Ubiquitination screen using protein microarrays for comprehensive identification of Rsp5 substrates in yeast
2007; Springer Nature; Volume: 3; Issue: 1 Linguagem: Inglês
10.1038/msb4100159
ISSN1744-4292
AutoresRonish Gupta, Bart Kus, Christopher Fladd, James D. Wasmuth, Raffi Tonikian, Sachdev S. Sidhu, Nevan J. Krogan, John Parkinson, Daniela Rotin,
Tópico(s)Advanced Proteomics Techniques and Applications
ResumoArticle5 June 2007Open Access Ubiquitination screen using protein microarrays for comprehensive identification of Rsp5 substrates in yeast Ronish Gupta Ronish Gupta Program in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Bart Kus Bart Kus Program in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Christopher Fladd Christopher Fladd Program in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author James Wasmuth James Wasmuth Program in Molecular Structure and Function, The Hospital for Sick Children, Toronto, Ontario, Canada Search for more papers by this author Raffi Tonikian Raffi Tonikian Banting & Best Department of Medical Research, University of Toronto, Canada Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Sachdev Sidhu Sachdev Sidhu Department of Protein Engineering, Genentech, South San Francisco, CA, USA Search for more papers by this author Nevan J Krogan Nevan J Krogan Department of Cellular and Molecular Pharmacology, University of California-San Francisco, San Francisco, CA, USA Search for more papers by this author John Parkinson John Parkinson Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada Program in Molecular Structure and Function, The Hospital for Sick Children, Toronto, Ontario, Canada Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Daniela Rotin Corresponding Author Daniela Rotin Program in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Ronish Gupta Ronish Gupta Program in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Bart Kus Bart Kus Program in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Christopher Fladd Christopher Fladd Program in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author James Wasmuth James Wasmuth Program in Molecular Structure and Function, The Hospital for Sick Children, Toronto, Ontario, Canada Search for more papers by this author Raffi Tonikian Raffi Tonikian Banting & Best Department of Medical Research, University of Toronto, Canada Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Sachdev Sidhu Sachdev Sidhu Department of Protein Engineering, Genentech, South San Francisco, CA, USA Search for more papers by this author Nevan J Krogan Nevan J Krogan Department of Cellular and Molecular Pharmacology, University of California-San Francisco, San Francisco, CA, USA Search for more papers by this author John Parkinson John Parkinson Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada Program in Molecular Structure and Function, The Hospital for Sick Children, Toronto, Ontario, Canada Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Daniela Rotin Corresponding Author Daniela Rotin Program in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Author Information Ronish Gupta1,2,‡, Bart Kus1,2,‡, Christopher Fladd1,2, James Wasmuth3, Raffi Tonikian4,5, Sachdev Sidhu6, Nevan J Krogan7, John Parkinson2,3,5 and Daniela Rotin 1,2 1Program in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada 2Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada 3Program in Molecular Structure and Function, The Hospital for Sick Children, Toronto, Ontario, Canada 4Banting & Best Department of Medical Research, University of Toronto, Canada 5Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada 6Department of Protein Engineering, Genentech, South San Francisco, CA, USA 7Department of Cellular and Molecular Pharmacology, University of California-San Francisco, San Francisco, CA, USA ‡These authors contributed equally to this work *Corresponding author. Program in Cell Biology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. Tel.: +1 416-813-5098; Fax: +1 416-813-8456; E-mail: [email protected] Molecular Systems Biology (2007)3:116https://doi.org/10.1038/msb4100159 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Ubiquitin-protein ligases (E3s) are responsible for target recognition and regulate stability, localization or function of their substrates. However, the substrates of most E3 enzymes remain unknown. Here, we describe the development of a novel proteomic in vitro ubiquitination screen using a protein microarray platform that can be utilized for the discovery of substrates for E3 ligases on a global scale. Using the yeast E3 Rsp5 as a test system to identify its substrates on a yeast protein microarray that covers most of the yeast (Saccharomyces cerevisiae) proteome, we identified numerous known and novel ubiquitinated substrates of this E3 ligase. Our enzymatic approach was complemented by a parallel protein microarray protein interaction study. Examination of the substrates identified in the analysis combined with phage display screening allowed exploration of binding mechanisms and substrate specificity of Rsp5. The development of a platform for global discovery of E3 substrates is invaluable for understanding the cellular pathways in which they participate, and could be utilized for the identification of drug targets. Synopsis Post-translational modification of proteins by the ubiquitin pathway has been implicated in numerous cellular processes. Substrates of this pathway are covalently modified by the attachment of a small protein called ubiquitin and as a result are targeted for degradation, endocytosis, protein sorting or subnuclear trafficking. An enzyme called E3, or ubiquitin ligase, is responsible for the specificity of the reaction, and associates with specific substrates in order to ensure their ubiquitination. Defects in the ability of the E3 to interact with substrates have been implicated in numerous diseases, including neurodegeneration, immunological disorders, hypertension and cancers. A significant fraction of the proteome is regulated by the ubiquitin pathway and eukaryotic genomes express hundreds of E3 enzymes to coordinate the ubiquitination of cellular proteins. Currently, most E3 enzymes have not been linked to any specific substrates despite advances in understanding the mechanics of the ubiquitin system and its role in the cell. We have developed a platform that allows systematic and high-throughput discovery of ubiquitinated E3 substrates. We expect that this application will be tremendously useful for gaining insights into cellular systems and will likely be exploited by the biomedical industry. In order to develop this platform, we used Rsp5, a yeast E3, as a model system. E3 enzymes from this family have been implicated in numerous cellular functions including protein degradation, endocytosis, sorting and trafficking. For example, Rsp5 regulates mitochondrial inheritance, drug resistance, intracellular pH, fatty acid biosynthesis, protein sorting at the trans-Golgi network and transcriptional regulation. Nedd4 (or Nedd4-2), the human Rsp5 homologue, prevents hypertension by ubiquitinating and regulating endocytosis of ENaC in the kidney. Our aim was to identify substrates of Rsp5 in the yeast proteome using the protein microrarray technology as our experimental platform. The arrays used in this study contain purified proteins immobilized at a high spatial density on standard sized slides and contain the majority of yeast proteins (Figure 1). Therefore, the yeast proteome can be readily exploited using traditional biochemical approaches. Recent studies have employed protein microarrays containing full-length proteins to discover calmodulin interacting proteins and to probe for a variety of other protein–protein interactions, as well as antibody–antigen, protein–small molecule, protein–lipid and protein–nucleotide associations. Protein microarrays are expected to provide excellent platforms for identifying post-translational modifications, but to date, only a few studies have assayed an enzymatic activity using this technology. In the current study, we have successfully used yeast (S. cerevisae) protein microarrays, covering most of the yeast proteome, to assay the enzymatic (ubiquitination) activity and binding ability of Rsp5 to substrate proteins, and we have identified previously reported and novel ubiquitinated substrates and interacting partners of this E3 ligase. The data generated in this study were integrated with published data from large-scale physical and genetic interaction studies to generate Rsp5 interaction networks. Our results demonstrate the feasibility of identifying substrates of E3 ligases and possibly other enzymes using a proteome microarray approach, and demonstrate how this approach can yield informative data regarding the binding mechanisms and substrate specificity of an E3 ligase enzyme. Introduction Substrates of the ubiquitin pathway are covalently modified by the attachment of a small protein called ubiquitin and as a result are targeted for degradation or other cellular fates (Pickart, 2001; Glickman and Ciechanover, 2002; Hicke and Dunn, 2003). Ubiquitination involves the sequential action of three enzymes: E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin-protein ligase) (Pickart, 2001). The E3 enzyme, which is responsible for the specificity of the reaction, associates with substrates (Hershko and Ciechanover, 1998; Pickart, 2001; Fang and Weissman, 2004), and defects in this interaction have been implicated in numerous diseases (Abriel et al, 1999; Dawson and Dawson, 2003; Liu, 2004; Nakayama and Nakayama, 2006). A significant fraction of the proteome is regulated by the ubiquitin pathway and eukaryotic genomes express hundreds of E3 ligases to coordinate the ubiquitination of cellular proteins (Peng et al, 2003; Willems et al, 2004). Currently, most E3 enzymes have not been linked to any specific substrate and any platform that would allow for the systematic discovery of enzymatic E3 substrates would be tremendously useful for advancing our understanding of the ubiquitin pathway. Rsp5 is a yeast E3 ubiquitin ligase that belongs to the Nedd4 family (Rotin et al, 2000). It contains a C2 domain, a catalytic HECT domain and three WW domains that can bind substrates directly by recognizing a (L/P)PxY sequence (PY motif) (Kanelis et al, 2001, 2006; Kasanov et al, 2001; Hu et al, 2004; Shcherbik et al, 2004). Ubiquitination of proteins by the Nedd4 E3 family has been implicated in numerous cellular functions, including endocytosis, sorting and trafficking (Rotin et al, 2000; Horak, 2003; Ingham et al, 2004). For example, Nedd4 (or Nedd4-2), the human Rsp5 homologue, ubiquitinates the epithelial sodium channel (ENaC) to regulate its endocytosis, and mutations that inhibit the Nedd4-2:ENaC interaction cause Liddle syndrome, a hereditary hypertension (Staub et al, 1996; Abriel et al, 1999; Lifton et al, 2001). Similarly, Rsp5 was demonstrated to regulate endocytosis and sorting of several yeast plasma membrane proteins (Horak, 2003; Dupre et al, 2004). Moreover, Rsp5 has been implicated in the regulation of several other cellular functions, including mitochondrial inheritance, drug resistance, intracellular pH, fatty acid biosynthesis and transcriptional control (see below). Despite the biological importance of the Nedd4/Rsp5 family of E3 ligases, only a few substrates have been identified to date for this ubiquitin ligase family. Thus, our goal was to globally identify Rsp5 substrates in the yeast proteome. For that, we chose to use protein microarray technology as our experimental platform. The arrays used in this study contain thousands of purified proteins (most of the Saccharomyces cerevisiae proteome) immobilized at a high spatial density on standard sized slides and can be readily used to probe the yeast proteome using traditional biochemical approaches (Zhu et al, 2001; Schweitzer et al, 2003; Zhu and Snyder, 2003; Bertone and Snyder, 2005; Ptacek et al, 2005; Smith et al, 2005). To date, few studies have assayed enzymatic activities using this technology. In the current study, we have successfully used yeast (S. cerevisiae) protein microarrays to assay the enzymatic (ubiquitination) activity and binding of Rsp5 to its substrates, and we have identified previously reported and novel ubiquitinated substrates and interacting partners of this E3 ligase. Our results also demonstrate how this approach can yield informative data regarding the binding mechanisms and substrate specificity of an E3 enzyme. Results Identification of proteins ubiquitinated by Rsp5 on a proteome array For this study, ubiquitinated Rsp5 substrates were identified using commercially available yeast protein microarrays (Invitrogen ProtoArray® Yeast Proteome Microarray). These protein microarrays are based on technology described previously (Zhu et al, 2001) and contain more than 4000 GST- and 6 × HIS-tagged yeast proteins from S. cerevisiae spotted in duplicate on nitrocellulose slides (ProtoArray® Yeast Proteome Microarray nc v1.1). Before assaying for ubiquitinated proteins on the protein microarray, we developed conditions in which Rsp5 could ubiquitinate one of its known substrates, the C-terminal domain of Rpb1 (CTD) (Beaudenon et al, 1999). The ubiquitination of CTD was dependent on the budding yeast E1 enzyme, an E2 enzyme (Ubc4), ubiquitin, Rsp5 and ATP, and was visualized by Western blotting. This control reaction was used to optimize conditions for Rsp5-dependent ubiquitination on nitrocellulose-coated glass slides. In these experiments, the CTD and other proteins were robotically spotted onto slides and incubated with a reaction mixture containing Rsp5 and FITC-labeled ubiquitin. The proteome array was then assayed for Rsp5-dependent ubiquitination using the optimized conditions (Figure 1A). Figure 1.Rsp5-mediated ubiquitination of the yeast proteome. (A) Assay development. To optimize ubiquitination conditions using protein microarrays, known substrates of Rsp5 (CTD and Ydl203c) and proteins not ubiquitinated by Rsp5 in vitro (Yer036c and GST alone) were robotically printed on slides and incubated in ubiquitination reactions containing Rsp5 and FITC-labeled ubiquitin. The fluorescent signal demonstrates CTD and Ydl203c ubiquitination in the presence of ATP (right panel), while negative control proteins are not ubiquitinated (left panel). Blue color represents ubiquitination (detected with FITC-Ub). GFP was used as a positive control, as it has the same excitation wavelength as FITC. The colors associated with the protein microarray spots indicate of the intensity of the signal (with light blue<bright blue<white). (B) Image of a scanned ubiquitinated protein microarray with an enlargement of one grid. All proteins are printed in duplicate and arrows indicate proteins that were identified as substrates after quantitative data analysis. Alexa dyes are spotted as controls in the left-hand corners of each grid. (C) Reproducibility. Two protein microarrays were ubiquitinated in separate experiments and the same grid from each array is shown. Arrows point to ubiquitinated proteins that were identified as substrates. Most spots producing significantly higher signal than background can be seen on both arrays, suggesting high reproducibility between slides. Download figure Download PowerPoint Following the reaction, protein microarray slides were washed, scanned and proteins modified by ubiquitin were identified by quantifying the intensity of the FITC signal produced compared with the background (Figure 1B). Although detection of protein–protein interactions on microarrays is generally highly reproducible (Zhu et al, 2001; Hesselberth et al, 2006) (Figure 1A and B), we repeated the Rsp5 ubiquitination reaction on two separate protein microarray slides to increase the quality of our data set. Based on selection criteria for identifying positive hits described in Materials and methods, we generated a data set of 150 Rsp5 substrates (henceforth referred to as the 'relaxed Rsp5 substrate set'). From this set of substrates, we selected a 'high-confidence' data set, which comprises the 40 proteins that produce the strongest signal. These proteins were considered for further study (Table I and Supplementary Table SI). Table 1. High-confidence Rsp5 substrate (unbiquitination) data set High-confidence Rsp5 substrate data set. The top 40 proteins and their PY motifs identified as Rsp5 substrates using the protein microarray are listed. A blue color in the column labeled 'Western' indicates proteins that were ubiquitinated in a Western blot. Proteins identified by protein microarray as interacting partners of Rsp5 in this study are shaded in blue in the column labeled 'Binding'. Boxes shaded in blue in the column labeled 'Hesselberth' indicate that the protein was identified in the microarray screen by Hesselberth and co-workers as an Rsp5 binding partner. The column labeled 'Known substrates' contains proteins that were previously described as Rsp5 substrates. Properties of the high-confidence Rsp5 substrate set PY motifs Since Rsp5-WW domains are known to bind PY motifs, we looked for the presence of these motifs in the sequences of proteins belonging to the Rsp5 substrate sets. As expected, proteins containing PY motifs were significantly enriched in the Rsp5 high-confidence substrate set (P<0.01—exact randomization test). In the yeast proteome, approximately 4% of proteins contain PPxY motifs, and 7% contain LPxY motifs. In the Rsp5 high-confidence substrate set, 72% of proteins had at least one of these motifs. Proteins with PPxY and LPxY motifs were significantly enriched both in the high-confidence and relaxed Rsp5 substrate data sets (P<0.01 for both—exact randomization test). Identification of known substrates Rsp5 has been implicated in a wide range of cellular pathways and a number of its substrates have previously been described. Eleven proteins in the high-confidence Rsp5 substrate set, and 17 proteins in the relaxed substrate set, have previously been identified in Rsp5 pathways through genetic and biochemical methods (Table I). The relatively large number of previously described Rsp5 substrates identified in this study suggests that the proteome microarray experimental approach is capable of discovering proteins ubiquitinated by Rsp5 in vivo and that many of the proteins in the high-confidence substrate set and the relaxed substrate set are likely novel biologically relevant substrates of Rsp5. Detection of substrate ubiquitination using Western blotting We used an established ubiquitination assay to confirm that the proteins identified as Rsp5 substrates on the protein microarray are modified by this E3. Traditional approaches for monitoring ubiquitination involve subjecting specific purified proteins to ubiquitination by an E3 in vitro and using a Western blot approach to visualize ubiquitination. Fifteen proteins from the Rsp5 high-confidence substrate set, and six proteins that were not identified as substrates of Rsp5, were purified from yeast using glutathione affinity purification, incubated in ubiquitination reactions containing Rsp5 and the above described E1 and E2 (Ubc4), and assayed for ubiquitination using anti-ubiquitin antibodies and Western blots. All of the proteins whose ubiquitination was detected on the protein microarray were verified to be ubiquitinated by Western blot analysis (Figure 2A; Table I). Most of the proteins were efficiently polyubiquitinated or ubiquitinated on multiple lysines. In contrast, the six proteins tested whose ubiquitination was not detected on the protein microarray did not appear to be ubiquitinated after Western blot analysis (Figure 2B), confirming that the enzymatic activity detected is specific and that the data generated by the protein microarray approach are consistent with established methods of detecting ubiquitination. Figure 2.Validation of substrate ubiquitination in vitro and in vivo. (A, B) In vitro ubiquitination: (A) 15 proteins identified as 'high-confidence' Rsp5 substrates using protein microarrays were expressed (fused to GST) in yeast, purified and incubated in ubiquitination reactions containing Rsp5. (B) Six randomly selected proteins that were not identified as Rsp5 substrates in the protein microarray experiments were used as negative controls. All 15 of the 'high-confidence' Rsp5 substrates and none of the negative control proteins were visibly ubiquitinated in the Western blots with anti-GST antibodies (arrows indicate the original size of the protein in the absence of ubiquitination (i.e. without ATP)). (C) In vivo ubiquitination: example of three Rsp5 substrates from the protein microarray exhibiting ubiquitination in vivo. The three proteins (HA tagged) were expressed in RSP5 (WT) or rsp5-1 mutant yeast cells. Following a shift to the non-permissive temperature (37°C), proteins were immunoprecipitated with anti-HA antibodies and immunoblotted with anti-ubiquitin antibodies. Note ubiquitination in the RSP5-WT but not the rsp5-1 cells. Download figure Download PowerPoint To further validate our in vitro data, we tested for in vivo ubiquitination of several putative substrates (known or suspected to be involved in sorting/endocytosis), by comparing ubiquitination of these proteins expressed in RSP5 (WT) or rsp5-1 mutant yeast cells. rsp5-1 is a temperature-sensitive mutant that reduces Rsp5 expression upon temperature shift to 37°C (an rsp5-null mutant is lethal). As shown in Figure 2C, Lsb1 and Sna3 (both known interactors or substrates of Rsp5; Ho et al, 2002; McNatt et al, 2007; Oestreich et al, 2007), as well as Sna4, were ubiquitinated in vivo by Rsp5. Although the function of Sna4 is unknown, it is a vacuolar resident protein, much like Sna3, and we thus anticipate that it too utilizes interactions with Rsp5 for vacuolar targeting. Our preliminary data also revealed in vivo ubiquitination of other substrates by Rsp5 (e.g. Yip5, Rcr1 and Rcr2—data not shown). Identification of Rsp5 interacting proteins To directly test Rsp5 substrate binding using the protein microarrays, and to compare these data to the ubiquitination data sets above, we screened the protein microarrays for proteins that bind Rsp5. Purified Rsp5 was labeled with Alexa 647 and incubated with the protein microarray in two separate experiments. After washing and scanning the slides, the data were analyzed and a data set of 155 Rsp5 binding proteins was generated (Table II and Supplementary Table SII). Table 2. High-confidence Rsp5 interaction data set High-confidence Rsp5 interaction data set. The top 40 proteins and their PY motifs identified as Rsp5 interacting partners using the protein microarray are listed. The columns are the same as in Table I, except the column labeled 'Ubiquitin'd' contains proteins that were identified in both ubiquitination and binding protein microarray assays. A sequence search revealed that the Rsp5 binding set was significantly enriched for proteins containing PY motifs (P<0.01—exact randomization test). Ten proteins in the Rsp5 binding set have previously been identified in Rsp5 pathways. Comparison between the Rsp5 substrate set and binding set Twelve proteins in the high-confidence Rsp5 substrate set and 52 proteins in the relaxed Rsp5 substrate set were also present in the Rsp5 interaction set. Conversely, 34% of the proteins in the Rsp5 interaction set were ubiquitinated by Rsp5. Eleven of the 12 proteins that both bound to and were ubiquitinated by Rsp5 contain PY motifs. Pro, Ser and Ala residues are enriched at the 'x' position of (L/P)PxY motifs We examined the amino-acid sequences of Rsp5 substrates to determine whether additional amino-acids residues in the PY motif ((L/P)PxY) may contribute to substrate specificity. A total of 38 PY motifs were present in 29 proteins of the high-confidence subset. Considering the third (x) position in the motif, the most frequent motifs were PPSY (ten), PPAY (five) and PPPY (five). Comparisons with sets of randomly selected proteins containing PY motifs showed that Ser and Ala (but not Pro) were both significantly overrepresented at the third position within our experimentally determined Rsp5 substrates (P<0.001; randomized exact test) (Figure 3). Figure 3.Sequence logos for substrates of Rsp5. PPxY motifs, together with six residues upstream and downstream of the motif from proteins identified as substrates of Rsp5, were aligned and used to generate sequence logos (Crooks et al, 2004). In each logo, stacks of letters indicate the relative frequency of certain amino acids at each position in the sequence. The overall height provides a guide to the level of sequence conservation associated at that position. Download figure Download PowerPoint To further confirm these findings, we performed a modified phage display screen to explore substrate specificity of each of the three WW domains of Rsp5. All peptides identified through this screen (over 300) were found to contain a PY motif. Consistent with data presented above, Ser and Ala were both found to be preferred at the third position (Figure 4). More interestingly, the most common amino-acid residue associated with the third position was Pro, suggesting an important biological role for this residue. Figure 4.Phage display logos. Peptides identified as substrates of Rsp5, using the phage display system, were aligned and are graphically displayed as logos, as shown in Figure 3 above. Download figure Download PowerPoint Discussion In this report, we demonstrate that protein microarrays can be used to identify, on a global scale, ubiquitinated substrates and binding partners of a yeast E3 ubiquitin ligase, Rsp5. A combination of techniques was used to validate the protein microarray data and contributes to our understanding of Rsp5 substrate interaction mechanisms. The high-confidence Rsp5 substrate set contains 12 proteins previously reported in Rsp5 pathways. Six of these (Ygr068c, Aly2, Lsb1, Ylr392c, Dia1 and Rim4) were reported in other HTP screens (Ito et al, 2001; Ho et al, 2002; Kus et al, 2005; Krogan et al, 2006), while the remaining six (Rod1, Rog3, Rvs167, Bul1, Sna3 and Ack1) were validated as substrates using a combination of genetic and biochemical approaches (Yashiroda et al, 1996; Andoh et al, 2002; Stamenova et al, 2004; Kus et al, 2005). Most of the high-confidence Rsp5 substrates contained at least one PY motif, usually PPxY (Table I). However, a few substrates did not (e.g. Sgt1, Cue5, Sip5). Sgt1, Cue1 and Sip5 are known to be involved in the ubiquitin pathway. The precise role of Sgt1 is not clear, but the association of this protein with Rsp5 is interesting, since it has been implicated as an activator of SCF E3 enzymes (Kitagawa et al, 1999; Spiechowicz and Filipek, 2005). Cue1 has a ubiquitin binding motif and its affinity for ubiquitin may facilitate its monoubiquitination (Kang et al, 2003). Alternatively, it is possible that it might have bound FITC-ubiquitin non-covalently; however, this is unlikely because its ubiquitination was also detected on a Western blot. Sip5 may not be an Rsp5 substrate, since it has a RING/U-box domain and likely produced a positive signal in the screen because it used the ubiquitin machinery present during the reaction for autoubiquitination. In addition to the known Rsp5 substrates described above, the relaxed Rsp5 substrate set contains six other proteins that were previously identified as Rsp5 substrates or implicated in Rsp5 pathways. These include, Rpb7 (Kus et al, 2005), Tef2 (Kwapisz et al, 2005), Ubi4, Uba1 (Huibregtse et al, 1995), Rpl40B (Kabir et al, 2005) and Rpl40A (Kabir et al, 2005; Kwapisz et al, 2005). The identification of 18 proteins known to participate in Rsp5 pathways or to be ubiquitinated directly by Rsp5 suggests that the protein microarray experimental approach is a valid tool for the discovery of ubiquitinated E3 substrates, and that this approach is capable of discovering physiological substrates of Rsp5. Not all known Rsp5 substrates were identified in our screen. First, some of the known substrates were not printed on the array (e.g. Mga2, Rpb1, Hpr1, Bsd2 and Pma1). Second, our approach is likely to have missed Rsp5 substrates that do not bind Rsp5 directly or require cofactors for their intera
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