Gillies Jennifer, Hickey Christopher M, Su Dan, Wu Zhiping, Peng Junmin, Hochstrasser Mark
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114.
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.
Genetics. 2016 Apr;202(4):1377-94. doi: 10.1534/genetics.116.187252. Epub 2016 Feb 2.
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.
在本报告中，我们鉴定了酿酒酵母两种小泛素相关修饰物（SUMO）蛋白酶之一Ulp2的细胞靶点，并研究了这些蛋白质SUMO修饰的功能。使用含有Slx5（Slx5/Slx8 SUMO靶向泛素连接酶（STUbL）的一个组分）的四个SUMO相互作用基序（SIMs）的工程化亲和试剂，从ulp2Δ和ulp2Δ slx5Δ细胞中分离出多聚SUMO缀合物。鉴定出的两种蛋白质Net1和Tof2调节核糖体DNA（rDNA）沉默，并且发现在ulp2Δ、slx5Δ和ulp2Δ slx5Δ细胞中被高度SUMO化。Net1和Tof2在ulp2Δ而非ulp1ts细胞中的SUMO化增加，表明这些核仁蛋白是Ulp2的特异性底物。基于定量染色质免疫沉淀分析，Net1和Tof2在ulp2Δ细胞中均失去与它们rDNA位点的结合，并且在ulp2Δ slx5Δ中这两种因子在很大程度上恢复了这种结合。对这些结果的一种简约解释是，这些蛋白质的高度SUMO化导致它们被Slx5/Slx8泛素化，损害了它们与rDNA的结合。Fob1是一种将Net1和Tof2都锚定到rDNA重复序列中的复制叉屏障（RFB）上的蛋白质，在野生型细胞中被SUMO化，并且其修饰水平在ulp2Δ细胞中特异性增加。Fob1在ulp2Δ细胞中与rDNA的结合减少了50%，这也通过消除Slx5得以挽救。此外，另一种与RFB相关的因子Sir2的过表达抑制了ulp2Δ细胞的生长缺陷。我们的数据表明，Ulp2和Slx5/Slx8 STUbL对rDNA调节蛋白的调控可能是ulp2Δ细胞多种缺陷的原因。
