A High Throughput Screen to Identify Substrates for the Ubiquitin Ligase Rsp5
2005; Elsevier BV; Volume: 280; Issue: 33 Linguagem: Inglês
10.1074/jbc.m502197200
ISSN1083-351X
AutoresBart Kus, Aaron S. Gajadhar, Karen Stanger, Rob Cho, Warren Sun, Nathalie Rouleau, Tammy Lee, Donovan Chan, Cheryl Wolting, A.M. Edwards, Roger Bossé, Daniela Rotin,
Tópico(s)Peptidase Inhibition and Analysis
ResumoUbiquitin-protein ligases (E3s) are implicated in various human disorders and are attractive targets for therapeutic intervention. Although most cellular proteins are ubiquitinated, ubiquitination cannot be linked directly to a specific E3 for a large fraction of these proteins, and the substrates of most E3 enzymes are unknown. We have developed a luminescent assay to detect ubiquitination in vitro, which is more quantitative, effective, and sensitive than conventional ubiquitination assays. By taking advantage of the abundance of purified proteins made available by genomic efforts, we screened hundreds of purified yeast proteins for ubiquitination, and we identified previously reported and novel substrates of the yeast E3 ligase Rsp5. The relevance of these substrates was confirmed in vivo by showing that a number of them interact genetically with Rsp5, and some were ubiquitinated by Rsp5 in vivo. The combination of this sensitive assay and the availability of purified substrates will enable the identification of substrates for any purified E3 enzyme. Ubiquitin-protein ligases (E3s) are implicated in various human disorders and are attractive targets for therapeutic intervention. Although most cellular proteins are ubiquitinated, ubiquitination cannot be linked directly to a specific E3 for a large fraction of these proteins, and the substrates of most E3 enzymes are unknown. We have developed a luminescent assay to detect ubiquitination in vitro, which is more quantitative, effective, and sensitive than conventional ubiquitination assays. By taking advantage of the abundance of purified proteins made available by genomic efforts, we screened hundreds of purified yeast proteins for ubiquitination, and we identified previously reported and novel substrates of the yeast E3 ligase Rsp5. The relevance of these substrates was confirmed in vivo by showing that a number of them interact genetically with Rsp5, and some were ubiquitinated by Rsp5 in vivo. The combination of this sensitive assay and the availability of purified substrates will enable the identification of substrates for any purified E3 enzyme. The ubiquitin pathway is conserved throughout eukaryotic evolution and is implicated in numerous cellular processes (1Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6959) Google Scholar). Proteins modified by the ubiquitin pathway are processed for degradation, endocytosis, protein sorting, and subnuclear trafficking (2Hicke L. Dunn R. Annu. Rev. Cell Dev. Biol. 2003; 19: 141-172Crossref PubMed Scopus (965) Google Scholar, 3Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2944) Google Scholar). Ubiquitination is catalyzed by three enzymes termed E1 1The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; b-Ub, biotinylated ubiquitin; GST, glutathione S-transferase; TCEP, tris(2-chloroethyl) phosphate; CTD, C-terminal domain. (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin protein ligase). E3 regulates the specificity of the reaction by binding directly to substrates (1Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6959) Google Scholar, 3Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2944) Google Scholar). The E3-substrate interaction is implicated in an increasing number of diseases, including neurodegeneration, immunological disorders, hypertension, and cancers. For example, numerous E3 enzymes such as Fbw7, Skp2, Mdm2, and VHL and their respective substrates, cyclin E, p27, p53, and HIF, have been linked to tumor progression (4Wong B.R. Parlati F. Qu K. Demo S. Pray T. Huang J. Payan D.G. Bennett M.K. Drug Discov. Today. 2003; 8: 746-754Crossref PubMed Scopus (61) Google Scholar, 5Burger A.M. Seth A.K. Eur. J. Cancer. 2004; 40: 2217-2229Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). The therapeutic importance of understanding ubiquitination has been underscored recently by the success of anticancer strategies that affect the ubiquitin pathway (6Orlowski R.Z. Expert Rev. Anticancer Ther. 2004; 4: 171-179Crossref PubMed Scopus (27) Google Scholar). Most, if not all, proteins are regulated by the ubiquitin pathway. A recent proteomic approach identified over a thousand proteins that are ubiquitinated in yeast under normal conditions (7Peng J. Schwartz D. Elias J.E. Thoreen C.C. Cheng D. Marsischky G. Roelofs J. Finley D. Gygi S.P. Nat. Biotechnol. 2003; 21: 921-926Crossref PubMed Scopus (1319) Google Scholar). This study, which likely did not detect many nonabundant proteins or proteins that are ubiquitinated under specific conditions (e.g. stress and nutrition), underlines the breadth of the ubiquitin system. Current estimates also predict that there are hundreds of E3 enzymes in eukaryotic genomes (8Willems A.R. Schwab M. Tyers M. Biochim. Biophys. Acta. 2004; 1695: 133-170Crossref PubMed Scopus (382) Google Scholar) whose role is to ubiquitinate these proteins. Despite the biomedical importance of E3 enzymes and great advances in understanding the mechanics of the ubiquitin system, a very small fraction of E3 enzymes has been linked to specific substrates, and currently, the scarcity of identified E3-substrate pairs in the literature is a major bottleneck in the ubiquitin field. Rsp5 is a yeast E3 enzyme, and many of its substrates have not yet been characterized. It belongs to the Nedd4 family of E3 ligases (9Rotin D. Staub O. Haguenauer-Tsapis R. J. Membr. Biol. 2000; 176: 1-17Crossref PubMed Google Scholar), which contain a C2 domain, WW domains, and a catalytic HECT domain (Fig. 1A). WW domains are protein-protein interaction modules that usually bind substrates directly by recognizing (L/P)PXY sequences (PY motifs) (10Kanelis V. Rotin D. Forman-Kay J.D. Nat. Struct. Biol. 2001; 8: 407-412Crossref PubMed Scopus (187) Google Scholar, 11Kasanov J. Pirozzi G. Uveges A.J. Kay B.K. Chem. Biol. 2001; 8: 231-241Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 12Hu H. Columbus J. Zhang Y. Wu D. Lian L. Yang S. Goodwin J. Luczak C. Carter M. Chen L. James M. Davis R. Sudol M. Rodwell J. Herrero J.J. Proteomics. 2004; 4: 643-655Crossref PubMed Scopus (114) Google Scholar, 13Shcherbik N. Kee Y. Lyon N. Huibregtse J.M. Haines D.S. J. Biol. Chem. 2004; 279: 53892-53898Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The best characterized role of this E3 family is to ubiquitinate transmembrane proteins and to regulate their endocytosis (9Rotin D. Staub O. Haguenauer-Tsapis R. J. Membr. Biol. 2000; 176: 1-17Crossref PubMed Google Scholar). In addition, Rsp5 has been implicated in various other biological functions, such as mini-chromosome maintenance, mitochondrial inheritance, actin cytoskeleton maintenance, drug resistance, regulation of intracellular pH, biosynthesis of fatty acids, and protein sorting at the trans-Golgi network (2Hicke L. Dunn R. Annu. Rev. Cell Dev. Biol. 2003; 19: 141-172Crossref PubMed Scopus (965) Google Scholar, 14Hein C. Springael J.Y. Volland C. Haguenauer-Tsapis R. Andre B. Mol. Microbiol. 1995; 18: 77-87Crossref PubMed Scopus (298) Google Scholar, 15Horak J. Biochim. Biophys. Acta. 2003; 1614: 139-155Crossref PubMed Scopus (63) Google Scholar). Rsp5 also regulates the activity of RNA polymerase II in response to DNA damage by ubiquitinating Rpb1, the large subunit of the holoenzyme (16Huibregtse J.M. Yang J.C. Beaudenon S.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3656-3661Crossref PubMed Scopus (182) Google Scholar, 17Beaudenon S.L. Huacani M.R. Wang G. McDonnell D.P. Huibregtse J.M. Mol. Cell. Biol. 1999; 19: 6972-6979Crossref PubMed Scopus (148) Google Scholar). The mammalian Rsp5 homolog, Nedd4 (or Nedd4-2), binds to the PY motifs of the plasma membrane-localized epithelial sodium channel and regulates its endocytosis (10Kanelis V. Rotin D. Forman-Kay J.D. Nat. Struct. Biol. 2001; 8: 407-412Crossref PubMed Scopus (187) Google Scholar, 18Staub O. Dho S. Henry P. Correa J. Ishikawa T. McGlade J. Rotin D. EMBO J. 1996; 15: 2371-2380Crossref PubMed Scopus (741) Google Scholar, 19Abriel H. Loffing J. Rebhun J.F. Pratt J.H. Schild L. Horisberger J.D. Rotin D. Staub O. J. Clin. Investig. 1999; 103: 667-673Crossref PubMed Scopus (329) Google Scholar, 20Kamynina E. Debonneville C. Bens M. Vandewalle A. Staub O. FASEB J. 2001; 15: 204-214Crossref PubMed Scopus (250) Google Scholar). Mutations in these PY motifs cause Liddle syndrome, a genetic form of hypertension (21Lifton R.P. Gharavi A.G. Geller D.S. Cell. 2001; 104: 545-556Abstract Full Text Full Text PDF PubMed Scopus (1376) Google Scholar). In this study, the yeast Rsp5 E3 ligase was used as a model to develop an in vitro ubiquitination assay in order to discover substrates for Rsp5 from among large numbers of purified GST-tagged proteins. Traditional immunoblotting (Western blot) assays used to monitor ubiquitination were unsuitable for this purpose because they are difficult to standardize, not quantitative, and not amenable to genome-scale analysis. We elected to develop a ubiquitination assay based on luminescence (AlphaScreen™ technology) in order to achieve higher sensitivity, greater throughput, and the potential for automation (22Warner G. Illy C. Pedro L. Roby P. Bosse R. Curr. Med. Chem. 2004; 11: 721-730Crossref PubMed Scopus (46) Google Scholar, 23Rouleau N. Turcotte S. Mondou M.H. Roby P. Bosse R. J. Biomol. Screen. 2003; 8: 191-197Crossref PubMed Scopus (27) Google Scholar). The assay measures the proximity of biotinylated ubiquitin (b-Ub) and GST-tagged substrate proteins (22Warner G. Illy C. Pedro L. Roby P. Bosse R. Curr. Med. Chem. 2004; 11: 721-730Crossref PubMed Scopus (46) Google Scholar, 23Rouleau N. Turcotte S. Mondou M.H. Roby P. Bosse R. J. Biomol. Screen. 2003; 8: 191-197Crossref PubMed Scopus (27) Google Scholar) (Fig. 1B). The luminescent assay was used to screen 188 purified yeast proteins for ubiquitination by Rsp5. We identified novel substrates as well as proteins already known to interact with Rsp5. The relevance of the novel substrates was confirmed in vivo by showing that their overexpression compromised the viability of strains deficient in Rsp5 function and by experiments demonstrating that a novel protein that produced a very strong signal in the luminescence assay was also ubiquitinated in vivo. Purification of Yeast E2 Enzymes—Yeast E2 genes UBC1 and UBC4 were cloned into pET15b plasmids as described (24Kus B.M. Caldon C.E. Andorn-Broza R. Edwards A.M. Proteins. 2004; 54: 455-467Crossref PubMed Scopus (63) Google Scholar). All proteins were expressed in Escherichia coli strain BL21 (DE3). Transformed cells were grown at 37 °C to an A590 of 0.6 in 2 liters of Luria broth, and expression was induced by addition of 1 mm isopropyl 1-thio-β-d-galactopyranoside. After 12 h of induction at 16 °C, the cells were harvested and lysed by sonication in binding buffer (20 mm HEPES, pH 8.0, 500 mm NaCl, 10% glycerol, 10 μm ZnCl2, 0.5 mm tris(2-chloroethyl) phosphate (TCEP)), and protease inhibitor tablets (1 tablet/50 ml of buffer, Roche Applied Science) containing 5 mm imidazole. Lysates were clarified by centrifugation at 100,000 × g for 1 h at 4 °C, and His-tagged proteins were purified from the clarified lysate on a 2-ml nickel-nitrilotriacetic acid superflow agarose column (Qiagen). Bound proteins were washed with binding buffer supplemented with 30 mm imidazole and eluted with binding buffer supplemented with 500 mm imidazole. Purification yielded 57 mg of Ubc4 and 22 mg of Ubc1 (at a concentration of 9.5 and 3.7 mg/ml, respectively). Purification of Yeast E3 Rsp5, Mutant Rsp5 C777A, and Rsp5 HECT Domain—The GST-Rsp5 expression plasmid (pGEX-6P2-RSP5) was a generous gift from Dr. Linda Hicke, and the GST-Rsp5 C777A expression plasmid (pGEX-6P2-RSP5 C777A) was a generous gift from Dr. Dale Haines. The HECT domain of Rsp5 was PCR-amplified from the GST-Rsp5 expression plasmid (pGEX-6P2-RSP5) using forward and reverse primers specific to the BamHI and EcoRI restriction sites. The PCR product was subcloned into the pGEX-6P1 plasmid to create GST-HECT. GST-Rsp5, GST-Rsp5 C777A, and GST-HECT were expressed in E. coli using the same method as described for the E2 enzymes except that imidazole was omitted from the binding buffer. The recombinant proteins were purified from the cell lysate on a column containing 3 ml of glutathione-Sepharose resin (Amersham Biosciences), washed once with 50 ml of binding buffer, followed by a wash with 25 ml of PreScission cleavage buffer (PCB: 50 mm Tris-HCl, pH 7.0, 150 mm NaCl, 1 mm TCEP, 10% glycerol). Rsp5, Rsp5 C777A, and HECT were proteolytically cleaved from the GST moiety by incubating the resin for 4 h with 1 ml of PCB containing 40 units of PreScission protease (Amersham Biosciences). Extensive cleavage reactions resulted in precipitation of Rsp5 and HECT. By using this protocol, we recovered 4.8 mg of Rsp5 (at 1.2 mg/ml), 3.2 mg of Rsp5 C777A (at 1 mg/ml), and 8 mg (at 0.8 mg/ml) of the HECT domain. Purification of Yeast GST-CTD—The GST-CTD expression vector (pET21a-GST-TEV-CTD) was constructed by Dr. Nova Fong 2N. Fong, unpublished data. and generously provided by Dr. David Bentley. GST-CTD was expressed in E. coli and purified using the same method as described for the GST-Rsp5 except that proteins were eluted with binding buffer containing 15 mm glutathione. The purification yielded 22 mg of GST-CTD (at 2 mg/ml). Purification of Yeast GST-tagged Substrates—The collection of yeast strains expressing GST proteins (25Zhu H. Bilgin M. Bangham R. Hall D. Casamayor A. Bertone P. Lan N. Jansen R. Bidlingmaier S. Houfek T. Mitchell T. Miller P. Dean R.A. Gerstein M. Snyder M. Science. 2001; 293: 2101-2105Crossref PubMed Scopus (1942) Google Scholar) was a generous gift from Dr. Michael Snyder. Culture of the yeast strains and expression of recombinant GST proteins were carried out as described previously (25Zhu H. Bilgin M. Bangham R. Hall D. Casamayor A. Bertone P. Lan N. Jansen R. Bidlingmaier S. Houfek T. Mitchell T. Miller P. Dean R.A. Gerstein M. Snyder M. Science. 2001; 293: 2101-2105Crossref PubMed Scopus (1942) Google Scholar). Proteins were purified from 50 ml of growth media using 100 μl of glutathione-Sepharose resin and eluted in 100 μl of binding buffer containing 15 mm glutathione (Amersham Biosciences). The final yield of purified proteins varied from 10 to 200 μg. Purified proteins at concentrations calculated to be greater than 1 μm were used in the ubiquitination assay. Protein Analysis—All purified proteins were resolved on 4–12% gradient SDS-polyacrylamide gels (NOVEX) and visualized by Coomassie Blue staining (Sigma B-7920) and immunoblotted using a mouse monoclonal α-GST antibody (B-14, Santa Cruz Biotechnology) or mouse monoclonal α-His antibodies (Amersham Biosciences). Protein yield was measured by Bradford assay (Bio-Rad) followed by gel densitometry using Gene Genius Bioimaging System, GeneSnap, and Genetools software (all from SynGene). Purified proteins were frozen in liquid nitrogen and stored at –80 °C. In Vitro Ubiquitination (Luminescence) Assays—Standard ubiquitination reactions contained 3 μlof5× assay buffer (250 mm HEPES, pH 7.4, 25 mm MgOAc, 2.5 mm TCEP, 500 mm NaCl, and 50% glycerol), 1 μg of biotinylated ubiquitin (b-Ub), 0.16 μg of yeast E1, 3.8 μg of Ubc4 E2, 4 μg of Ubc1 E2, 1.2 μg of Rsp5 E3, 8 pmol of GST-tagged substrate, and 3.3 mm ATP (Sigma). E1 and b-Ub were purchased from Boston Biochem. Where indicated, Rsp5 was substituted with an equal amount of Rsp5 C777A or 0.52 μg of HECT domain. Water was added to each of the reactions to bring the final volume of all reactions to 15 μl. ATP was either omitted or added last in order to minimize autocatalytic ubiquitination reactions by the ubiquitination enzymes. Reactions were allowed to proceed for 4 h at room temperature and stopped by boiling in 5 μl of SDS-PAGE sample buffer containing 3 m urea, or by diluting the samples 100–1000-fold with AlphaScreen assay buffer (25 mm HEPES, pH 7.4, 100 mm NaCl, 0.1% Tween 20, and 1 mm dithiothreitol). To prepare the ubiquitination reactions for analysis using AlphaScreen, the samples were diluted 270-fold with AlphaScreen assay buffer (unless otherwise indicated) and transferred to 384-well OptiPlate-NEW microplates containing 10 μl of AlphaScreen anti-GST acceptor and streptavidin donor beads (both at a final concentration of 20 μg/ml). This dilution step brings the concentration of b-Ub and GST proteins into the nanomolar range, which corresponds to the binding capacities of AlphaScreen beads. Plates were incubated at 23 °C for 1 h and analyzed on an AlphaQuest HTS analyzer. A homogenous assay format was also developed in which similar conditions were used (not shown). AlphaScreen GST detection kits, analyzer, and microplates were purchased from PerkinElmer Life Sciences. Detection of Ubiquitination by Immunoblotting—Completed ubiquitination reactions (15 μl) were boiled in 3× SDS-PAGE sample buffer and resolved by gel electrophoresis on 4–12% gradient SDS-polyacrylamide gels or 12% polyacrylamide gels. GST-tagged substrate proteins were detected using a 1:2000 dilution of the α-GST antibody. In Vivo Overexpression Yeast Experiments—The rsp5-1 strain and the corresponding wild-type strain (FY56 and FW1808, respectively) were generously provided by Dr. Andrew Emili and were transformed with purified plasmids obtained from the GST fusion protein overexpression library. To assay lethality at restrictive and permissive temperatures, strains were grown in 1-ml 96-well plates (Ultident) in liquid Ura– synthetic drop-out media containing 2% glucose and then spotted onto solid plates containing 2% glucose or 2% galactose using a 96-well pin (Ultident). Plates were grown at the indicated temperatures for 40 h. In Vivo Ubiquitination Experiments—The rsp5-1 and the corresponding wild-type strains expressing the desired GST proteins were grown to log phase in Ura–synthetic drop-out media containing 2% raffinose. The temperature was then shifted to 37 °C; the expression of the GST proteins was induced by the addition of galactose to 2%, and growth was continued at the restrictive temperature for 2 h. The cells were then harvested, and the GST proteins were purified from the cells as described except that 50 μm LLNL (Sigma), 1 μm chloroquine (Sigma), and 1 μm MG-132 (Boston Biochem) was added to the lysis buffers in addition to benzamidine and phenylmethylsulfonyl fluoride. The proteins extracted from the mutant and wild-type strains were analyzed using SDS-PAGE and probed with α-GST and α-ubiquitin (Covance) antibodies. Development of the Luminescent Ubiquitination Assay—The new luminescent assay was designed to monitor the ubiquitination of GST fusion proteins in reactions containing purified E1, E2, and E3 (Rsp5) enzymes, b-Ub, and ATP (Fig. 1B). As a starting point for assay development, we measured Rsp5-dependent ubiquitination of the C-terminal domain (CTD) of Rpb1, a known Rsp5 substrate, by using Western blots. Once these assay conditions were established, we developed the initial luminescent assay conditions using CTD as the substrate. Furthermore, the ubiquitination of four other in vitro GST-tagged substrates of Rsp5 (Ybr196c, Ypr084w, Ynl136w, and Ydr203c, which were discovered in the course of the study) and five proteins not ubiquitinated by Rsp5 in vitro (GST alone, Yer177w, Ycl040w, Yor057w, and Yar015w) was also reconstituted and monitored using both Western blot and luminescent assays. Fig. 2A shows the detection of ubiquitination using the luminescent assay, and Fig. 2B shows the ubiquitination of these same proteins as detected by Western blot. Ubiquitination of the Rsp5 substrates was 6–11-fold greater in the presence of Rsp5 (Fig. 2A). Significantly lower luminescence was observed in the negative controls, and no ubiquitination was seen on Western blots (Fig. 2B). To ensure that the luminescent signal was dependent on the ubiquitination enzyme cascade, we independently titrated each of the assay components. There was a dose-dependent response to all assay components (Fig. 2C). Reactions containing Ycl040w and Yer177w, which were not ubiquitinated by Rsp5 in vitro according to Western blot analysis, did not produce significant signal over background (not shown). Compared with Western blots, the luminescent assay required 270-fold less biotinylated product to produce an optimal signal (Fig. 3). Finally, we observed a time-dependent response until the reactions containing GST-CTD and Ybr196c were completed after ∼2 h (Fig. 2C). On average, the standard error of measurement in the luminescence assay was ±9.8% (Fig. 4). The greatest source of error was the variability in the enzymatic reaction and not the detection reaction or substrate concentrations (Fig. 4).Fig. 4Assay variability. A, the purification of 14 proteins was repeated between four and six times, and each separately purified protein was incubated in a ubiquitination reaction. The luminescent signal generated by each reaction is represented by a point on the graph to show the reproducibility of the assay. On average, the S.E. is 9.8% of the mean. B, ubiquitination of Ybr196c and Ypr084w was performed, and each reaction was divided into 8 aliquots. Independent detection reactions were then performed (n = 8) for each substrate (the error bars are not seen due to low variability). C, 10 independent ubiquitination and detection reactions were performed for Ybr196c and Ypr084w by using the luminescent assay (n = 10). D, 10 independent ubiquitination detection reactions were performed with Ybr196c and Ypr084w, and for each experiment the luminescent signal was normalized to the signal generated by Ybr196c. Inter-assay variability was determined by using these normalized results. A and D, the absolute luminescence values were normalized to the signal produced by the substrate Ybr196c.View Large Image Figure ViewerDownload (PPT) Screen for Ubiquitinated Substrates of Rsp5—The luminescence assay allows for a direct comparison of the relative level of ubiquitination between different proteins and provides sensitivity and speed at low cost, although in its current format it cannot distinguish between mono- and polyubiquitination. To explore the feasibility of using the assay for substrate discovery, we screened for substrates of Rsp5. 188 GST fusion proteins were purified from a yeast overexpression library (25Zhu H. Bilgin M. Bangham R. Hall D. Casamayor A. Bertone P. Lan N. Jansen R. Bidlingmaier S. Houfek T. Mitchell T. Miller P. Dean R.A. Gerstein M. Snyder M. Science. 2001; 293: 2101-2105Crossref PubMed Scopus (1942) Google Scholar). 130 of these were selected at random, and 58 were selected because they contained a PY motif, which is a potential target for the Rsp5 WW domains. Equal amounts of purified substrate proteins were subjected to standardized reactions, and Rsp5-dependent ubiquitination was monitored. In the presence of Rsp5, nine substrates generated signal that was higher than CTD (supplemental Table I). GST alone generated a low background signal (0.01 units, Fig. 5 and supplemental Table I). As expected, most of the proteins that generated strong luminescent signal contain a PY motif (Fig. 5), and on average, these proteins produced higher signal (p > 0.01, t test). The WW domains of Rsp5 therefore appeared to retain specificity for PY motifs in the in vitro assays. The nine proteins generating signal higher than CTD were designated as potential substrates of Rsp5. To provide evidence that Rsp5 recognizes substrates through its WW domain in the screen, we selected six of these substrates and tested their ubiquitination by a construct of Rsp5 harboring the HECT domain alone, and lacking the C2 and WW domains, or with a mutant Rsp5 in which the catalytic cysteine was mutated to alanine (Rsp5-C777A) to render it catalytically inactive. The full-length Rsp5 was able to ubiquitinate the six substrates much more efficiently than did either the HECT domain alone or the Rsp5 mutant (Rsp5-C777A) (26Dunn R. Hicke L. Mol. Biol. Cell. 2001; 12: 421-435Crossref PubMed Scopus (120) Google Scholar) (Fig. 6). The HECT domain alone and the wild-type Rsp5 possessed equal specific activity as monitored by their auto-ubiquitination, a property of some E3 enzymes (not shown). These results suggest that substrate recognition (likely via the Rsp5-WW domains), as well as catalytic activity of Rsp5, is required for efficient substrate ubiquitination in these assays. Monitoring Poly- or Mono-ubiquitination Using a Western Blot Assay—In order to provide further evidence for covalent attachment of ubiquitin to the novel substrates, and to determine the ubiquitin chain topology, we used a Western blot assay. Fig. 2B demonstrates that all the proteins whose ubiquitination was detected in the luminescent assay were visibly ubiquitinated on the Western blot. Most of the proteins were polyubiquitinated or ubiquitinated on multiple lysines, except for Ybr196c, and possibly Ydr404c, which appeared to be either mono- or di-ubiquitinated. Proteins whose ubiquitination was not detected by the luminescent assay did not appear to be ubiquitinated on the Western blot (Fig. 2B). Genetic Interactions between Novel Substrates and Rsp5 in Vivo—To provide genetic evidence for the relevance of the substrates discovered in the biochemical screens, we tested the genetic interaction of strains overexpressing the putative substrates in a strain harboring a temperature-sensitive mutant of Rsp5 (rsp5-1) (27Wang G. Yang J. Huibregtse J.M. Mol. Cell. Biol. 1999; 19: 342-352Crossref PubMed Scopus (147) Google Scholar). 34 wild-type and rsp5-1 strains were transformed with GST fusion protein expression plasmids. All of the transformed rsp5-1 strains grew normally at 30 °C, and none were able to grow at 37 °C (not shown). Colony growth was monitored at the weakly permissive temperature (35 °C) at which overexpression of the Rsp5 substrates would be predicted to be lethal. 11 of the overexpressed proteins caused lethality in the rsp5-1 strains (Fig. 7). 8 of these 11 toxic proteins were from the group representing highly ubiquitinated Rsp5 substrates; 2 were from the moderately ubiquitinated proteins, and 1 was from the weakly ubiquitinated proteins, revealing a positive correlation between the extent of substrate ubiquitination in vitro and toxicity in yeast cells. Rsp5-dependent in Vivo Ubiquitination of a Novel Substrate, Ydl203c—Several of the substrates identified in our in vitro luminescence screen had been described previously as in vivo substrates for Rsp5 (e.g. CTD, Bul1, and Hpr1). To test whether a novel in vitro substrate identified in our screen, Ydl203c (which gave a high luminescence signal similar to the above listed known substrates, see Fig. 5 and Table I) is also an in vivo substrate of Rsp5, we investigated in vivo ubiquitination of Ydl203c in Rsp5 and rsp5-1 mutant strains, and we compared it to a protein that gave a very weak signal in the luminescence assay, GST. We opted to focus on in vivo ubiquitination rather than on protein stability because not all ubiquitinated proteins in the cell are degraded. In addition, ubiquitination of a small fraction of a cellular pool of a protein can be detected, even if its total cellular level may not be altered significantly. We thus monitored the in vivo ubiquitination of GST-Ydl203c or GST alone in rsp5-1 and wild-type yeast cells at the restrictive temperature. These proteins (and any attached ubiquitins) were purified in the presence of proteasome and lysosome inhibitors, and equal amounts were analyzed by SDS-PAGE and Western blotting with anti-ubiquitin antibodies. As seen in Fig. 8, GST-Ydl203c, but not GST alone, was strongly ubiquitinated in the Rsp5 but not rsp5-1 mutant cells, suggesting Ydl203c is an in vivo substrate for Rsp5.Table IEvidence for physiological relevance of novel substrates†The level of ubiquitination as measured by the biochemical assay, see Fig. 5 and supplemental Table S1.‡Polyubiquitination or monoubiquitination as measured by Western blot, see Fig. 6C.ζBased on the inability of the HECT domain alone mutant to ubiquitinate the novel substrates, see Fig. 6 A and B.ξGenetic interaction, see Fig. 7.§Rsp5-substrate interaction detected in large scale proteome-wide screens.*Evidence (from this study and other studies) supporting that the putative substrate is part of Rsp5-dependent pathways (also see Fig. 8). Open table in a new tab Fig. 8In vivo ubiquitination of the Rsp5 substrate and Ydl203c in wild-type and rsp5-1 strains. GST-tagged Ydl203c, or GST alone, were purified from wild-type and rsp5-1 strains in the presence of proteasome and lysosome inhibitors. Equal loading of purified proteins was confirmed using Ponceau S staining (not shown) and anti-GST immunoblotting (top panel). Ubiquitination was monitored using anti-ubiquitin antibodies (bottom panel). To show that the anti-ubiquitin antibody can detect ubiquitin and polyubiquitin chains, purified GST-Ub and ubiquitinated GST-CTD were visualized using the anti-ubiquitin antibody (bottom, right). Ubiquitinated Ydl203c is marked with an asterisk.View Large Image Figure ViewerDownload (PPT) †The level of ubiquitination as measured by the biochemical assay, see Fig. 5 and supplemental Table S1. ‡Polyubiquitination or monoubiquitination as measured by Western blot, see Fig. 6C. ζBased on the inability of the HECT domain alone mutant to ubiquitinate the novel substrates, see Fig. 6 A
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