Recruitment of the Ulp2 protease to the inner kinetochore prevents its hyper-sumoylation to ensure accurate chromosome segregation

The kinetochore is the central molecular machine that drives chromosome segregation in all eukaryotes. Genetic studies have suggested that protein sumoylation plays a role in regulating the inner kinetochore; however, the mechanism remains elusive. Here, we show that Saccharomyces cerevisiae Ulp2, an evolutionarily conserved SUMO specific protease, contains a previously uncharacterized kinetochore-targeting motif that recruits Ulp2 to the kinetochore via the Ctf3(CENP-I)-Mcm16(CENP-H)-Mcm22(CENP-K) complex (CMM). Once recruited, Ulp2 selectively targets multiple subunits of the kinetochore, specifically the Constitutive Centromere-Associated Network (CCAN), via its SUMO-interacting motif (SIM). Mutations that impair the kinetochore recruitment of Ulp2 or its binding to SUMO result in an elevated rate of chromosome loss, while mutations that affect both result in a synergistic increase of chromosome loss rate, hyper-sensitivity to DNA replication stress, along with a dramatic accumulation of hyper-sumoylated CCAN. Notably, sumoylation of CCAN occurs at the kinetochore and is perturbed by DNA replication stress. These results indicate that Ulp2 utilizes its dual substrate recognition to prevent hyper-sumoylation of CCAN, ensuring accurate chromosome segregation during cell division. Author summary The kinetochore plays a central role in controlling accurate chromosome segregation in all eukaryotes. Defects in the kinetochore are a frequent cause of aneuploidy, which leads to birth defects and cancer in humans. Although the composition of the kinetochore has been extensively studied, how it is regulated remains poorly understood. Prior genetic studies have implicated an important role for protein sumoylation, the attachment of the Small Ubiquitin-like MOdifier (SUMO) to cellular proteins, in preventing aneuploidy. Here we identify a novel kinetochore-targeting motif in the SUMO specific protease Ulp2 and uncover the mechanism by which it selectively targets the inner kinetochore. We further show that the sumoylation level of the inner kinetochore is tightly regulated, and this regulation is necessary to prevent the aberrant loss of chromosomes during cell division. These findings uncover a critical molecular link between protein sumoylation and the suppression of aneuploidy.