Quantitative Profiling of Ubiquitylated Proteins Reveals Proteasome Substrates and the Substrate Repertoire Influenced by the Rpn10 Receptor Pathway
2007; Elsevier BV; Volume: 6; Issue: 11 Linguagem: Inglês
10.1074/mcp.m700264-mcp200
ISSN1535-9484
AutoresThibault Mayor, Johannes Graumann, Jennifer Bryan, Michael J. MacCoss, Raymond J. Deshaies,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoThe ubiquitin proteasome system (UPS) comprises hundreds of different conjugation/deconjugation enzymes and multiple receptors that recognize ubiquitylated proteins. A formidable challenge to deciphering the biology of ubiquitin is to map the networks of substrates and ligands for components of the UPS. Several different receptors guide ubiquitylated substrates to the proteasome, and neither the basis for specificity nor the relative contribution of each pathway is known. To address how broad of a role the ubiquitin receptor Rpn10 (S5a) plays in turnover of proteasome substrates, we implemented a method to perform quantitative analysis of ubiquitin conjugates affinity-purified from experimentally perturbed and reference cultures of Saccharomyces cerevisiae that were differentially labeled with 14N and 15N isotopes. Shotgun mass spectrometry coupled with relative quantification using metabolic labeling and statistical analysis based on q values revealed ubiquitylated proteins that increased or decreased in level in response to a particular treatment. We first identified over 225 candidate UPS substrates that accumulated as ubiquitin conjugates upon proteasome inhibition. To determine which of these proteins were influenced by Rpn10, we evaluated the ubiquitin conjugate proteomes in cells lacking either the entire Rpn10 (rpn10Δ) (or only its UIM (ubiquitin-interacting motif) polyubiquitin-binding domain (uimΔ)). Twenty-seven percent of the UPS substrates accumulated as ubiquitylated species in rpn10Δ cells, whereas only one-fifth as many accumulated in uimΔ cells. These findings underscore a broad role for Rpn10 in turnover of ubiquitylated substrates but a relatively modest role for its ubiquitin-binding UIM domain. This approach illustrates the feasibility of systems-level quantitative analysis to map enzyme-substrate networks in the UPS. The ubiquitin proteasome system (UPS) comprises hundreds of different conjugation/deconjugation enzymes and multiple receptors that recognize ubiquitylated proteins. A formidable challenge to deciphering the biology of ubiquitin is to map the networks of substrates and ligands for components of the UPS. Several different receptors guide ubiquitylated substrates to the proteasome, and neither the basis for specificity nor the relative contribution of each pathway is known. To address how broad of a role the ubiquitin receptor Rpn10 (S5a) plays in turnover of proteasome substrates, we implemented a method to perform quantitative analysis of ubiquitin conjugates affinity-purified from experimentally perturbed and reference cultures of Saccharomyces cerevisiae that were differentially labeled with 14N and 15N isotopes. Shotgun mass spectrometry coupled with relative quantification using metabolic labeling and statistical analysis based on q values revealed ubiquitylated proteins that increased or decreased in level in response to a particular treatment. We first identified over 225 candidate UPS substrates that accumulated as ubiquitin conjugates upon proteasome inhibition. To determine which of these proteins were influenced by Rpn10, we evaluated the ubiquitin conjugate proteomes in cells lacking either the entire Rpn10 (rpn10Δ) (or only its UIM (ubiquitin-interacting motif) polyubiquitin-binding domain (uimΔ)). Twenty-seven percent of the UPS substrates accumulated as ubiquitylated species in rpn10Δ cells, whereas only one-fifth as many accumulated in uimΔ cells. These findings underscore a broad role for Rpn10 in turnover of ubiquitylated substrates but a relatively modest role for its ubiquitin-binding UIM domain. This approach illustrates the feasibility of systems-level quantitative analysis to map enzyme-substrate networks in the UPS. The classical function of ubiquitylation is to direct substrates for proteolysis via the ubiquitin proteasome system (UPS). 1The abbreviations used are: UPS, ubiquitin proteasome system; VWA, von Willebrand A; UIM, ubiquitin-interacting motif; FDR, false discovery rate; TAP, tandem affinity purification; MudPIT, multidimensional protein identification technology; WT, wild-type; DUB, deubiquitylating. Recognition of proteasome substrates is specifically mediated by several receptor proteins (1Madura K. Rad23 and Rpn10: perennial wallflowers join the melee.Trends Biochem. Sci. 2004; 29: 637-640Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). In yeast, there are at least five potential receptors (Ddi1, Dsk2, Rad23, Rpn10, and Rpt5) plus a set of Cdc48-based complexes, including the Cdc48-Npl4-Ufd1 heterotrimer, that may possess receptor function (2Elsasser S. Chandler-Militello D. Muller B. Hanna J. Finley D. Rad23 and Rpn10 serve as alternative ubiquitin receptors for the proteasome.J. Biol. Chem. 2004; 279: 26817-26822Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 3Ivantsiv Y. Kaplun L. Tzirkin-Goldin R. Shabek N. Raveh D. Unique role for the UbL-UbA protein Ddi1 in turnover of SCFUfo1 complexes.Mol. Cell. Biol. 2006; 26: 1579-1588Crossref PubMed Scopus (51) Google Scholar, 4Medicherla B. Kostova Z. Schaefer A. Wolf D.H. A genomic screen identifies Dsk2p and Rad23p as essential components of ER-associated degradation.EMBO Rep. 2004; 5: 692-697Crossref PubMed Scopus (170) Google Scholar, 5Verma R. Oania R. Graumann J. Deshaies R.J. Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system.Cell. 2004; 118: 99-110Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar, 6Lam Y.A. Lawson T.G. Velayutham M. Zweier J.L. Pickart C.M. A proteasomal ATPase subunit recognizes the polyubiquitin degradation signal.Nature. 2002; 416: 763-767Crossref PubMed Scopus (369) Google Scholar, 7Hartmann-Petersen R. Wallace M. Hofmann K. Koch G. Johnsen A.H. Hendil K.B. Gordon C. The Ubx2 and Ubx3 cofactors direct Cdc48 activity to proteolytic and nonproteolytic ubiquitin-dependent processes.Curr. Biol. 2004; 14: 824-828Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). This diversity of postubiquitylation targeting pathways is mystifying. Currently it is not known which subset of proteasome substrates is targeted by a given receptor or what features govern the allocation of substrates to a particular receptor pathway. The yeast Rpn10 protein is a stoichiometric component of the 26 S proteasome and was the first protein found to bind polyubiquitin chains (8Deveraux Q. Ustrell V. Pickart C. Rechsteiner M. A 26 S protease subunit that binds ubiquitin conjugates.J. Biol. Chem. 1994; 269: 7059-7061Abstract Full Text PDF PubMed Google Scholar). Its amino-terminal domain consists of a conserved von Willebrand A (VWA) motif that docks Rpn10 to the proteasome. Recruitment of ubiquitin chains to Rpn10 is mediated by the 20-amino acid ubiquitin-interacting motif (UIM) domain located near its carboxyl terminus (9Hofmann K. Falquet L. A ubiquitin-interacting motif conserved in components of the proteasomal and lysosomal protein degradation systems.Trends Biochem. Sci. 2001; 26: 347-350Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar). S5a protein, the human Rpn10 ortholog, contains a second UIM domain that is thought to mediate the recruitment of other receptor proteins (10Hiyama H. Yokoi M. Masutani C. Sugasawa K. Maekawa T. Tanaka K. Hoeijmakers J.H. Hanaoka F. Interaction of hHR23 with S5a. The ubiquitin-like domain of hHR23 mediates interaction with S5a subunit of 26 S proteasome.J. Biol. Chem. 1999; 274: 28019-28025Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). The general impact of Rpn10 on the turnover of proteasome substrates is not known. Given that budding yeast rpn10Δ mutants are viable (11van Nocker S. Sadis S. Rubin D.M. Glickman M. Fu H. Coux O. Wefes I. Finley D. Vierstra R.D. The multiubiquitin-chain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate-specific role in protein turnover.Mol. Cell. Biol. 1996; 16: 6020-6028Crossref PubMed Scopus (356) Google Scholar, 12Fu H. Sadis S. Rubin D.M. Glickman M. van Nocker S. Finley D. Vierstra R.D. Multiubiquitin chain binding and protein degradation are mediated by distinct domains within the 26 S proteasome subunit Mcb1.J. Biol. Chem. 1998; 273: 1970-1981Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), Rpn10 may be required for the turnover of only a small subset of ubiquitylated proteins, or Rpn10 may target a large number of substrates that, in its absence, are targeted by other proteasomal receptors (e.g. Rad23 or Dsk2). Even less well understood is the contribution of the two domains of Rpn10 to substrate turnover. Complete deletion of RPN10 (i.e. rpn10Δ) stabilizes the cell cycle regulator Sic1 and the transcription factor Gcn4. Paradoxically removal of the UIM domain by itself (i.e. uimΔ) has no discernable effect on either of these substrates (5Verma R. Oania R. Graumann J. Deshaies R.J. Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system.Cell. 2004; 118: 99-110Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar) 2J. R. Lipford, personal communication. suggesting that Rpn10 function may rely solely on an uncharacterized biochemical activity associated with its VWA domain. To understand fully the biological roles of protein ubiquitylation and the functions of individual components of the UPS such as Rpn10, it will be necessary to identify UPS substrates on a proteome-wide scale. Several studies have started to address this challenge using mass spectrometry to analyze the ubiquitin proteome (13Kirkpatrick D.S. Weldon S.F. Tsaprailis G. Liebler D.C. Gandolfi A.J. Proteomic identification of ubiquitinated proteins from human cells expressing His-tagged ubiquitin.Proteomics. 2005; 5: 2104-2111Crossref PubMed Scopus (81) Google Scholar, 14Matsumoto M. Hatakeyama S. Oyamada K. Oda Y. Nishimura T. Nakayama K.I. Large-scale analysis of the human ubiquitin-related proteome.Proteomics. 2005; 5: 4145-4151Crossref PubMed Scopus (160) Google Scholar, 15Mayor T. Lipford J.R. Graumann J. Smith G.T. Deshaies R.J. Analysis of polyubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets.Mol. Cell. Proteomics. 2005; 4: 741-751Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 16Peng J. Schwartz D. Elias J.E. Thoreen C.C. Cheng D. Marsischky G. Roelofs J. Finley D. Gygi S.P. A proteomics approach to understanding protein ubiquitination.Nat. Biotechnol. 2003; 21: 921-926Crossref PubMed Scopus (1319) Google Scholar, 17Tagwerker C. Flick K. Cui M. Guerrero C. Dou Y. Auer B. Baldi P. Huang L. Kaiser P.A. A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivo cross-linking.Mol. Cell Proteomics. 2006; 5: 737-748Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 18Hitchcock A.L. Auld K. Gygi S.P. Silver P.A. A subset of membrane-associated proteins is ubiquitinated in response to mutations in the endoplasmic reticulum degradation machinery.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12735-12740Crossref PubMed Scopus (134) Google Scholar). Although these seminal studies illustrate that shotgun mass spectrometry is a powerful tool that can provide a systems-level view of the ubiquitin proteome, it is clear that application of this technology to the ubiquitin system remains in an embryonic state. For example, no proteomics study has yet succeeded in identifying even one of the 11 yeast G1 and mitotic cyclins that are well known substrates of the UPS. Indeed many ubiquitin conjugates identified in proteomics experiments might be stably accumulating species that are not substrates of the UPS. To obtain more focused information from shotgun mass spectrometry experiments, we and others have previously applied subtractive approaches to identify conjugates that accumulate in rpn10Δ (15Mayor T. Lipford J.R. Graumann J. Smith G.T. Deshaies R.J. Analysis of polyubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets.Mol. Cell. Proteomics. 2005; 4: 741-751Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) and in npl4ts but not ubc7Δ mutants (18Hitchcock A.L. Auld K. Gygi S.P. Silver P.A. A subset of membrane-associated proteins is ubiquitinated in response to mutations in the endoplasmic reticulum degradation machinery.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12735-12740Crossref PubMed Scopus (134) Google Scholar). Although this strategy allowed the identification of several ubiquitylated proteins, by its nature the subtractive approach excludes substrates whose accumulation is only partially dependent upon a given factor. This is a major concern given the redundancy of many UPS pathways (5Verma R. Oania R. Graumann J. Deshaies R.J. Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system.Cell. 2004; 118: 99-110Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar, 19Chi Y. Huddleston M.J. Zhang X. Young R.A. Annan R.S. Carr S.A. Deshaies R.J. Negative regulation of Gcn4 and Msn2 transcription factors by Srb10 cyclin-dependent kinase.Genes Dev. 2001; 15: 1078-1092Crossref PubMed Scopus (262) Google Scholar). No fewer than six ubiquitin ligases (Mdm2, Pirh2, p300, PARC, Cul7, and Cop1) have been implicated in p53 regulation (20Andrews P. He Y.J. Xiong Y. Cytoplasmic localized ubiquitin ligase cullin 7 binds to p53 and promotes cell growth by antagonizing p53 function.Oncogene. 2006; 25: 4534-4548Crossref PubMed Scopus (82) Google Scholar, 21Brooks C.L. Gu W. p53 ubiquitination: Mdm2 and beyond.Mol. Cell. 2006; 21: 307-315Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar, 22Grossman S.R. Deato M.E. Brignone C. Chan H.M. Kung A.L. Tagami H. Nakatani Y. Livingston D.M. Polyubiquitination of p53 by a ubiquitin ligase activity of p300.Science. 2003; 300: 342-344Crossref PubMed Scopus (386) Google Scholar), and at least three different ubiquitin chain receptors contribute to turnover of ubiquitylated Sic1 (5Verma R. Oania R. Graumann J. Deshaies R.J. Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system.Cell. 2004; 118: 99-110Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar). Clearly a method that allows for more subtle quantitative comparisons is needed. In this study, we adapted stable isotope labeling techniques that have been used previously to address a variety of biological problems to perform relative quantitative analysis of polyubiquitylated proteins in two distinct cell cultures. By applying a statistical approach based on p and q values, we were able to identify ubiquitylated proteins whose levels are altered in response to a specific perturbation (chemical or genetic). After validating the approach, we used this method to identify putative substrates of the proteasome and to determine the contribution of the Rpn10 proteasome receptor pathway in the targeting of UPS substrates. We further dissected the function of Rpn10 by assessing the role of its UIM domain. All Saccharomyces cerevisiae strains used in this study are listed in Supplemental Table 1. RJD2997, which constitutively expresses His6-ubiquitin, was described previously (23Mayor T. Deshaies R.J. Two-step affinity purification of multiubiquitylated proteins from Saccharomyces cerevisiae.Methods Enzymol. 2005; 399: 385-392Crossref PubMed Scopus (23) Google Scholar). PDR5 was deleted to increase sensitivity to the proteasome inhibitor MG132 (24Fleming J.A. Lightcap E.S. Sadis S. Thoroddsen V. Bulawa C.E. Blackman R.K. Complementary whole-genome technologies reveal the cellular response to proteasome inhibition by PS-341.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1461-1466Crossref PubMed Scopus (165) Google Scholar). To obtain a prototrophic strain for labeling with heavy nitrogen, we reverted auxotrophic markers by homologous recombination. The following genes were PCR-amplified using the indicated pair of primers: ADE2 (5′-TATTAGTGAGAAGCCGAGA, 5′-GATCTTATGTATGAAATTCTT), LEU2 (5′-TGGTTGTTTGGCCGAGCGG, 5′-TCGACTACGTCGTTAAGGCC), and URA3 (5′-TCTTAACCCAACTGCACAG, 5′-GTGAGTTTAGTATACATGC). The PCR-amplified fragments were purified and transformed into W303 cells, and revertants were identified by applying the corresponding selection. RJD3313 and -3314 were obtained by successive crosses until all the markers were reverted. Both rpn10Δ and uimΔ (5Verma R. Oania R. Graumann J. Deshaies R.J. Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system.Cell. 2004; 118: 99-110Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar) were crossed with RJD3314 to obtain RJD3315 and RJD3318, respectively. Cells were grown in YNB-D (0.17% yeast nitrogen base without amino acids and ammonium sulfate, 5% dextrose, 20 μg/ml ampicillin) supplemented with 0.5% ammonium sulfate (14N) at 25 °C to an A600 between 0.5 and 1, washed three times with YNB-D only, and then diluted to an A600 of 0.008 in 2 liters of YNB-D with 0.5% 14N- or 15N-labeled (>98%, Cambridge Isotope Laboratories) ammonium sulfate. The two different isotopically labeled cell cultures were grown in parallel at 25 °C to an A600 of 1 (seven generations), and a drug treatment was applied for 30 min where indicated. Each isotopically labeled culture was then harvested by centrifugation and resuspended in 400 ml of ice-cold TBS. The A600 was carefully measured, and equal amounts of cells for each isotope variant were mixed together, centrifuged, resuspended in 200 ml of ice-cold TBS with 1 mm 1,10-phenanthroline and 10 mm iodoacetamide, recentrifuged, and snap frozen in liquid nitrogen. The two-step purification and tryptic digestion of ubiquitin conjugates was carried out as described previously (23Mayor T. Deshaies R.J. Two-step affinity purification of multiubiquitylated proteins from Saccharomyces cerevisiae.Methods Enzymol. 2005; 399: 385-392Crossref PubMed Scopus (23) Google Scholar) with the following minor changes: cells were broken in 40 ml of lysis buffer after which 1.8 mg each of recombinant GST-Rad23 and GST-Dsk2 coupled to Sepharose were used for the first step and 100 μl of nickel magnetic bead slurry (Promega) were used for the second. Typically only one-half of the sample was analyzed in a single LCQ analysis (corresponding to 1 liter of each labeled cell sample), whereas with the LTQ, material corresponding to 500 ml of each labeled culture was analyzed. An aliquot (10 μl) of cleared total cell extract was collected after the first centrifugation, precipitated (25Wessel D. Flugge U.I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids.Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3191) Google Scholar), and resuspended in UB buffer (8 m urea, 100 mm NaH2PO4, 10 mm Tris-HCl, pH 8.0). 5 μg of protein were subjected to tryptic digest, mass spectrometry, and quantitative analysis to calculate the average ratio of isotopically labeled proteins in the total cell extract (median of 14N/15N ratio values). The proteolytically digested proteins analyzed in Fig. 1 were evaluated by multidimensional chromatography coupled in line to ESI-MS using an LCQ-Deca mass spectrometer (ThermoFisher) as described previously (15Mayor T. Lipford J.R. Graumann J. Smith G.T. Deshaies R.J. Analysis of polyubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets.Mol. Cell. Proteomics. 2005; 4: 741-751Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 26Graumann J. Dunipace L.A. Seol J.H. McDonald W.H. Yates III, J.R. Wold B.J. Deshaies R.J. Applicability of tandem affinity purification MudPIT to pathway proteomics in yeast.Mol. Cell. Proteomics. 2004; 3: 226-237Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Samples in Fig. 3, Fig. 4, Fig. 5 were analyzed with an LTQ ion trap (ThermoFisher) using a modified protocol (supplemental information). Sequest and DTASelect (27Eng J.K. McCormack A.L. Yates III, J.R. An approach to correlate tandem mass-spectral data of peptides with amino-acid-sequences in a protein database.J. Am. Soc. Mass Spectrom. 1994; 5: 976-989Crossref PubMed Scopus (5472) Google Scholar, 28Tabb D.L. McDonald W.H. Yates III, J.R. DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics.J. Proteome Res. 2002; 1: 21-26Crossref PubMed Scopus (1144) Google Scholar) were used to analyze the mass spectrometry profiles using two sets of parameters for 14N and 15N searches (supplemental information). Proteins identified by at least two peptides (Supplemental Table 2) were retained for the quantitative analysis, and peptides (14N and 15N) were separately analyzed with RelEx (versions 0.91 and 0.92 (29MacCoss M.J. Wu C.C. Liu H. Sadygov R. Yates III, J.R. A correlation algorithm for the automated quantitative analysis of shotgun proteomics data.Anal. Chem. 2003; 75: 6912-6921Crossref PubMed Scopus (249) Google Scholar)) using the following parameters: (i) width option was ±75 scans with LCQ and ±25 scans with LTQ data; (ii) for purified material, ratio correction was applied using the (median ratio)−1 value derived from total cell extract; (iii) regression filter was applied (minimum correlation, 0.7 at 1 and 0.4 at 10); and (iv) signal to noise filter was applied (minimum of 3 for LCQ and 5 for LTQ data). The files from the two separate analyses (14N and 15N) were merged and sorted using RelEx (Supplemental Table 3).Fig. 3The UPS proteome.A, frequency distributions of log ratios in the MG132-treated 15N cell experiment (dark blue) and reference experiment (light blue; see also Fig. 4A) using the LTQ ion trap. Truncation of the graph excluded some log ratios with extreme values. B, pie diagram representing the distribution of proteasome substrate functions (with indicated percentage). Gene ontologies were retrieved from the Saccharomyces Genome Database (www.stanford.edu/Saccharomyces). C, histograms representing the percentage of proteins in the whole (light blue) and UPS (dark blue) proteomes that are localized in one of four major cellular compartments: cytoplasm (cytoplasm, bud, bud neck, microtubule, and actin), nucleus (nucleus, nucleolus, nuclear periphery, spindle pole, and microtubule), membrane-associated organelles (endoplasmic reticulum, Golgi, endosome, vacuole, lipid particle, cell periphery, and punctate composite), and mitochondria plus peroxisomes. Information was retrieved from a global study based on localization of green fluorescent protein fusion proteins expressed from endogenous loci (39Huh 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 (3326) Google Scholar). Note that some proteins localized in more than one compartment. D, table representing different categories of protein abundances. Protein abundances were retrieved from a previous study based on a genome-wide collection of TAP tagged open reading frames (40Ghaemmaghami 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 (3008) Google Scholar), and categories were arbitrarily established using abundance values (column a). Of the 4102 proteins that were successfully quantified, the number of proteins in each category is listed (column b) as is the percentage of total proteins represented by that category (column c). Of the 225 putative UPS substrates identified in the MG132 analysis, 169 were quantified by Ghaemmaghami et al. (40Ghaemmaghami 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 (3008) Google Scholar). Of these, the total number that fall into each abundance category is listed (column d) as is the corresponding percentage (column e). Column f equals column e divided by column c and thus represents the degree of enrichment for proteins in each abundance category. resp., response; mito., mitochondria; pero., peroxisomes; assoc., associated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Analysis of the impact of Rpn10 on the UPS proteome.A, frequency distributions of log ratios. All analyses were performed using the LTQ ion trap. The reference experiment (displayed on the left) was performed by comparing 14N WT versus15N WT cells (370 proteins). Using the same method applied to the reference experiment in Fig. 1, the variance (σ2) was estimated at 0.46. In the 14N rpn10Δ versus 15N WT analysis, 530 proteins were quantified (middle), and in the 14N uimΔ versus 15N WT analysis, 344 proteins were quantified (right). The bell-shaped distribution obtained in the reference experiment is depicted in the three panels as a dotted line. Only frequencies for |log ratio| √n× log (ratio)/σref) where Z is N(0, 1)(Eq. 1) To permit the control of the false discovery rate (FDR) (30Benjamini Y. Hochberg Y. Controlling the false discovery rate—a practical and powerful approach to multiple testing.J. R. Stat. Soc. Ser. B Stat. Methodol. 1995; 57: 289-300Google Scholar) in subsequent lists of putative proteasome substrates, we computed adjusted p values, also known as "q values" (31Storey J.D. Tibshirani R. Statistical significance for genomewide studies.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9440-9445Crossref PubMed Scopus (7169) Google Scholar), q value (p(i)) = p(i) × π0 × m/i(Eq. 2) where p(i) is the ith order statistic of the observed p values, π0 is the (estimated) proportion of unresponsive proteins, and m is the number of proteins quantified. To calculate the recovery rate associated with a q value cutoff equal to q(i), note that, on average, i × q(i) of the i"discoveries" are false and conversely that i × (1 − q(i)) of the i discoveries are true. Overall m × (1 − π0) ORFs are thought to be truly responsive; therefore we can estimate the fractio
Referência(s)