Role of the Spc105 Complex in Organization and Microtubule‐Binding Activity of the Budding Yeast Kinetochore

다국어 초록 (Multilingual Abstract)

Equally partitioning duplicated chromosomes during mitosis is of paramount importance; failure can cause aneuploidy, which predisposes individuals to cancer and birth defects. Chromosome segregation is orchestrated by kinetochores, a super‐complex of proteins that assemble onto centromeric DNA. Kinetochores must strongly couple chromosomes to spindle microtubules and they must sense and correct aberrant microtubule attachments. The Spc105 complex is an essential part of the budding yeast kinetochore, as are its homologues found widely across eukaryotes; however, it has yet to be completely defined. Spc105 complex plays an important role in the mitotic checkpoint, which produces a soluble ‘wait’ signal to delay anaphase in the absence of proper kinetochore‐microtubule attachments. The Spc105 complex is also proposed to contribute directly to kinetochore‐microtubule coupling. However, the extent to which the Spc105 complex and its homologues in other organisms contribute to microtubule coupling is unclear because it has been extremely difficult to study the purified complex in vitro owing to its biochemical instability. Molecular understanding of the binding of Spc105 complex to other complexes within the kinetochore is also lacking. We purified the recombinant Spc105 complex for the first time through co‐expression of its two protein components, Spc105p and Kre28p. Chemical cross‐linking and mass spectrometry (XL/MS) reveal that Spc105p and Kre28p interact through a region of about 200 amino acids. Additional XL/MS and pulldown experiments suggest regions through which the Spc105 complex interacts with the microtubule‐binding Ndc80 complex. Total internal reflection fluorescence (TIRF) microscopy shows that the Spc105‐GFP complex binds directly on microtubules. The ability to recombinantly purify the Spc105 complex will allow molecular details of its organization at the kinetochore and its contribution to the kinetochore function to be studied directly.
Support or Funding Information
National Institutes of Health (R01 GM040506) ‐ Trisha N Davis
This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.