A High Throughput Screen to Identify Substrates for the Ubiquitin Ligase Rsp5

Abstract

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 (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 E1 1 (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 ligase...

Dissecting the ubiquitin pathway by mass spectrometry

“…It is also shown to use liquid chromatography coupled with multiple reaction monitoring (MRM) to quantify K48 and K63 linkages. Table 1 Comparison of multiple analyses of ubiquitinated proteins by mass spectrometry Yeast Peng et al [9] Hitchcock et al [31] Mayor et al [63] Tagwerker et al [25] Ub tags (His) 6 …”

“…Indeed, a similar analysis was used to identify many substrates of the BRCA1-BARD1 Ub ligase [30]. Alternatively, a high throughput screen was developed to screen for substrates of specific ligases using recombinant proteins in reconstituted ubiquitination reactions [31].…”

Biochemical and genetic analysis of the yeast proteome with a movable ORF collection

“…High-throughput genomic analyses have provided unprecedented views of gene function and of networks of interactions connecting genes (Uetz et al 2000;Gavin et al 2002;Giaever et al 2002;Ho et al 2002;Tong et al 2004). The study of entire genomes has also highlighted some of the complexities involved in gene and protein characterization, such as the difficulties in identification of bona fide genes from genomic sequence (Basrai et al 1997;Blandin et al 2000;Kumar et al 2002;Oshiro et al 2002;Cliften et al 2003;Kellis et al 2003;Kessler et al 2003) and the need to examine proteins for processing and multiple post-translational modifications (Zhu et al 2000;Huang et al 2004;Kus et al 2005). …”

Genetic and Expression Aberrations of E3 Ubiquitin Ligases in Human Breast Cancer

“…Interestingly, the ubiquitination of p27 KIP1 by SCF skp2 requires an accessory protein Cks1 (89). Recent structural studies show that Cks1 binds to the phosphorylated Thr 187 side chain of p27 KIP1 (90). Expression of Cks1 is associated with decreased tumor differentiation and poor disease-free and overall survival outcome in breast cancer (91).…”

“…Several strategies, including bioinformatics, in vitro expressing cloning, fusion protein, RNA interference, and arrays of synthetic phosphopeptides, certainly facilitate the identification of E3 substrates (184)(185)(186). A high-throughput screening to identify substrates for the yeast ubiquitin ligase Rsp5 has been reported recently (187). Because the E3s interact with their substrate proteins, maps of the interactome network generated from yeast (188), Drosophila (189), Caenorhabditis elegans (190), and human proteome (191,192) by high-throughput yeast two-hybrid assays and newly developed mass spectrometric analysis technology will also facilitate the identification of the specific substrates for E3s.…”