title: SUMO Pathway Modulation of Regulatory Protein Binding at the Ribosomal DNA Locus in Saccharomyces cerevisiae.
abstract: In this report, we identify cellular targets of Ulp2, one of two Saccharomyces cerevisiae small ubiquitin-related modifier (SUMO) proteases, and investigate the function of SUMO modification of these proteins. PolySUMO conjugates from ulp2Δ and ulp2Δ slx5Δ cells were isolated using an engineered affinity reagent containing the four SUMO-interacting motifs (SIMs) of Slx5, a component of the Slx5/Slx8 SUMO-targeted ubiquitin ligase (STUbL). Two proteins identified, Net1 and Tof2, regulate ribosomal DNA (rDNA) silencing and were found to be hypersumoylated in ulp2Δ,slx5Δ, and ulp2Δ slx5Δ cells. The increase in sumoylation of Net1 and Tof2 in ulp2Δ, but not ulp1ts cells, indicates that these nucleolar proteins are specific substrates of Ulp2 Based on quantitative chromatin-immunoprecipitation assays, both Net1 and Tof2 lose binding to their rDNA sites in ulp2Δ cells and both factors largely regain this association in ulp2Δ slx5Δ A parsimonious interpretation of these results is that hypersumoylation of these proteins causes them to be ubiquitylated by Slx5/Slx8, impairing their association with rDNA. Fob1, a protein that anchors both Net1 and Tof2 to the replication-fork barrier (RFB) in the rDNA repeats, is sumoylated in wild-type cells, and its modification levels increase specifically in ulp2Δ cells. Fob1 experiences a 50% reduction in rDNA binding in ulp2Δ cells, which is also rescued by elimination of Slx5 Additionally, overexpression of Sir2, another RFB-associated factor, suppresses the growth defect of ulp2Δ cells. Our data suggest that regulation of rDNA regulatory proteins by Ulp2 and the Slx5/Slx8 STUbL may be the cause of multiple ulp2Δ cellular defects.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Gillies
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Hickey
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114.
authors.forename: Dan
authors.initials: D
authors.lastname: Su
authors.affiliation: Department of Structural Biology, St. Jude Proteomics Facility, St. Jude Children's Research Hospital, Memphis, Tennessee 38105 Department of Developmental Neurobiology, St. Jude Proteomics Facility, St. Jude Children's Research Hospital, Memphis, Tennessee 38105.
authors.forename: Zhiping
authors.initials: Z
authors.lastname: Wu
authors.affiliation: Department of Structural Biology, St. Jude Proteomics Facility, St. Jude Children's Research Hospital, Memphis, Tennessee 38105 Department of Developmental Neurobiology, St. Jude Proteomics Facility, St. Jude Children's Research Hospital, Memphis, Tennessee 38105.
authors.forename: Junmin
authors.initials: J
authors.lastname: Peng
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114 mark.hochstrasser@yale.edu.
authors.forename: Mark
authors.initials: M
authors.lastname: Hochstrasser
mesh_terms: D001665:Binding Sites; D059447:Cell Cycle Checkpoints / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D025842:SUMO-1 Protein / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D017384:Sequence Deletion; D015398:Signal Transduction; D013379:Substrate Specificity; D058207:Sumoylation
chemical_list: D004275:DNA, Ribosomal; D025842:SUMO-1 Protein; D029701:Saccharomyces cerevisiae Proteins; D010450:Endopeptidases; C405949:ULP2 protein, S cerevisiae
keywords: Fob1; RENT complex; SUMO; Ulp2; rDNA
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