genes in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein.Mol. Biol. Cell. 1996; 7: 2029-2044Crossref PubMed Scopus (448) Google Scholar, 20Ward C.L. Omura S. Kopito R.R. Degradation of CFTR by the ubiquitin-proteasome pathway.Cell. 1995; 83: 121-127Abstract Full Text PDF PubMed Scopus (1107) Google Scholar, 21Jensen T.J. Loo M.A. Pind S. Williams D.B. Goldberg A.L. Riordan J.R. Multiple proteolytic systems, including the proteasome, contribute to CFTR processing.Cell. 1995; 83: 129-135Abstract Full Text PDF PubMed Scopus (757) Google Scholar). Select ERAD substrates were also identified via MS. For example, a transporter assembly factor, CD147, was uncovered as an ERAD substrate in human cells by isolating partners of an ER lectin in cells lacking a downstream component (22Tyler R.E. Pearce M.M. Shaler T.A. Olzmann J.A. Greenblatt E.J. Kopito R.R. Unassembled CD147 is an endogenous endoplasmic reticulum-associated degradation substrate.Mol. Biol. Cell. 2012; 23: 4668-4678Crossref PubMed Scopus (57) Google Scholar). In another example, Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) of yeast containing or lacking components of the ER-associated ubiquitination machinery led to the isolation of Erg1, an enzyme in the ergosterol pathway, as a regulated ERAD substrate (23Foresti O. Ruggiano A. Hannibal-Bach H.K. Ejsing C.S. Carvalho P. Sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase Doa10/Teb4.Elife. 2013; 2: e00953Crossref PubMed Scopus (115) Google Scholar). A homolog of Erg1 that acts in the sterol biosynthetic pathway in higher cells was targeted by homologous components of the ubiquitination machinery in human cells, validating the power of the yeast-based screen. Moreover, the capture of ubiquitinated proteins after treating human cells with a retrotranslocation inhibitor and SILAC analysis identified other ERAD substrates (24Huang E.Y. To M. Tran E. Dionisio L.T.A. Cho H.J. Baney K.L.M. Pataki C.I. Olzmann J.A. A VCP inhibitor substrate trapping approach (VISTA) enables proteomic profiling of endogenous ERAD substrates.Mol. Biol. Cell. 2018; 29: 1021-1030Crossref PubMed Scopus (17) Google Scholar). Many of these more recently identified substrates are enzymes whose steady-state levels are controlled by ERAD(25Stevenson J. Huang E.Y. Olzmann J.A. Endoplasmic Reticulum-Associated Degradation and Lipid Homeostasis.Annu. Rev. Nutr. 2016; 36: 511-542Crossref PubMed Scopus (60) Google Scholar, 26Printsev I. Curiel D. Carraway 3rd, K.L. Membrane Protein Quantity Control at the Endoplasmic Reticulum.J. Membr. Biol. 2017; 250: 379-392Crossref PubMed Scopus (25) Google Scholar, 27Goder, V., Alanis-Dominguez, E., and Bustamante-Sequeiros, M., (2020) Lipids and their (un)known effects on ER-associated protein degradation (ERAD). Biochim Biophys Acta Mol. Cell Biol. Lipids, 1865(1), 158488.Google Scholar). Nevertheless, there are undoubtedly undiscovered ERAD substrates because >7,000 proteins in a human cell associate at some point during their biogenesis with the ER (28Venter J.C. Adams M.D. Myers E.W. Li P.W. Mural R.J. Sutton G.G. Smith H.O. Yandell M. Evans C.A. Holt R.A. Gocayne J.D. Amanatides P. Ballew R.M. Huson D.H. Wortman J.R. Zhang Q. Kodira C.D. Zheng X.H. Chen L. Skupski M. Subramanian G. Thomas P.D. Zhang J. Gabor Miklos G.L. Nelson C. Broder S. Clark A.G. Nadeau J. McKusick V.A. Zinder N. Levine A.J. Roberts R.J. Simon M. Slayman C. Hunkapiller M. Bolanos R. Delcher A. Dew I. Fasulo D. Flanigan M. Florea L. Halpern A. Hannenhalli S. Kravitz S. Levy S. Mobarry C. Reinert K. Remington K. Abu-Threideh J. Beasley E. Biddick K. Bonazzi V. Brandon R. Cargill M. Chandramouliswaran I. Charlab R. Chaturvedi K. Deng Z. Francesco V.D. Dunn P. Eilbeck K. Evangelista C. Gabrielian A.E. Gan W. Ge W. Gong F. Gu Z. Guan P. Heiman T.J. Higgins M.E. Ji R.-R. Ke Z. Ketchum K.A. Lai Z. Lei Y. Li Z. Li J. Liang Y. Lin X. Lu F. Merkulov G.V. Milshina N. Moore H.M. Naik A.K. Narayan V.A. Neelam B. Nusskern D. Rusch D.B. Salzberg S. Shao W. Shue B. Sun J. Wang Z.Y. Wang A. Wang X. Wang J. Wei M.-H. Wides R. Xiao C. Yan C. Yao A. Ye J. Zhan M. Zhang W. Zhang H. Zhao Q. Zheng L. Zhong F. Zhong W. Zhu S.C. Zhao S. Gilbert D. Baumhueter S. Spier G. Carter C. Cravchik A. Woodage T. Ali F. An H. Awe A. Baldwin D. Baden H. Barnstead M. Barrow I. Beeson K. Busam D. Carver A. Center A. Cheng M.L. Curry L. Danaher S. Davenport L. Desilets R. Dietz S. Dodson K. Doup L. Ferriera S. Garg N. Gluecksmann A. Hart B. Haynes J. Haynes C. Heiner C. Hladun S. Hostin D. Houck J. Howland T. Ibegwam C. Johnson J. Kalush F. Kline L. Koduru S. Love A. Mann F. May D. McCawley S. McIntosh T. McMullen I. Moy M. Moy L. Murphy B. Nelson K. Pfannkoch C. Pratts E. Puri V. Qureshi H. Reardon M. Rodriguez R. Rogers Y.-H. Romblad D. Ruhfel B. Scott R. Sitter C. Smallwood M. Stewart E. Strong R. Suh E. Thomas R. Tint N.N. Tse S. Vech C. Wang G. Wetter J. Williams S. Williams M. Windsor S. Winn-Deen E. Wolfe K. Zaveri J. Zaveri K. Abril J.F. Guigó R. Campbell M.J. Sjolander K.V. Karlak B. Kejariwal A. Mi H. Lazareva B. Hatton T. Narechania A. Diemer K. Muruganujan A. Guo N. Sato S. Bafna V. Istrail S. Lippert R. Schwartz R. Walenz B. Yooseph S. Allen D. Basu A. Baxendale J. Blick L. Caminha M. Carnes-Stine J. Caulk P. Chiang Y.-H. Coyne M. Dahlke C. Mays A.D. Dombroski M. Donnelly M. Ely D. Esparham S. Fosler C. Gire H. Glanowski S. Glasser K. Glodek A. Gorokhov M. Graham K. Gropman B. Harris M. Heil J. Henderson S. Hoover J. Jennings D. Jordan C. Jordan J. Kasha J. Kagan L. Kraft C. Levitsky A. Lewis M. Liu X. Lopez J. Ma D. Majoros W. McDaniel J. Murphy S. Newman M. Nguyen T. Nguyen N. Nodell M. Pan S. Peck J. Peterson M. Rowe W. Sanders R. Scott J. Simpson M. Smith T. Sprague A. Stockwell T. Turner R. Venter E. Wang M. Wen M. Wu D. Wu M. Xia A. Zandieh A. Zhu X. The sequence of the human genome.Science. 2001; 291: 1304-1351Crossref PubMed Scopus (10198) Google Scholar). In addition, due to the expansion of genome datasets, a plethora of mutations and polymorphisms have been identified in secreted and membranes proteins, and many of these variants may also be targeted for ERAD. Together, there is a growing appreciation that metabolic pathway components residing in the ER as well as aberrant proteins are subject to ERAD. In this study, we wished to identify previously uncharacterized UPS substrates using an approach that, to our knowledge, had not previously been pursued in yeast. First, we created a yeast strain that lacked a drug efflux pump so a specific inhibitor of the proteasome could be applied. Next, a proteasome subunit was tagged with an epitope and the gene encoding the modified subunit was integrated into the chromosome in the drug-sensitive yeast strain. We then performed a series of pulldown assays using experimental and control strains treated with a proteasome inhibitor, and label-free differential MS was used to compare isolated proteins from each condition. Data obtained in the presence of a proteasome inhibitor identified known UPS substrates along with an ER resident protein, Erg25, which had not previously been directly characterized as a UPS target. Subsequent biochemical and genetic analyses confirmed that Erg25 associates with the proteasome, is polyubiquitinated, and is stabilized in a yeast strain lacking ERAD-associated E3 ligases, thereby establishing Erg25 as a bona fide ERAD substrate. Because this enzyme plays a critical role in the biosynthesis of a yeast sterol (29Bard M. Bruner D.A. Pierson C.A. Lees N.D. Biermann B. Frye L. Koegel C. Barbuch R. Cloning and characterization of ERG25, the Saccharomyces cerevisiae gene encoding C-4 sterol methyl oxidase.Proc. Natl. Acad. Sci. U S A. 1996; 93: 186-190Crossref PubMed Scopus (130) Google Scholar), our data expand the number of regulated, WT enzymes directed to the ERAD pathway and highlight the ER as a dynamic regulator of both protein synthesis and degradation. Six biological replicates per condition were analyzed by GFP-affinity purification. MaxQuant software suite (version 1.6.6.0) (30Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (8086) Google Scholar, 31Tyanova S. Temu T. Sinitcyn P. Carlson A. Hein M.Y. Geiger T. Mann M. Cox J. The Perseus computational platform for comprehensive analysis of (prote)omics data.Nat. Methods. 2016; 13: 731-740Crossref PubMed Scopus (2714) Google Scholar) was used to analyze the MS raw files. The statistical significances of the differences were determined using a two-sample Student's t test on the log2-transformed intensity values. A combination of statistical significance and fold change differences were used to select significant candidates. A yeast strain expressing GFP-tagged Pre8 from its endogenous promoter was obtained from the Yeast GFP Clone Collection (32Huh W.K. Falvo J.V. Gerke L.C. Carroll A.S. Howson R.W. Weissman J.S. O'Shea E.K. Global analysis of protein localization in budding yeast.Nature. 2003; 425: 686-691Crossref PubMed Scopus (3189) Google Scholar) (Thermo Fisher Scientific, Waltham, MA). The parental WT strain used for the construction of the GFP clones was obtained from ATCC (ATCC 201388: MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0). Homologous recombination was used to disrupt the PDR5 locus in these two yeast backgrounds. In brief, a PDR5 deletion cassette was generated by PCR amplifying the NATMX cassette from the pFA6a–NAT-MX6 cassette, with primers containing homology to the 5‘ and 3‘ regions of the PDR5 locus (33Hecht K.A. Wytiaz V.A. Ast T. Schuldiner M. Brodsky J.L. Characterization of an M28 metalloprotease family member residing in the yeast vacuole.FEMS Yeast Res. 2013; 13: 471-484Crossref PubMed Scopus (8) Google Scholar). The resulting PCR product was purified and transformed into the yeast strain described above and plated on media containing the antibiotic, nourseothricin (NAT; clonNAT, Werner BioAgents, Jena, Germany) to select for integration of the NATMX cassette at the PDR5 locus. The genotypes of the resulting strains were confirmed by PCR amplification of the NAT cassette at the PDR5 locus. The Δpdr5 phenotype was confirmed by assessing the inhibition of the degradation of a known proteasome substrate, NBD2*, in response to MG132 treatment, as described (34Guerriero C.J. Weiberth K.F. Brodsky J.L. Hsp70 targets a cytoplasmic quality control substrate to the San1p ubiquitin ligase.J. Biol. Chem. 2013; 288: 18506-18520Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). A plasmid containing a GFP-tagged control protein, SZ*, was used as described (35Sun Z. Brodsky J.L. The degradation pathway of a model misfolded protein is determined by aggregation propensity.Mol Biol Cell. 2018; 12: 1422-1434Crossref Scopus (18) Google Scholar). Unless indicated otherwise, all yeast manipulations and growth conditions employed standard methods (36Adams A. Gottschling D.E. Kaiser C.A. Stearns T. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, 1997Google Scholar). The following yeast strains were used for the MS analysis: (1) those containing a WT copy of PDR5 and an untagged proteasome subunit (PRE8 cells), (2) those containing the proteasome-tagged subunit as well as the WT copy of PDR5 (PRE8:GFP cells), and (3) those lacking PDR5 but harboring the tagged proteasome subunit (PRE8:GFP Δpdr5; Fig. 1A). All three strains also expressed NBD2* (see above) from an introduced vector. The strains were grown to late log phase in selective media at 30 °C and MG132 (Selleckchem, Houston, TX) was added to a final concentration of 100 μm to all cultures for 1 h prior to harvesting. Approximately 20 OD600 units of cells were used for each GFP affinity purification. After incubation, cells were harvested by centrifugation at 2000 × g for 5 min and the cell pellets were washed twice with 5 ml of ice-cold PBS. Cell lysis was carried out in 1 ml of ice-cold lysis buffer (10 mM TrisCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 5 mM MgCl2, 10 mM ATP, 0.5% Nonidet P-40, and an ATP regeneration mix (37Brodsky J.L. Hamamoto S. Feldheim D. Schekman R. Reconstitution of protein translocation from solubilized yeast membranes reveals topologically distinct roles for BiP and cytosolic Hsc70.J. Cell Biol. 1993; 120: 95-102Crossref PubMed Scopus (129) Google Scholar)) in the presence of freshly added protease and phosphatase inhibitors (Thermo Fisher Scientific) using the FastPrep24 instrument and Lysing Matrix C (MP Biomedical, LLC, Santa Ana, CA). The disruption was achieved with 3 rounds of vigorous Vortex mixing at a speed setting of 6 meters/sec for 60 s. After centrifugation for 5 min at 4 °C at 16000 × g, the supernatants were transferred to new Eppendorf tubes containing 20 μl GFP-Trap®_A bead slurry (ChromoTek GmbH, 82152 Planegg-Martinsried, Germany) pre-washed with washing buffer (10 mM TrisCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA). After 2 h of end-over-end rotation at 4 °C, the GFP-Trap®_A beads were collected by centrifugation at 2000 × g for 2 min and then washed twice with 500 µL of ice-cold washing buffer. Bound proteins were eluted at 95 °C in 25 µL 2 × NuPAGE™ LDS Sample Buffer (Thermo Fisher Scientific) for 10 min. All 18 GFP affinity purifications (6 purifications per transformed cell type; Fig. 1A) were performed simultaneously on the same day. In-gel trypsin digestion was carried out as previously described (38Braganza A. Li J. Zeng X. Yates N.A. Dey N.B. Andrews J. Clark J. Zamani L. Wang X.H. St Croix C. O'Sullivan R. Garcia-Exposito L. Brodsky J.L. Sobol R.W. UBE3B is a calmodulin-regulated, mitochondrion-associated E3 ubiquitin ligase.J. Biol. Chem. 2017; 292: 2470-2484Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). In brief, eluates from the affinity purification were resolved by SDS-PAGE (NuPAGE™ 4–12% Bis-Tris Protein Gels, 1.0 mm, 10-well; Thermo Fisher Scientific) at 150 V for 10 min and stained with SimplyBlueTM SafeStain (Thermo Fisher Scientific). The stained region was excised, washed with HPLC water, and destained with 50% acetonitrile (ACN)/25 mM ammonium bicarbonate until no visible staining remained. Gel slices were dehydrated with 100% ACN and reduced with 10 mM DTT (DTT) at 56 °C for 1 h, which was followed by alkylation with 55 mM iodoacetamide (IAA) at room temperature for 45 min in the dark. The gel pieces were then dehydrated with 100% ACN to remove excess DTT and IAA. Next, 50 µL of 20 ng/µL trypsin in 25 mM ammonium bicarbonate was added for overnight digestion at 37 °C. The resulting tryptic peptides were extracted with 70% ACN/5% formic acid (FA), vacuum dried, and reconstituted in 18 µL 0.1% FA. A pooled instrument control sample was generated by combining equal volumes of the individual digests and analyzed repeatedly to monitor nLC-MS/MS performance throughout the duration of the experiment. To minimize systemic bias, a randomized block design was used to balance the sample analysis order by sample type (supplemental Table S1). Tryptic peptides were analyzed with a nLC-MS/MS system consisting of a nanoACQUITY (Waters Corporation) nano-flow HPLC coupled to an Orbitrap Velos Pro hybrid ion trap mass spectrometer (Thermo Fisher Scientific, Inc.). For each nLC-MS/MS analysis, 1 µL of each peptide digest was injected onto a 0.075 × 250 mm PicoChipTM column (New Objective, Inc.) packed with 3 μm 120 Å Reprosil C18 chromatography media and terminated with an integrated 15 μm electrospray emitter. Reverse-phase separation was achieved with a 66 min linear gradient composition of 2–35% ACN/0.1% formic acid and a flow rate of 300 nL/min. A positive electrospray ionization voltage of 1.9 kV and a capillary temperature of 275 °C was used to ionize and desolvate the eluted peptides, respectively. The mass spectrometer was operated in the data-dependent acquisition mode that records one high-resolution mass spectrum (MS1) followed by low-resolution tandem mass spectra (MS2) for each of the 13 most abundant precursor ions detected in the MS1. All high-resolution MS1 spectra were acquired with a m/z range of 375–1800 Da, an Orbitrap resolution setting of 60,000, and an automatic gain control (AGC) target value of 1.0E06. Low resolution MS2 spectra were acquired using collision-induced dissociation (CID) and an AGC target value of 5.0E06. The acquisition of replicate MS2 spectra was minimized using a dynamic exclusion time of 90 s. The nLC-MS/MS data were analyzed with the MaxQuant software suite (version 1.6.6.0) (30Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (8086) Google Scholar, 31Tyanova S. Temu T. Sinitcyn P. Carlson A. Hein M.Y. Geiger T. Mann M. Cox J. The Perseus computational platform for comprehensive analysis of (prote)omics data.Nat. Methods. 2016; 13: 731-740Crossref PubMed Scopus (2714) Google Scholar). The Andromeda protein identification search engine and SwissProt Saccharomyces cerevisiae protein database (downloaded June, 2019 with 6,721 entries) was utilized with default settings for Orbitrap instruments unless specified. Briefly, the parameters used included a precursor mass tolerance of 20 ppm for the first search and 4.5 ppm for the main search, a product ion mass tolerance of 0.8 Da, and a minimum peptide length of 7 amino acids. Trypsin was set as the proteolytic enzyme with a maximum of two missed cleavages allowed. The enzyme specifically cleaves peptide bonds C-terminal of Arginine (R) and Lysine (K) if they are not followed by Proline (P). Carbamidomethylation of Cysteine (C) was set as a fixed modification. Oxidation of Methionine (M), deamination of Asparagine (N) and Glutamine (Q), GlyGly modification of Lysine (K), and acetylation of the protein N terminus were set as variable modification. Both isotope time correlation and theoretical isotope correlation were set at 0.4. Advanced peak splitting was turned on. The alignment time window was set at 20 min and match time window at 1.5 min. A 1% false discovery rate (FDR) was used to filter the peptide identification results. Quantification of the MS1 peptide intensity was performed for all identified peptides following retention time alignment and peak matching across samples, and these values are provided in supplemental Tables S2, S3, and S4 (protein groups with single peptide identification were excluded). The MS proteomics data have been deposited to the ProteomXchange (39Vizcaino J.A. Deutsch E.W. Wang R. Csordas A. Reisinger F. Rios D. Dianes J.A. Sun Z. Farrah T. Bandeira N. Binz P.A. Xenarios I. Eisenacher M. Mayer G. Gatto L. Campos A. Chalkley R.J. Kraus H.J. Albar J.P. Martinez-Bartolome S. Apweiler R. Omenn G.S. Martens L. Jones A.R. H" @default.
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• W3082078584 date "2020-11-01" @default.
• W3082078584 modified "2023-10-03" @default.
• W3082078584 title "The Capture of a Disabled Proteasome Identifies Erg25 as a Substrate for Endoplasmic Reticulum Associated Degradation" @default.
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• W3082078584 doi "https://doi.org/10.1074/mcp.ra120.002050" @default.
• W3082078584 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/7664122" @default.
• W3082078584 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/32868373" @default.
• W3082078584 hasPublicationYear "2020" @default.
• W3082078584 type Work @default.

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