Ohkuni Kentaro, Takahashi Yoshimitsu, Fulp Alyona, Lawrimore Josh, Au Wei-Chun, Pasupala Nagesh, Levy-Myers Reuben, Warren Jack, Strunnikov Alexander, Baker Richard E, Kerscher Oliver, Bloom Kerry, Basrai Munira A
Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institute of Health, Bethesda, MD 20892.
Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
Mol Biol Cell. 2016 Mar 9;27(9):1500-10. doi: 10.1091/mbc.E15-12-0827.
Centromeric histone H3, CENP-A, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-A restricts its localization to centromeres. Mislocalization of CENP-A is associated with aneuploidy in yeast, flies and tumorigenesis in human cells; thus, defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast has shown that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here, we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and in vitro. Ubiquitination of Cse4 by Small Ubiquitin-related Modifier (SUMO)-Targeted Ubiquitin Ligase (STUbL) Slx5 plays a critical role in proteolysis of Cse4 and prevents mislocalization of Cse4 to euchromatin under normal physiological conditions. Accumulation of sumoylated Cse4 species and increased stability of Cse4 in slx5∆ strains suggest that sumoylation precedes ubiquitin-mediated proteolysis of Cse4. Slx5-mediated Cse4 proteolysis is independent of Psh1 since slx5∆ psh1∆ strains exhibit higher levels of Cse4 stability and mislocalization compared to either slx5∆ or psh1∆ strains. Our results demonstrate a role for Slx5 in ubiquitin-mediated proteolysis of Cse4 to prevent its mislocalization and maintain genome stability.
着丝粒组蛋白H3（CENP - A）对于准确的染色体分离至关重要。对CENP - A细胞水平的严格调控将其定位限制在着丝粒。在酵母、果蝇中，CENP - A的错误定位与非整倍体相关，在人类细胞中则与肿瘤发生相关；因此，确定调节CENP - A水平的途径对于理解CENP - A的错误定位如何导致人类癌症中的非整倍体至关重要。先前在芽殖酵母中的研究表明，E3连接酶Psh1对过表达的Cse4进行泛素化，这部分促成了Cse4的蛋白水解。在此，我们提供了首个证据，证明Cse4在体内和体外被E3连接酶Siz1和Siz2进行SUMO化修饰。小泛素相关修饰物（SUMO）靶向泛素连接酶（STUbL）Slx5对Cse4的泛素化在Cse4的蛋白水解中起关键作用，并在正常生理条件下防止Cse4错误定位于常染色质。在slx5Δ菌株中，SUMO化修饰的Cse4物种积累以及Cse4稳定性增加，这表明SUMO化修饰先于泛素介导的Cse4蛋白水解。Slx5介导的Cse4蛋白水解独立于Psh1，因为与单独的slx5Δ或psh1Δ菌株相比，slx5Δ psh1Δ菌株表现出更高水平的Cse4稳定性和错误定位。我们的结果证明了Slx5在泛素介导的Cse4蛋白水解中发挥作用，以防止其错误定位并维持基因组稳定性。
