Global Analysis of Protein Sumoylation in Saccharomyces cerevisiae

Abstract

Although the modification of cellular factors by SUMO is an essential process in Saccharomyces cerevisiae, the identities of the substrates remain largely unknown. Using a mass spectrometry-based approach, we have identified 271 new SUMO targets. These substrates play roles in a diverse set of biological processes and greatly expand the scope of SUMO regulation in eukaryotic cells. Transcription appears to be the most prevalent process associated with sumoylation with novel SUMO substrates found in basal transcription machinery for RNA polymerases I, II, and III, pol II transcriptional elongation complexes, and a variety of chromatin remodeling, chromatin modifying, and chromatin silencing complexes. Additionally, our global analysis has revealed a number of interesting biological patterns in the list of SUMO targets including a clustering of sumoylation targets within macromolecular complexes.Posttranslational modification of proteins is a central mechanism by which biological processes are regulated. One such modification involves the covalent attachment of the small ubiquitin-related polypeptide SUMO 1 (Smt3p in budding yeast) to different cellular substrates (1, 2). SUMO conjugation is carried out by a multistep enzymatic pathway consisting of the heterodimeric SUMO-activating enzyme Aos1p/Uba2p, the SUMO-conjugating enzyme Ubc9p, and several different SUMO ligases including Siz1p and Siz2p in yeast (3-7). The end result of this enzymatic cascade is a covalent isopeptide bond linking the C-terminal glycine of SUMO to the ⑀-amino group of specific lysines in the target protein. Sumoylation of a target protein in this manner can regulate protein function by a number of different mechanisms including altering its subcellular localization, modulating its interaction with other proteins, or by antagonizing its attachment to ubiquitin or other lysine-targeting modifications. Desumoylation of substrates by SUMO isopeptidases, such as Ulp1p and Ulp2p in yeast, is also a central feature in regulation of protein function by . Genetic data in both budding and fission yeast, as well as the variety of known SUMO targets in mammalian cells, point to a broad role for sumoylation and desumoylation in the regulation of many biological processes including transcription, cell cycle progression, DNA damage response, and signal transduction (11,12).It is known that a large number of proteins are sumoylated in Saccharomyces cerevisiae, but very few of these substrates have been identified to date. This is thought to be due to the low abundance of the sumoylated targets and the fact that only a small fraction of a substrate is sumoylated under a given set of conditions. A critical step in the effort to gain a more complete picture of the role of SUMO in eukaryotic biology will be the identification and characterization of a wider range of SUMO targets. To this end, we have performed a global proteomics analysis of sumoylation in budding yeast. EXPERIMENTAL PROCEDURESYeast Strains-EJ337 contains at the chromosomal SMT3 locus a vers...

In vivo modeling of polysumoylation uncovers targeting of Topoisomerase II to the nucleolus via optimal level of SUMO modification

“…Among them are the existence of a nucleolus-resident E3 SUMO ligase Mms21p (Zhao and Blobel 2005), the localization of two mammalian SUMOisopeptidases to the nucleolus (Mukhopadhyay and Dasso 2007), and especially by proteomic data in budding yeast (Wohlschlegel et al 2004;Hannich et al 2005). Indeed, among the 196 nuclear sumoylated proteins that have at least two sumoylated sites in vivo (Wohlschlegel et al 2004), 39 are nucleolar (according to GO), 41 are confirmed to be partially nucleolar, 90 are chromatin components that can potentially have a nucleolar subset, six are SUMO pathway components, and only 20 are uncharacterized or unlikely to be nucleolar. Although the hypothesis of polysumoylation's role in nucleolar targeting needs additional extensive validation, its emergence highlights the usefulness of the improved SUMO fusion approach (CSM), both for practical application to a given SUMO target and for advancement of general knowledge about SUMO biology.…”

“…Elimination of all three E3 activities (Siz1, Siz2, and Mms21) is synthetically lethal (Reindle et al 2006). While recent proteomic studies demonstrated that several hundreds of proteins can be modified in vivo (Panse et al 2004;Wohlschlegel et al 2004;Zhou et al 2004;Denison et al 2005;Hannich et al 2005;Wykoff and O'Shea 2005), direct evidence of biological significance and/or functional importance of these modifications is lacking for most substrates. Two main obstacles prevent the elucidation of the sumoylation's role for a given target protein using straightforward biochemical or genetic methods.…”

SUMO-targeted ubiquitin ligases in genome stability

“…Among these targets might be components of the sumoylation machinery, i.e. E1-3, as they are known to be SUMO modified (Wohlschlegel et al, 2004). This may partially explain the global deregulation of sumoylation in STUbL mutants.…”

Histone sumoylation is a negative regulator in Saccharomyces cerevisiae and shows dynamic interplay with positive-acting histone modifications

“…However, while HDAC sumoylation regulates a number of gene regulatory pathways in mammalian cells, in S. cerevisiae, proteomic analyses of sumoylated proteins indicate that HDACs are not a robust target (Wohlschlegel et al 2004;Zhou et al 2004;Hannich et al 2005), although Hda1 was detected in one SUMO proteomic analysis (Panse et al 2004). Our findings that substitution mutations in histone sumoylation sites increase transcription, and that SUMO fused to histones is repressive to transcription, strengthen the first two models of direct effect of histone sumoylation through either competition with acetylation or recruitment of HDACs (i.e., the first and second proposed mechanisms), rather than an indirect effect through altering HDAC activity (i.e., the third mechanism).…”

“…Recent genomic studies aimed at identifying cellular substrates for sumoylation indicate that many are involved in transcription regulation and include transcription factors, transcription machinery proteins, and components that modify chromatin structure (Manza et al 2004;Wohlschlegel et al 2004;Zhou et al 2004;Hannich et al 2005). In most cases, sumoylation of transcriptional activators results in transcriptional repression (Gill 2003;Hay 2005).…”