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Regulation of budding yeast kinetochore proteins by SUMO modification

University of British Columbia

Genome stability is a fundamental requirement for the stable propagation of eukaryotic cells, maintenance of phenotype, and cell viability. For example, aneuploidy (abnormal chromosome number), is a hallmark of most human cancers, and can be attributed to increased rates of chromosome instability in cancer cells. The multiprotein kinetochore complex contributes to faithful chromosome segregation by mediating the attachment of a specialized chromosomal region, the centromere, to the mitotic spindle. A dysfunctional kinetochore represents one possible source for chromosome instability and the generation of aneuploid cancer cells, due to a failure to properly mediate this attachment. To better understand the regulation of kinetochore proteins and their role in chromosome segregation, a series of genomic screens were performed with known kinetochore components in the budding yeast Saccharomyces. cerevisiae, to identify genetic/physical interactions and novel functions that are critical for proper chromosome segregation. This work lead to the study of two distinct relationships: (1) between the kinetochore and the nuclear envelope, and (2) between kinetochore proteins and the ubiquitin-like protein SUMO. In the first study, genes that are linked to chromosome stability were identified by performing genome-wide synthetic lethal screens using a series of novel temperature sensitive mutations in essential genes encoding a central (SPC24) and outer, (SPC34) kinetochore protein. By performing these screens using different mutant alleles of each gene, we aimed to identify genetic interactions that revealed diverse pathways affecting chromosome stability. This study, which is the first example of genome-wide synthetic lethal screening with multiple alleles of a single gene, demonstrated that functionally distinct mutants uncover different cellular processes required for chromosome maintenance. Two of our screens identified APQ12, a gene that encodes a nuclear envelope protein required for proper nucleocytoplasmic transport of mRNA, which was subsequently characterized with respect to chromosome stability. We found that apql2 mutants are delayed in anaphase, re-replicate their DNA and re-bud prior to completion of cytokinesis, suggesting a defect in controlling mitotic progression. Overall, this analysis revealed a novel relationship between nucleocytoplasmic transport and chromosome stability. In the second study, functional genomics lead to the identification of the kinetochore proteins Ndc10, Bir1 , Ndc80, and Cep3 as being sumoylated substrates in budding yeast. This work demonstrated that Ndc10, Bir1, and Cep3, but not Ndc80, are differentially modified upon exposure to nocodazole, indicating distinct roles for SUMO modification in modulating kinetochore protein function and providing a potential link between sumoylation of kinetochore proteins and mitotic checkpoint function. Specific lysine to arginine mutations, were shown to eliminate sumoylation of Ndc10and to cause chromosome instability, mis-localization of Ndc10 from the mitotic spindle, and abnormal anaphase spindles, suggesting that sumoylation of Ndc10 and other kinetochore proteins plays a critical role during the mitotic process. These results support the recent findings that post-translational modifications by the ubiquitin-like protein SUMO is an important regulator of many cellular processes including genome integrity.

Text

2011-01-28

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http://hdl.handle.net/2429/30958

Biochemistry and Molecular Biology

University of British Columbia

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