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

Abstract

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 Ctf3CENP-I-Mcm16CENP-H-Mcm22CENP-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.

The structural basis for kinetochore stabilization by Cnn1/CENP-T

“…The sample was eluted by isocratic flow at 0.1 mL/min, and fractions were collected at 30 second intervals. For the sample reported here, excess recombinant NH2-biotin-Ulp2-CCR peptide (Suhandynata et al, 2019) was included but not visualized in the final density.…”

“…When the Ctf19c is purified from extract, Ctf3c proteins are required for Cnn1-Wip1 recovery, while the reverse is not true (Pekgoz Altunkaya et al, 2016). Ctf3 itself is required in cells for recruitment of the replicative kinase DDK and the SUMO protease Ulp2 (Hinshaw et al, 2017;Suhandynata et al, 2019). The molecular details of DDK and Ulp2 recruitment are not yet clear.…”

SUMO orchestrates multiple alternative DNA-protein crosslink repair pathways

“…Nano-flow LC-MS/MS analysis was performed on a Thermo Scientific Ultimate 3000 Nano-LC System and a Thermo Scientific Orbitrap Fusion Lumos mass spectrometer; acquired via NIH S10 OD023498. Data analysis for SILAC-labeled samples was performed as previously described, with the exception that all proteins were required to have a minimum of 3 unique peptides (Suhandynata et al, 2019). A list of "DNA repair" genes (GO:0006281) was extracted from the R package "org.Sc.sgd.db", v.3.7.0.…”

The structural basis for Ulp2 recruitment to the kinetochore

“…The digested peptides were then processed for analysis using a Thermo Scientific Orbitrap Fusion LUMOS Tribrid mass spectrometer. Methods used for mass spectrometry analysis are described elsewhere (Suhandynata et al, 2019). To quantify Ctf3-associated kinetochore proteins, several additional data filtering criteria were applied, including: 1) a cutoff score for peptide identification probability was set to at 0.9; 2) parental ion mass accuracy was < 10 ppm; and 3) the spectral intensity was >1000.…”

“…SIM-dependence implies homeostatic regulation; excessive substrate sumoylation enhances Ulp2 recruitment, and cleavage of the chains by Ulp2 itself releases the enzyme from its substrates. Sumoylated inner kinetochore proteins accumulate in cells expressing Ulp2 kinetochore interaction mutants (Ulp2-SIM-3A-KIM-3A) (Suhandynata et al, 2019). These cells, like ulp2∆ cells, frequently missegregate chromosomes (Ryu et al, 2016;Suhandynata et al, 2019).…”