Defining the SUMO-modified Proteome by Multiple Approaches in Saccharomyces cerevisiae* (original) (raw)
Related papers
A Proteomic Strategy for Gaining Insights into Protein Sumoylation in Yeast
Molecular & Cellular Proteomics, 2005
Sumoylation represents a vital post-translational modification that pervades numerous aspects of cell biology, including protein targeting, transcriptional regulation, signal transduction, and cell division. However, despite its broad reaching effects, most biological outcomes of protein sumoylation remain poorly understood. In an effort to provide further insight into this complex process, a proteomics approach was undertaken to identify the targets of sumoylation en mass. Specifically, SUMO-conjugated proteins were isolated by a double-affinity purification procedure from a Saccharomyces cerevisiae strain engineered to express tagged SUMO. The components of the isolated protein mixture were then identified by subsequent LC-MS/MS analysis using an LTQ FT mass spectrometer. In this manner, 159 candidate sumoylated proteins were identified by two or more peptides. Furthermore, the high accuracy of the instrument, combined with stringent search criteria, enabled the identification of an additional 92 putative candidates by only one peptide. The validity of this proteomics approach was confirmed by performing subsequent Western blot experiments for numerous proteins and determining the actual sumoylation sites for several other substrates. These data combine with recent works to further our understanding of the breadth and impact of protein sumoylation in a diverse array of biological processes.
Nucleic Acids Research, 2017
The genome of the cell is often exposed to DNA damaging agents and therefore requires an intricate wellregulated DNA damage response (DDR) to overcome its deleterious effects. The DDR needs proper regulation for its timely activation, repression, as well as appropriate choice of repair pathway. Studies in Saccharomyces cerevisiae have advanced our understanding of the DNA damage response, as well as the mechanisms the cell employs to maintain genome stability and how these mechanisms are regulated. Eukaryotic cells utilize post-translational modifications as a means for fine-tuning protein functions. Ubiquitylation and SUMOylation involve the attachment of small protein molecules onto proteins to modulate function or protein-protein interactions. SUMO in particular, was shown to act as a molecular glue when DNA damage occurs, facilitating the assembly of large protein complexes in repair foci. In other instances, SUMOylation alters a protein's biochemical activities, and interactions. SUMO-targeted ubiquitin ligases (STUbLs) are enzymes that target SUMOylated proteins for ubiquitylation and subsequent degradation, providing a function for the SUMO modification in the regulation and disassembly of repair complexes. Here, we discuss the major contributions of SUMO and STUbLs in the regulation of DNA damage repair pathways as well as in the maintenance of critical regions of the genome, namely rDNA regions, telomeres and the 2 m circle in budding yeast.
Proteotoxic stress reprograms the chromatin landscape of SUMO modification
Science Signaling, 2015
The small ubiquitin-like modifier 2 (SUMO-2) is required for survival when cells are exposed to treatments that induce proteotoxic stress by causing the accumulation of misfolded proteins. Exposure of cells to heat shock or other forms of proteotoxic stress induces the conjugation of SUMO-2 to proteins in the nucleus. Here, we investigated the chromatin landscape of SUMO-2 modifications in response to heat stress. Through chromatin immunoprecipitation assays coupled to high-throughput DNA sequencing and with mRNA sequencing, we showed that in response to heat shock, SUMO-2 accumulated at nucleosome-depleted, active DNA-regulatory elements, which represented binding sites for large protein complexes and were predominantly associated with active genes. However, SUMO did not act as a direct transcriptional repressor or activator of these genes during heat shock. Instead, integration of our results with published proteomics data on heat shock-induced SUMO-2 substrates supports a model in which the conjugation of SUMO-2 to proteins acts as an acute stress response that is required for the stability of protein complexes involved in gene expression and posttranscriptional modification of mRNA. We showed that the conjugation of SUMO-2 to chromatin-associated proteins is an integral component of the proteotoxic stress response, and propose that SUMO-2 fulfills its essential role in cell survival by contributing to the maintenance of protein complex homeostasis.
SUMO and transcriptional regulation
Seminars in Cell & Developmental Biology, 2004
The small ubiquitin-like modifier (SUMO) is covalently attached to lysine residues in target proteins and in doing so changes the properties of the modified protein. Here we examine the role of SUMO modification in transcriptional regulation. SUMO addition to components of the transcriptional apparatus does not have a common consequence as it can both activate and repress transcription. In most cases, however, SUMO modification of transcription factors leads to repression and various models to explain this, ranging from retention in nuclear bodies to recruitment of histone deacetylases are discussed. (R.T. Hay). modified by specific SUMO types and SUMO-2/-3 are thought to be conjugated to protein targets in response to a variety of cellular stress events .
Ubc9 Sumoylation Controls SUMO Chain Formation and Meiotic Synapsis in Saccharomyces cerevisiae
Posttranslational modification with the small ubiquitin-related modifier SUMO depends on the sequential activities of E1, E2, and E3 enzymes. While regulation by E3 ligases and SUMO proteases is well understood, current knowledge of E2 regulation is very limited. Here, we describe modification of the budding yeast E2 enzyme Ubc9 by sumoylation (Ubc9*SUMO). Although less than 1% of Ubc9 is sumoylated at Lys153 at steady state, a sumoylation- deficient mutant showed significantly reduced meiotic SUMO conjugates and abrogates synaptonemal complex formation. Biochemical analysis revealed that Ubc9*SUMO is severely impaired in its classical activity but promoted SUMO chain assembly in the presence of Ubc9. Ubc9*SUMO cooperates with charged Ubc9 (Ubc9SUMO) by noncovalent backside SUMO binding and by positioning the donor SUMO for optimal transfer. Thus, sumoylation of Ubc9 converts an active enzyme into a cofactor and reveals a mechanism for E2 regulation that orchestrates catalytic (Ubc9SUMO) and noncatalytic (Ubc9*SUMO) functions of Ubc9.
A SUMO-interacting motif activates budding yeast ubiquitin ligase Rad18 towards SUMO-modified PCNA
Nucleic Acids Research, 2012
SUMO-targeted ubiquitin ligases (STUbLs) recognize sumoylated proteins as substrates for ubiquitylation and have been implicated in several aspects of DNA repair and the damage response. However, few physiological STUbL substrates have been identified, and the relative importance of SUMO binding versus direct interactions with the substrate remains a matter of debate. We now present evidence that the ubiquitin ligase Rad18 from Saccharomyces cerevisiae, which monoubiquitylates the sliding clamp protein proliferating cell nuclear antigen (PCNA) in response to DNA damage, exhibits the hallmarks of a STUbL. Although not completely dependent on sumoylation, Rad18's activity towards PCNA is strongly enhanced by the presence of SUMO on the clamp. The stimulation is brought about by a SUMO-interacting motif in Rad18, which also mediates sumoylation of Rad18 itself. Our results imply that sumoylated PCNA is the physiological ubiquitylation target of budding yeast Rad18 and suggest a new mechanism by which the transition from S phase-associated sumoylation to damage-induced ubiquitylation of PCNA is accomplished.
Biochemical Journal, 2003
The phenotypes of mutants defective in the Schizosaccharomyces pombe SUMO (small, ubiquitin-like modifier)-conjugating enzyme Hus5 (the homologue of Ubc9) show that it is required for recovery from S-phase arrest. Unlike the case with ubiquitination, where ligases are required, SUMO-conjugating enzymes are sufficient for substrate recognition and conjugation of SUMO on to target proteins, at least in vitro. Thus SUMO-conjugating enzymes are likely to be important regulators of sumoylation. Here, we report on the characterization of two hus5 alleles. Although hus5.17 and hus5.62 respond in a similar manner to UV and ionizing radiation, they have different responses to the DNAsynthesis inhibitor, hydroxyurea. In addition, SUMO (Pmt3) is mislocalized in hus5.17 cells, but not in hus5.62 mutant cells. The mutations in hus5.62 and hus5.17 map to Ala 129 and the 5 splice site of intron 2 respectively. We have characterized the Hus5.62 protein and shown, in vitro, that it still interacts with SUMO and at least one protein, Rad22, which is a SUMO-modified target. The Hus5.62 protein is also capable of forming a thioester link with SUMO, but it does not function in sumoylation assays, either in the modification of Rad22 or in SUMO chain formation. When overexpressed in wild-type S. pombe cells, the Hus5.62 protein has a dominant-negative effect on sumoylation.
A Universal Strategy for Proteomic Studies of SUMO and Other Ubiquitin-like Modifiers
Molecular & Cellular Proteomics, 2004
Post-translational modification by the conjugation of small ubiquitin-like modifiers is an essential mechanism to affect protein function. Currently, only a limited number of substrates are known for most of these modifiers, thus limiting our knowledge of their role and relevance for cellular physiology. Here, we report the development of a universal strategy for proteomic studies of ubiquitin-like modifiers. This strategy involves the development of stable transfected cell lines expressing a double-tagged modifier under the control of a tightly negatively regulated promoter, the induction of the expression and conjugation of the tagged modifier to cellular proteins, the tandem affinity purification of the pool of proteins covalently modified by the tagged-modifier, and the identification of the modified proteins by liquid chromatography and mass spectrometry. By applying this methodology to the proteomic analysis of SUMO-1 and SUMO-3 we determined that SUMO-1 and SUMO-3 are stable proteins exhibiting half-lives of over 20 h, demonstrated that sumoylation with both SUMO-1 and SUMO-3 is greatly stimulated by MG-132 and heat shock treatment, demonstrated the preferential usage of either SUMO-1 or SUMO-3 for some known SUMO substrates, and identified 122 putative SUMO substrates of which only 27 appeared to be modified by both SUMO-1 and SUMO-3. This limited overlapping in the subset of proteins modified by SUMO-1 and SUMO-3 supports that the SUMO paralogues are likely to be functionally distinct. Three of the novel putative SUMO substrates identified, namely the polypyrimidine tract-binding protein-associated splicing factor PSF, the structural microtubular component alpha-Tubulin, and the GTP-binding nuclear protein Ran, were confirmed as authentic SUMO substrates. The application of this universal strategy to the identification of the pool of cellular substrates modified by other ubiquitin-like modifiers will dramatically increase our knowledge of the biological role of the different ubiquitin-like conjugations systems in the cell.