A Degenerate Cohort of Yeast Membrane Trafficking DUBs Mediates Cell Polarity and Survival*
2015; Elsevier BV; Volume: 14; Issue: 12 Linguagem: Inglês
10.1074/mcp.m115.050039
ISSN1535-9484
AutoresJanelR. Beckley, Jun‐Song Chen, Yanling Yang, Junmin Peng, Kathleen L. Gould,
Tópico(s)Cellular transport and secretion
ResumoDeubiquitinating enzymes (DUBs), cysteine or metallo- proteases that cleave ubiquitin chains or protein conjugates, are present in nearly every cellular compartment, with overlapping protein domain structure, localization, and functions. We discovered a cohort of DUBs that are involved in membrane trafficking (ubp4, ubp5, ubp9, ubp15, and sst2) and found that loss of all five of these DUBs but not loss of any combination of four, significantly impacted cell viability in the fission yeast Schizosaccharomyces pombe (1.Kouranti I. McLean J.R. Feoktistova A. Liang P. Johnson A.E. Roberts-Galbraith R.H. Gould K.L. A global census of fission yeast deubiquitinating enzyme localization and interaction networks reveals distinct compartmentalization profiles and overlapping functions in endocytosis and polarity.PLoS Biol. 2010; 8: e1000471Crossref PubMed Scopus (65) Google Scholar). Here, we delineate the collective and individual functions and activities of these five conserved DUBs using comparative proteomics, biochemistry, and microscopy. We find these five DUBs are degenerate rather than redundant at the levels of cell morphology, substrate selectivity, ubiquitin chain specificity, and cell viability under stress. These studies reveal the complexity of interplay among these enzymes, providing a foundation for understanding DUB biology and providing another example of how cells utilize degeneracy to improve survival. Deubiquitinating enzymes (DUBs), cysteine or metallo- proteases that cleave ubiquitin chains or protein conjugates, are present in nearly every cellular compartment, with overlapping protein domain structure, localization, and functions. We discovered a cohort of DUBs that are involved in membrane trafficking (ubp4, ubp5, ubp9, ubp15, and sst2) and found that loss of all five of these DUBs but not loss of any combination of four, significantly impacted cell viability in the fission yeast Schizosaccharomyces pombe (1.Kouranti I. McLean J.R. Feoktistova A. Liang P. Johnson A.E. Roberts-Galbraith R.H. Gould K.L. A global census of fission yeast deubiquitinating enzyme localization and interaction networks reveals distinct compartmentalization profiles and overlapping functions in endocytosis and polarity.PLoS Biol. 2010; 8: e1000471Crossref PubMed Scopus (65) Google Scholar). Here, we delineate the collective and individual functions and activities of these five conserved DUBs using comparative proteomics, biochemistry, and microscopy. We find these five DUBs are degenerate rather than redundant at the levels of cell morphology, substrate selectivity, ubiquitin chain specificity, and cell viability under stress. These studies reveal the complexity of interplay among these enzymes, providing a foundation for understanding DUB biology and providing another example of how cells utilize degeneracy to improve survival. Eukaryotic cells integrate signaling pathways to modulate their response to environmental changes, predominately through dynamic protein posttranslational modifications like ubiquitination (Ub'n) (2.Reyes-Turcu F.E. Ventii K.H. Wilkinson K.D. Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes.Annu. Rev. Biochem. 2009; 78: 363-397Crossref PubMed Scopus (1063) Google Scholar, 3.Clague M.J. Barsukov I. Coulson J.M. Liu H. Rigden D.J. Urbé S. Deubiquitylases from genes to organism.Physiol. Rev. 2013; 93: 1289-1315Crossref PubMed Scopus (313) Google Scholar). Cycles of Ub'n modulate protein stability, localization, and/or binding partners while maintaining cellular ubiquitin (Ub) homeostasis (3.Clague M.J. Barsukov I. Coulson J.M. Liu H. Rigden D.J. Urbé S. Deubiquitylases from genes to organism.Physiol. Rev. 2013; 93: 1289-1315Crossref PubMed Scopus (313) Google Scholar). Ub'n of substrate proteins is catalyzed by a linear sequence of enzymes (E1, E2, E3) and reversed by deubiquitinases (DUBs 1The abbreviations used are:DUBdeubiquitinaseUbubiquitinUb'nubiquitinationODoptical densityGOGene ontologyMSmass spectrometryLCliquid chromatographyEMMEdinburgh Minimal MediumAMSHAssociated molecule with the SH3 domain of STAMANOVAanalysis of varianceDICDifferential interference contrast. 1The abbreviations used are:DUBdeubiquitinaseUbubiquitinUb'nubiquitinationODoptical densityGOGene ontologyMSmass spectrometryLCliquid chromatographyEMMEdinburgh Minimal MediumAMSHAssociated molecule with the SH3 domain of STAMANOVAanalysis of varianceDICDifferential interference contrast.). Ub chains can be formed through any of Ub's seven lysines (K6, K11, K27, K29, K33, K48, K63) or its N terminus (M1), generating a wide variety of Ub chain architectures that mediate specific cellular signals (4.Ikeda F. Dikic I. Atypical ubiquitin chains: New molecular signals. Protein modifications: Beyond the usual suspects' review series.EMBO Rep. 2008; 9: 536-542Crossref PubMed Scopus (654) Google Scholar, 5.Komander D. Rape M. The ubiquitin code.Annu. Rev. Biochem. 2012; 81: 203-229Crossref PubMed Scopus (2225) Google Scholar). DUBs have been implicated in multiple essential cellular roles, including chromatin remodeling, DNA damage repair, kinase activation, endocytosis, ribosomal maturation, and immune responses (2.Reyes-Turcu F.E. Ventii K.H. Wilkinson K.D. Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes.Annu. Rev. Biochem. 2009; 78: 363-397Crossref PubMed Scopus (1063) Google Scholar, 3.Clague M.J. Barsukov I. Coulson J.M. Liu H. Rigden D.J. Urbé S. Deubiquitylases from genes to organism.Physiol. Rev. 2013; 93: 1289-1315Crossref PubMed Scopus (313) Google Scholar). deubiquitinase ubiquitin ubiquitination optical density Gene ontology mass spectrometry liquid chromatography Edinburgh Minimal Medium Associated molecule with the SH3 domain of STAM analysis of variance Differential interference contrast. deubiquitinase ubiquitin ubiquitination optical density Gene ontology mass spectrometry liquid chromatography Edinburgh Minimal Medium Associated molecule with the SH3 domain of STAM analysis of variance Differential interference contrast. Surprisingly, while multiple Ub'n enzymes (E1, E2, and E3) are essential in yeast (6.Kim D.U. Hayles J. Kim D. Wood V. Park H.O. Won M. Yoo H.S. Duhig T. Nam M. Palmer G. Han S. Jeffery L. Baek S.T. Lee H. Shim Y.S. Lee M. Kim L. Heo K.S. Noh E.J. Lee A.R. Jang Y.J. Chung K.S. Choi S.J. Park J.Y. Park Y. Kim H.M. Park S.K. Park H.J. Kang E.J. Kim H.B. Kang H.S. Park H.M. Kim K. Song K. 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Shafer B. Shoemaker D.D. Sookhai-Mahadeo S. Storms R.K. Strathern J.N. Valle G. Voet M. Volckaert G. Wang C.Y. Ward T.R. Wilhelmy J. Winzeler E.A. Yang Y. Yen G. Youngman E. Yu K. Bussey H. Boeke J.D. Snyder M. Philippsen P. Davis R.W. Johnston M. Functional profiling of the Saccharomyces cerevisiae genome.Nature,. 2002; 418: 387-391Crossref PubMed Scopus (3238) Google Scholar), only a single DUB is essential for viability of both budding and fission yeasts (6.Kim D.U. Hayles J. Kim D. Wood V. Park H.O. Won M. Yoo H.S. Duhig T. Nam M. Palmer G. Han S. Jeffery L. Baek S.T. Lee H. Shim Y.S. Lee M. Kim L. Heo K.S. Noh E.J. Lee A.R. Jang Y.J. Chung K.S. Choi S.J. Park J.Y. Park Y. Kim H.M. Park S.K. Park H.J. Kang E.J. Kim H.B. Kang H.S. Park H.M. Kim K. Song K. Song K.B. Nurse P. Hoe K.L. Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe.Nat. Biotechnol. 2015; 28: 617-623Crossref Scopus (509) Google Scholar, 7.Hayles J. Wood V. Jeffery L. Hoe K.L. Kim D.U. Park H.O. Salas-Pino S. Heichinger C. Nurse P. A genome-wide resource of cell cycle and cell shape genes of fission yeast.Open Biol. 2013; 3: 130053Crossref PubMed Scopus (107) Google Scholar, 8.Giaever G. Chu A.M. Ni L. Connelly C. Riles L. Véronneau S. Dow S. Lucau-Danila A. Anderson K. André B. Arkin A.P. Astromoff A. El-Bakkoury M. Bangham R. Benito R. Brachat S. Campanaro S. Curtiss M. Davis K. Deutschbauer A. Entian K.D. Flaherty P. Foury F. Garfinkel D.J. Gerstein M. Gotte D. Güldener U. Hegemann J.H. Hempel S. Herman Z. Jaramillo D.F. Kelly D.E. Kelly S.L. Kötter P. LaBonte D. Lamb D.C. Lan N. Liang H. Liao H. Liu L. Luo C. Lussier M. Mao R. Menard P. Ooi S.L. Revuelta J.L. Roberts C.J. Rose M. Ross-Macdonald P. Scherens B. Schimmack G. Shafer B. Shoemaker D.D. Sookhai-Mahadeo S. Storms R.K. Strathern J.N. Valle G. Voet M. Volckaert G. Wang C.Y. Ward T.R. Wilhelmy J. Winzeler E.A. Yang Y. Yen G. Youngman E. Yu K. Bussey H. Boeke J.D. Snyder M. Philippsen P. 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In contrast, in metazoans, knockdown or loss of individual DUBs often results in developmental defects or disease states (3.Clague M.J. Barsukov I. Coulson J.M. Liu H. Rigden D.J. Urbé S. Deubiquitylases from genes to organism.Physiol. Rev. 2013; 93: 1289-1315Crossref PubMed Scopus (313) Google Scholar, 12.Tsou W.L. Sheedlo M.J. Morrow M.E. Blount J.R. McGregor K.M. Das C. Todi S.V. Systematic analysis of the physiological importance of deubiquitinating enzymes.PLoS ONE. 2012; 7: e43112Crossref PubMed Scopus (52) Google Scholar, 13.McCloskey R.J. Kemphues K.J. Deubiquitylation machinery is required for embryonic polarity in Caenorhabditis elegans.PLoS Genet. 2012; 8: e1003092Crossref PubMed Scopus (15) Google Scholar). Consistent with this possibility, we previously found that loss of five DUBs (5DUB delete: ubp4Δ1 ubp5Δ ubp9Δ ubp15Δ sst2Δ) but not any combination of four intracellular membrane trafficking DUBs significantly impacted cell polarity, Ub conjugate accumulation, and viability in S. pombe (1.Kouranti I. McLean J.R. Feoktistova A. Liang P. Johnson A.E. Roberts-Galbraith R.H. Gould K.L. A global census of fission yeast deubiquitinating enzyme localization and interaction networks reveals distinct compartmentalization profiles and overlapping functions in endocytosis and polarity.PLoS Biol. 2010; 8: e1000471Crossref PubMed Scopus (65) Google Scholar). To begin to make sense of this functional overlap, here we dissected the shared and specific functions of these five DUBs on multiple levels, defining their contributions to cell polarity, Ub chain specificities, shared and specific putative substrates, and individual and combined effects of DUB loss on cell survival under stress. We find that this cohort of five DUBs is degenerate (different elements that have overlapping but not fully redundant roles), forming a robust functional module for maintenance of cell polarity and viability. S. pombe and human DUB protein sequences (FASTA files) were downloaded from the UniprotKB database (September 2014) and analyzed in MEGA6 (14.Tamura K. Stecher G. Peterson D. Filipski A. Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0.Mol. Biol. Evol. 2013; 30: 2725-2729Crossref PubMed Scopus (33496) Google Scholar). The final image was colored in Adobe Illustrator (CS6). The Ub expression vector used for all large-scale purifications was constructed as follows. Ub (processed coding sequence) was amplified from a Xenopus Ub vector (15.Shih S.C. Katzmann D.J. Schnell J.D. Sutanto M. Emr S.D. Hicke L. Epsins and Vps27p/Hrs contain ubiquitin-binding domains that function in receptor endocytosis.Nat. Cell Biol. 2002; 4: 389-393Crossref PubMed Scopus (360) Google Scholar) by PCR using primers containing NdeI(5′)/XmaI(3′) and cloned into the pREP1 (16.Maundrell K. Thiamine-repressible expression vectors pREP and pRIP for fission yeast.Gene. 1993; 123: 127-130Crossref PubMed Scopus (928) Google Scholar) vector using these sites. The His-biotin-His (HBH) tag (17.Tagwerker C. Flick K. Cui M. Guerrero C. Dou Y. Auer B. Baldi P. Huang L. Kaiser P. A tandem affinity tag for two-step purification under fully denaturing conditions: Application in ubiquitin profiling and protein complex identification combined with in vivocross-linking.Mol. Cell. Proteomics. 2006; 5: 737-748Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar) was amplified from the pFa6-HBH-kanR (KLG p3589) vector using primers containing NdeI restriction sites on both the 5′ and 3′ ends and cloned into the pREP1 vector containing Ub, yielding the pREP1-HBH-Ub construct (KLG p4954). Strain construction and tetrad analysis were accomplished through standard methods. WT, DUB deletion strains, and endogenously tagged strains (Supplemental Table S1) were grown in rich YE media or Edinburgh minimal media (EMM) with appropriate supplements. For overexpression of Flag-Ub (KLG p3729) or HBH-Ub, strains were transformed with pREP1 expression vectors (containing a thiamine repressible promoter) using a standard sorbitol transformation procedure (18.Prentice H.L. High efficiency transformation of Schizosaccharomyces pombe by electroporation.Nucleic Acids Res. 1992; 20: 621Crossref PubMed Scopus (214) Google Scholar). Transformed strains were first grown in EMM containing thiamine to suppress expression and then in EMM lacking thiamine for 20–22 h (19.Moreno S. Klar A. Nurse P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe.Methods Enzymol. 1991; 194: 795-823Crossref PubMed Scopus (3137) Google Scholar). Cell pellets were frozen in a dry ice/ethanol bath. Pellets for large-scale experiments (for LC-MS/MS) were harvested from 8 liters of EMM (∼6,500 optical density pellets). Pellets for midscale experiments (for Western blots) were harvested from 1 liter of EMM (∼800 OD). To assay the response of various DUB deletion strains to stress (Fig. 5 and Supplemental Figs. S5 and S6), serial 10-fold dilutions of each strain were spotted onto EMM agar plates or YE agar plates in the absence or presence of the following drugs (from Sigma, except as noted): 25 μm Brefeldin A (Molecular Probes); 1 μg/ml bleomycin (Bleo); 0.5 mg/ml calcofluor; 10 μg/ml cycloheximide; 5 mm ethylene glycol tetraacetic acid; 5 mm hydroxyurea; 1 m KCl; 0.25 μm Latrunculin A (Cayman Chemical, Ann Arbor, MI); 10 μg/ml methyl benzimidazol-2-yl-carbamate; 0.01% methyl methanesulfonate; 0.005% sodium dodecyl sulfate (SDS, Fisher Scientific); 1.2 m sorbitol; 12.5 μg/ml thiabendazole; 100 μm CdCl2 (Fluka); 10 μg/ml canavanine, 1 mm H2O2. Plates were incubated at 32 °C for 2–6 days prior to colony imaging. Purifications were performed as previously described (17.Tagwerker C. Flick K. Cui M. Guerrero C. Dou Y. Auer B. Baldi P. Huang L. Kaiser P. A tandem affinity tag for two-step purification under fully denaturing conditions: Application in ubiquitin profiling and protein complex identification combined with in vivocross-linking.Mol. Cell. Proteomics. 2006; 5: 737-748Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 20.Elmore Z.C. Beckley J.R. Chen J.S. Gould K.L. Histone H2B ubiquitination promotes the function of the anaphase-promoting complex/cyclosome in Schizosaccharomyces pombe.G3. 2014; 4: 1529-1538Crossref PubMed Scopus (10) Google Scholar) using equal amounts of cell pellets and affinity resin. For Western blots, 2X SDS gel loading buffer was added and samples were boiled prior to gel loading. For MS analysis, proteins were digested off streptavidin beads as described below. Streptavidin beads bound to HBH-ubiquitin were washed three times with Tris-urea buffer (100 mm Tris, pH 8.5, 8 m urea). Proteins were reduced with 3 mm Tris(2-carboxyethyl)phosphine hydrochloride, alkylated with 10 mm chloroacetamide, and digested with trypsin (0.4 μg of Trypsin Gold, Promega). The digest supernatant and washes were combined, concentrated, and desalted (Zebra spin column, Pierce). 2D-LC-MS/MS was performed as follows: Peptides were loaded onto 26-cm columns with a bomb pressure cell and then separated and analyzed by three-phase multidimensional protein identification technology on an linear trap quadrupole (LTQ) or Velos LTQ (Thermo Scientific, West Palm Beach, FL) coupled to a nanoHPLC (NanoAcquity; Waters Corporation). The NanoAcquity autosampler was used to inject 2 μl of varying concentrations of ammonium acetate (0, 10, 25, 50, 100, 200, 300, 400, 600, 800, 1,000, 5,000 mm) for 11 salt elution steps. Each injection was followed by elution of peptides with a 0–40% acetonitrile gradient (60 min) except the first and last injections, in which a 0–90% acetonitrile gradient was used. One full precursor MS scan (400–2,000 mass-to-charge ratio) and five tandem MS scans of the most abundant ions detected in the precursor MS scan under dynamic exclusion were performed. Ions with a neutral loss of 98 Da (singly charged), 49 Da (doubly charged), or 32.7 Da (triply charged) from the parent ions during MS2 were subjected to MS3 fragmentation. RAW and processed LC-MS/MS files have been deposited in the PRIDE partner repository (ProteomeXchange Consortium) with the dataset identifier PXD001767. RAW files containing more than 20 peaks were converted to DTA files using Scansifter software (21.Ma Z.Q. Tabb D.L. Burden J. Chambers M.C. Cox M.B. Cantrell M.J. Ham A.J. Litton M.D. Oreto M.R. Schultz W.C. Sobecki S.M. Tsui T.Y. Wernke G.R. Liebler D.C. Supporting tool suite for production proteomics.Bioinformatics. 2011; 27: 3214-3215Crossref PubMed Scopus (28) Google Scholar) (v2.1.25). Each DTA file was searched using the SEQUEST algorithm (TurboSequest v.27 rev12) against the S. pombe protein database (created in May 2011 from pombase.org). Common contaminants were added and all sequences were reversed to estimate the false discovery rate, yielding 10,352 total entries. Variable modifications (C+57, M+16, K+42, K+114, [STY]+80), strict tryptic cleavage, 13 (22.Beausoleil S.A. Villén J. Gerber S.A. Rush J. Gygi S.P. A probability-based approach for high-throughput protein phosphorylation analysis and site localization.Nat. Biotechnol. 2006; 24: 1285-1292Crossref PubMed Scopus (1205) Google Scholar) are reported (1,200 di-gly modified peptides were identified in 494 proteins, which is ∼25% of all proteins identified in all HBH purifications). Gene ontology (GO) annotation categories for each substrate are listed in Supplemental Table S8. The analysis of all polyUb linkages in the yeast samples was performed as reported previously (23.Na C.H. Jones D.R. Yang Y. Wang X. Xu Y. Peng J. Synaptic protein ubiquitination in rat brain revealed by antibody-based ubiquitome analysis.J. Proteome Res. 2012; 11: 4722-4732Crossref PubMed Scopus (104) Google Scholar) with a few modifications. Briefly, JMP024 (24.Xu P. Duong D.M. Seyfried N.T. Cheng D. Xie Y. Robert J. Rush J. Hochstrasser M. Finley D. Peng J. Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation.Cell. 2009; 137: 133-145Abstract Full Text Full Text PDF PubMed Scopus (847) Google Scholar) was metabolically labeled with heavy lysine (Lys + 8.0142, Arg + 10.0083) and used as internal standard. Fission yeast (WT and 5DUB delete) were cultured in regular (light) media, harvested, and then each strain was separately mixed with an equal amount of the internal standard. Following mixing, yeast cells were lysed in lysis buffer (10 mm Tris, pH 8.0; 8 m urea, 0.02% SDS; 10 mm iodoacetamide; and Roche protease inhibitor mixture) with 0.5 mm glass beads (Biospec Products, Inc.). The sample was vortexed at the highest speed for 30 s with 30 s break on ice 10X, followed by cycles of sonication for a 1 s pulse and 30 s at 4 °C. The lysate was clarified by centrifugation at 21,000 g for 5 min. Samples were loaded on an 8% SDS gel and stained with Coomassie blue G250. The gel lane was excised for in-gel digestion by trypsin (12.5 ng/μl) at 37 °C overnight. The resulting peptides were extracted with a buffer of 5% formic acid and 50% acetonitrile at room temperature, dried, and reconstituted in 1x IAP buffer (50 mm MOPS/NaOH, pH7.2; 10 mm Na2HPO4; 50 mm NaCl). Then the peptide samples were transferred to K-ε-GG antibody coupled to protein A agarose beads (Cell Signaling Technology, Inc.), and the mixture was incubated for 1.5 h at 4 °C with gentle shaking. The antibody beads were washed three times with 1X IAP (Cell Signaling Technology, Inc.) buffer containing 0.15% sodium deoxycholate at 4 °C. Tryptic diGly-containing peptides were eluted from beads by incubating with 0.15% TFA at room temperature for 10 min. Eluted peptides were desalted using StageTips (Thermo Fisher Scientific, Inc.) and analyzed by LC-MS/MS on a Thermo Q-Exactive mass spectrometer. Peptides were loaded onto a 75 μm inner diameter × 10 cm PicoFrit capillary column packed with C18 resins (2.7 μm HALO beads) (New Objective, Inc.) by the autosampler and eluted with a 40 min gradient with 6–40% of buffer B (buffer A, 0.2% formic acid; buffer B, 0.2% formic acid and 70% acetonitrile; flow rate: ca. 400 nl/min). The eluted peptides were detected in a precursor MS scan by Q Exactive (400–900 m/z, 140,000 resolution at m/z 200, 1 μscan, and 1 × 106 for automatic gain control), followed by targeted MS/MS scans of Ub linkage-specific GG peptide ions (isolation width of 2 m/z, 1 μscan, target value of 500,000 for automatic gain control; see Supplemental Table S9). The Ub-specific diGly-containing peptides from fission yeast and budding yeast were eluted together and separated in the mass spectrometer due to mass difference, enabling relative quantification. The quantitation of polyUb linkages in WT and DUB mutant yeast cells was carried out following a previously reported protocol (23.Na C.H. Jones D.R. Yang Y. Wang X. Xu Y. Peng J. Synaptic protein ubiquitination in rat brain revealed by antibody-based ubiquitome analysis.J. Proteome Res. 2012; 11: 4722-4732Crossref PubMed Scopus (104) Google Scholar). The intensities of the tryptic peptides (GG-signature peptides) derived from Ub were manually analyzed by ion chromatograms using Xcalibur v2.2.0 software (Thermo Fisher Scientific, Inc.) and used to estimate the Ub linkage changes between WT and 5DUB delete. The peak height of each precursor was calculated using Genesis peak algorithm with a mass tolerance of 20 ppm. The relative abundance of each peptide was obtained by calculating the ratio of peak height of the light form of each peptide and the corresponding peak height of the heavy form (internal standard). HBH purifications and GST pull-down experiments analyzed by SDS-PAGE on 4–12% Tris-Glycine gels (Life Technologies) were blotted on Immobilon P (Millipore) and probed with the following primary antibodies: anti-Flag (Sigma M2 monoclonal antibody) and secondary antibodies/conjugates: streptavidin (Li-COR, 680 nm or 800 nm conjugates), goat-anti rabbit or goat anti-mouse 680 nm or 800 nm conjugates (Life Technologies). Membranes were imaged on a Li-COR instrument using Odyssey software. Di-Ub cleavage assays were performed using M1, K6, K11, K27, K29, K33, K48, and K63 linkages. For each reaction, lysates of strains containing TAP-tagged DUBs were made from 40 OD pellets in native lysis buffer (6 mm Na2HPO4, 4 mm NaH2PO4·H2O, 1% Nonidet P-40, 300 mm NaCl, 2 mm EDTA, 50 mm NaF, 0.1 mm Na3VO4 plus Roche complete protease mixture, 1 μm benzamidine (Sigma) and 0.1 mm diisopropyl fluorophosphate (Sigma)). Cleared lysates were incubated with 10 μl slurry of M280 tosylactivated dynabeads (Life Technologies) coated with rabbit IgG (MP Biomedicals) to isolate DUB-TAPs. After washing with lysis buffer, the dynabeads were equilibrated with reaction buffer (20 mm HEPES-KOH [pH 8], 20 mm NaCl, 0.1 mg/ml BSA 0.5 mm EDTA, and 10 mm DTT). Each cleavage reaction was started by adding 1 μg of a di-Ub chain (Boston Biochem, Boston, MA, USA) in reaction buffer to the dynabeads containing the DUB-TAPs complexes and incubating with shaking at 30 °C for 1–18 h. Negative controls using strains lacking DUB-TAPs were included for each set of reactions to control for proteases nonspecifically bound to the beads. All images of S. pombe cells were acquired using either: (1) a spinning disk confocal microscope (Ultraview LCI; PerkinElmer), which is equipped with a Zeiss Axiovert 200m microscope, 100X NA 1.40 PlanApo oil immersion objective, a 488-nm argon ion laser (GFP), a 594-nm helium neon laser (RFP, mCherry), a charge-coupled device camera (Orca-ER; Hamamatsu Phototonics), and Metamorph 7.1 software (MDS Analytical Technologies; Molecular Devices) or (2) a Personal DeltaVision microscope system (Applied Precision), which includes an Olympus IX71 microscope, 60X NA 1.42 PlanApo and 100X NA 1.40 UPlanSApo objectives, fixed- and live-cell filter wheels, a Photometrics CoolSnap HQ2 camera, and softWoRx imaging software. Images were processed in Image J (Fiji) (25.Schindelin J. Arganda-Carreras I. Frise E. Kaynig V. Longair M. Pietzsch T. Preibisch S. Rueden C. Saalfeld S. Schmid B. Tinevez J.Y. White D.J. Hartenstein V. Eliceiri K. Tomancak P. Cardona A. Fiji: An open-source platform for biological-image analysis.Nat. Methods. 2012; 9: 676-682Crossref PubMed Scopus (30116) Google Scholar), vacuolar volume was calculated using the 3D object counter plugin (26.Bolte S. Cordelieres F.P. A guided tour into subcellular colocalization analysis in light microscopy.J. Microscopy. 2006; 224: 213-232Crossref PubMed Scopus (3218) Google Scholar), and final figures were assembled in Adobe Creative Suite (CS6). These five membrane trafficking DUBs–Ubp4, Ubp5, Ubp9, Ubp15, and Sst2–span two of the four Ub protease domain families found in S. pombe–Ub-specific protease (USP: Ubp4, Ubp5, Ubp9, and Ubp15) and Jab1/MPN domain-associated metallo-isopeptidase (JAMM: Sst2) (Fig. 1A). Although four of the five enzymes contain USP domains, they differ in sequence, metal binding capacity (Ubp5 and Ubp15 lack a functional metal binding site), and domain architecture (Fig. 1A) (1.Kouranti I. McLean J.R. Feoktistova A. Liang P. Johnson A.E. Roberts-Galbraith R.H. Gould K.L. A global census of fission yeast deubiquitinating enzyme localization and interaction networks reveals distinct compartmentalization profiles and overlapping functions in endocytosis and polarity.PLoS Biol. 2010; 8: e1000471Crossref PubMed Scopus (65) Google Scholar, 27.Ye Y. Sc
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