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Originally published In Press as doi:10.1074/jbc.M502197200 on June 13, 2005

J. Biol. Chem., Vol. 280, Issue 33, 29470-29478, August 19, 2005
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A High Throughput Screen to Identify Substrates for the Ubiquitin Ligase Rsp5*{boxs}

Bart Kus{ddagger}§, Aaron Gajadhar{ddagger}, Karen Stanger{ddagger}, Rob Cho{ddagger}, Warren Sun{ddagger}, Nathalie Rouleau¶, Tammy Lee{ddagger}, Donovan Chan¶, Cheryl Wolting||, Aled Edwards{ddagger}, Roger Bosse¶, and Daniela Rotin||**

From the {ddagger}Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, PerkinElmer Life Sciences, Biosignal, Montreal, QuebecH3J 1R4, and ||The Hospital for Sick Children and Department of Biochemistry, University of Toronto, Toronto, Ontario M5G 1X8, Canada

Received for publication, February 25, 2005 , and in revised form, June 8, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The ubiquitin pathway is conserved throughout eukaryotic evolution and is implicated in numerous cellular processes (1). Proteins modified by the ubiquitin pathway are processed for degradation, endocytosis, protein sorting, and subnuclear trafficking (2, 3). Ubiquitination is catalyzed by three enzymes termed E11 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin protein ligase). E3 regulates the specificity of the reaction by binding directly to substrates (1, 3). 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 (4, 5). The therapeutic importance of understanding ubiquitination has been underscored recently by the success of anticancer strategies that affect the ubiquitin pathway (6).

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 (7). 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 (8) 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 (9), 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) (10–13). The best characterized role of this E3 family is to ubiquitinate transmembrane proteins and to regulate their endocytosis (9). 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 (2, 14, 15). Rsp5 also regulates the activity of RNA polymerase II in response to DNA damage by ubiquitinating Rpb1, the large subunit of the holoenzyme (16, 17). 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 (10, 18–20). Mutations in these PY motifs cause Liddle syndrome, a genetic form of hypertension (21).

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 (AlphaScreenTM technology) in order to achieve higher sensitivity, greater throughput, and the potential for automation (22, 23). The assay measures the proximity of biotinylated ubiquitin (b-Ub) and GST-tagged substrate proteins (22, 23) (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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Purification of Yeast E2 Enzymes—Yeast E2 genes UBC1 and UBC4 were cloned into pET15b plasmids as described (24). 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-{beta}-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 x 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 2 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 (25) 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 (25). 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 {alpha}-GST antibody (B-14, Santa Cruz Biotechnology) or mouse monoclonal {alpha}-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 µlof5x 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 3x 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 {alpha}-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 {alpha}-GST and {alpha}-ubiquitin (Covance) antibodies.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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).



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  		FIG. 1.
Rsp5 and the AlphaScreen platform. A, schematic diagram of Rsp5 structure. B, substrate GST-tagged fusion proteins are ubiquitinated in an enzymatic process that utilizes b-Ub and Rsp5 as the E3 ubiquitin ligase enzyme, as well as E1, E2, and ATP. Anti-GST acceptor and streptavidin donor beads simultaneously capture ubiquitinated GST fusion proteins. This proximity of acceptor and donor beads induced by the production of ubiquitinated GST-tagged proteins generates the luminescent signal upon irradiation at 680 nm.

 
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).

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 (25). 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) (26) (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) (27). 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.


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  		TABLE I
Evidence for physiological relevance of novel substrates

This table summarizes properties of the in vitro substrates discovered in this study. Yellow color highlights data that are consistent with a physiological interaction of the substrate with Rsp5. GST alone, which is not ubiquitinated by Rsp5, is also shown for comparison.

 



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  		FIG. 2.
Development of the luminescent ubiquitination assay (AlphaScreen). A, the luminescent assay is used to detect the ubiquitination of GST-CTD and the four other proteins that were visibly ubiquitinated on a Western blot (see B). Five proteins that were not ubiquitinated on a Western blot and do not generate significant luminescent signal are also shown. The corresponding Western blots, which show the ubiquitination of the same proteins, is described in B. B, monitoring the ubiquitination of the substrates using anti-GST Western blot assays. All the proteins that produced ubiquitination signal in the luminescent assay appear to be ubiquitinated on the Western blot. All the substrates except for Ybr196c, and to a lesser degree Ydr404c, appear to be polyubiquitinated or ubiquitinated on multiple lysines. Substrates whose ubiquitination was not detected by the luminescent assay do not appear to be ubiquitinated on the Western blot (A). All reactions were reconstituted in the presence or absence of Rsp5. C, titration of assay components and time course. Each panel shows how the luminescent signal produced by CTD and Ybr196c is affected by various parameters of the ubiquitination reaction including substrates (CTD and Ybr196c), E1, E2, Rsp5, b-Ub, ATP, and time. Maximum signal was achieved with 1.5 µM Ybr196c, 2 µM CTD, 0.1 µM E1, 0.25 µM E2, 1 µM Rsp5, 10 µM b-Ub, and 6 µM ATP.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Six lines of evidence support the hypothesis that some or all of the Rsp5 substrates identified in our screen have physiological relevance. (i) In addition to Rpb1, other known substrates of Rsp5 were identified in the screen, including Bul1 and Hpr1. Both of these proteins were among the most strongly ubiquitinated proteins that scored as positive in the screen and have been validated in vivo as Rsp5 substrates. Bul1 interacts with Rsp5 in vivo through its PY motif and modifies its activity (28). Hpr1 is a member of the transcription-export complex and couples mRNA transcription to nuclear export. It was discovered recently that Hpr1 is also a substrate of Rsp5 and is targeted for ubiquitination by this E3 at high temperatures (29). The fact that known substrates of Rsp5 were uncovered in our screen provides strong evidence that our approach is capable of discovering biologically relevant substrates for this E3 enzyme. (ii) Three other potential substrates that we identified (Ydl203c, Ybr196c, and Ypr084w) have been described as Rsp5 substrates in large scale protein-protein interaction screens (30, 31). (iii) A greater proportion of heavily ubiquitinated proteins in our screen contained PY motifs; eight of the nine novel substrates have PY motifs, and proteins containing these motifs produced, on average, higher luminescent signal in the assay. This is consistent with the accepted model of Rsp5 substrate recognition and suggests that this mechanism is retained in vitro. (iv) A mutant of Rsp5 lacking its protein-protein interaction (WW) domains failed to ubiquitinate at least six of the new substrates. (v) Seven of the new substrates interacted genetically with Rsp5. (vi) In vivo ubiquitination assays suggest that a highly ubiquitinated novel substrate in the luminescence assay, Ydl203c, is also ubiquitinated in vivo. Collectively, these data suggest that the application of this screen on a larger scale could identify many new Rsp5 as well as other E3 ligase substrates. Evidence that the nine in vitro substrates may be physiological substrates of Rsp5 is summarized in Table I.



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  		FIG. 3.
Detection of protein ubiquitination using Western blot and the luminescent assay. GST-tagged fusion proteins were incubated at increasing concentrations in standard ubiquitination assay conditions in the presence or absence of ATP. Ubiquitination of Ybr196c (A and E), GST-CTD (B and F), Ycl040w (C and G), and Yer177w (D and H) was monitored using Western blots and the luminescent assay. The sensitivity of the luminescent assay was at least 270 times greater than the Western blot because in order to optimally detect the ubiquitination using AlphaScreen technology, the completed reactions were diluted 270-fold (see "Experimental Procedures").

 
Genetic Evidence of Substrate-Rsp5 Interactions—The genetic experiments in this study provide evidence for the physiological interaction of some of the substrates with Rsp5. Two models can explain why specific overexpressed proteins cause lethality in the temperature-sensitive rsp5-1 strain. One possibility is that at 35 °C, the overexpressed proteins, which are normally degraded by Rsp5, are toxic to the cell because they are stabilized as a result of low Rsp5 activity. We have shown experimentally, however, that stability itself does not account for the toxicity of at least three proteins and that these proteins appear to be stable when overexpressed in both rsp5-1 and wild-type cells at restrictive temperatures (data not shown). Perhaps a more plausible explanation is that overexpressed proteins with affinity for Rsp5 act as a "molecular sink" and sequester active Rsp5 away from its essential functions of ubiquitinating cellular substrates. Although cells can survive this added stress at permissive temperatures, at 35 °C, Rsp5 activity is already compromised and results in cell death.

One interesting candidate protein, Rpb7 (Ydr404c), interacted with Rsp5 in both genetic and biochemical assays and merits further attention. Both Rpb1, which is ubiquitinated and degraded in the presence of DNA damage (16, 17),3 and Rpb7 are members of the RNA polymerase II complex. The crystal structure of RNA polymerase II predicts that Rpb1 is adjacent to Rpb7 and that the PY motif of Rpb7 is exposed (32). Recent evidence suggests that in humans both Rpb1 and Rpb7 subunits are targeted for ubiquitination by a single E3 ligase, the VHL complex (33, 34). Thus, it is possible that in yeast, both subunits are also ubiquitinated by a single E3 ligase, Rsp5. Although Rpb7 is stable in the presence of DNA damage,3 Rpb7 may be ubiquitinated under other conditions, and Rpb7 may play a role in Rpb1 ubiquitination because it mediates DNA damage responses (35). Finally, the interaction between Rpb7 and Rsp5 was predicted in data base searches for proteins containing optimal Rsp5-binding sequences (36).



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  		FIG. 4.
Assay 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.

 



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  		FIG. 5.
Biochemical (luminescent) screen for Rsp5 substrates. Each bar in the histogram represents a unique protein that was purified, ubiquitinated, and monitored in the biochemical screen. The height of the bar represents the level of luminescent signal generated in the reaction (all absolute values were normalized to the signal produced by one of the novel substrates, Ybr196c). The average (normalized) signal generated by PY motif and non-PY motif-containing proteins was 0.30 and 0.17, respectively. Proteins containing a PY motif are shown as shaded bars, and bars representing Bul1, CTD, Ydl203c, and GST alone are marked with an arrow.

 
In Vivo Evidence for Ubiquitination of Substrates by Rsp5— The physiological roles of most substrates discovered in this study are not well characterized and have yet to be placed in Rsp5-dependent pathways in the cell. Upon the completion of the in vitro luminescent screen, however, we realized that it would be difficult to validate all the in vitro substrates using simple in vivo ubiquitination (or stability) assays because: (i) ubiquitination and degradation of many Rsp5 substrates likely require specific environmental cues (14), such as DNA damage, temperature changes, nutrient availability, or other stresses. For example, two known in vivo substrates of Rsp5 that were also identified in our screen, Rpb1 and Hpr1, require specific environmental cues to be ubiquitinated in vivo. (ii) In vivo, subcellular localization of Rsp5 may prevent it from accessing its substrates (until the appropriate environmental signal is provided). (iii) Cellular inhibitors absent in vitro may negatively regulate Rsp5-substrate interactions in vivo. (iv) Our in vitro luminescent assay is much more sensitive than the in vivo ubiquitination assay, rendering the latter more difficult to detect. Nevertheless, we were able to provide evidence for physiological interactions between some of the novel substrates and Rsp5, including data suggesting genetic interactions, as well as Rsp5-dependent in vivo ubiquitination of one of the novel substrates Ydl203c.



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  		FIG. 6.
Validation of biochemical screen. A, six of the substrates generating high luminescent signal in the screen were titrated in increasing concentrations into reactions containing either full-length Rsp5 or just the HECT domain itself (HECT domain alone). Absolute luminescent values were normalized to the signal produced by Ybr196c (when incubated at 0.53 µM). B, the same six substrates were ubiquitinated in standard conditions in the presence/absence of Rsp5, catalytically inactive Rsp5 C777A, or the HECT domain alone.

 



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  		FIG. 7.
Genetic validation of the biochemical screen. Wild type (WT) and rsp5-1 mutant strains transformed with plasmids coding for GST fusion proteins were grown at 35 °C on glucose (to repress overexpression of proteins) and galactose (to induce protein overexpression). rsp5-1 strains unable to form colonies when grown with galactose are marked with a box. The strains are placed into one of three groups based on whether they produced high, moderate, or weak signal in the biochemical (luminescent) screen.

 
Ydl203c, a Novel Rsp5 Substrate—Ydl203c is an interesting candidate for further study as a potential physiological substrate of Rsp5. Evidence for its role in Rsp5 pathways includes the following: (i) the presence of a PY motif in the N-terminal portion of the protein; (ii) failure of the HECT domain alone construct to ubiquitinate Ydl203c; (iii) a genetic interaction between Rsp5 and Ydl203c in the overexpression screen; (iv) Rsp5-dependent ubiquitination of Ydl203c in vivo (Fig. 8); and finally (vi) the identification of Ydl203c as a substrate of Rsp5 in a large scale yeast two-hybrid screen (30).

Ydl203c is an otherwise uncharacterized protein with seven C-terminal Sel1 domains downstream of its PY motif. This domain is repeated in the C terminus of a number of other proteins, including Hdr1, which is a yeast ubiquitin ligase, and Sel1, a Caenorhabditis elegans protein whose role is to negatively regulate Notch. A human protein, Sel1L, also contains C-terminal Sel1 repeats that are necessary for its anti-tumorigenic activity (37). The precise function of this domain has not been elucidated, but a possible role in mediating protein-protein interactions has been hypothesized (37).



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  		FIG. 8.
In 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.

 
In summary, we developed and used a luminescent platform to test 188 purified GST-tagged yeast proteins as potential substrates of Rsp5, and we identified 7 new proteins that were specifically ubiquitinated and that showed a genetic interaction with Rsp5. As might have been predicted, a high proportion of the heavily ubiquitinated substrates contained PY motifs, in keeping with the known specificity of Rsp5. More importantly, this assay may also allow for the identification of novel Rsp5-interacting sequences in substrates lacking PY motifs. Taken together, the highly sensitive luminescent assay we developed to detect and quantify ubiquitination will serve a means to identify novel E3 substrates and perhaps as a platform to screen for inhibitors of E3-dependent ubiquitination.


   FOOTNOTES
 
* This work was supported in part by the National Cancer Institute of Canada with funds from the Canadian Cancer Society (to A. E. and D. R.), the Canadian Institute for Health Research (to D. R.), and the Canadian Foundation for Innovation (to D. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains Table S1. Back

§ Supported by Ontario Graduate Scholarship. Back

** To whom correspondence should be addressed: Program in Cell Biology, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-5098; Fax: 416-813-5771; E-mail: drotin{at}sickkids.ca.

1 The 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. Back

2 N. Fong, unpublished data. Back

3 B. Kus, A. Gajadhar, K. Stanger, R. Cho, W. Sun, N. Rouleau, T. Lee, D. Chan, C. Wolting, A. Edwards, R. Bosse, and D. Rotin, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Michael Snyder for supplying the yeast overexpression library and Drs. Linda Hicke, Rosine Haguenauer-Tsapis, and Dale Haines for providing vectors and strains. We thank Richelle Sopko and Dr. Susan McCracken for helpful discussions and technical help.



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 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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