Role of Ame1, a central component of the Saccharomyces cerevisiae kinetochore, in kinetochore function and structure

Abstract

SUMMARY The present work provides an analysis of kinetochore function and structure with respect to Ame1, a central component of the yeast Saccharomyces cerevisiae kinetochore. Applying mutant analyses (ame1-2) and biochemical binding assays the following results were obtained: 1. Classical kinetochore functions, chromosome attachment, chromosome segregation, and the supervision of these events by the spindle assembly checkpoint were analyzed in the ame1-2 mutant: • The Ame1 protein is needed for the establishment of bipolar attachments to microtubules emanating from opposing poles. Under tension one of the attachments is lost due to a structural weakening of the kinetochore, which results in monopolar segregation of sister chromatides. • Checkpoint analysis revealed that checkpoint functions are not impaired in the mutant, as judged by the inducement of the occupancy checkpoint and the ability of ame1-2 cells to sense their own attachment / tension defect. 2. ame1-2, as also other kinetochore mutants, causes a severe defect in the stability of interpolar microtubules (mitotic spindle). This effect is somewhat surprising, since kinetochores and interpolar microtubules do not interact with each other. Failure in spindle formation caused by kinetochore defects can be explained in two alternative ways: First, kinetochore proteins can also function as spindle stabilizing MAPs (microtubule associated proteins). Second, spindle defects are also observed if a separation of spindle poles occurs in absence of Esp1 and Cdc14 activity. Following experimental evidence suggests that the spindle defect in ame1-2 is not due to the above mentioned causes, but rather results from an alternative function of the kinetochore that is compromised in the mutant in a cell cycle dependent manner: • Ame1 does not locate to the mitotic spindle and thus is not a MAP. • As mentioned above, the ame1-2 mutant fails to achieve a stable bipolar attachment leading to the separation of spindle poles. In parallel the mutant senses this defect and maintains an active spindle assembly checkpoint which inhibits Esp1 and Cdc14 activation. As described (Higuchi and Uhlmann, 2005), such a spindle defect can be rescued by overexpression of Esp1 or Cdc14. The main cause for the spindle defect in ame1-2 however, is not a spindle pole separation in presence of low Esp1 or Cdc14 activity: i) Overexpression of Cdc14 in ame1-2 does not rescue the spindle defect of the mutant. ii) Overexpression of Esp1 in ame1-2 does only allow for a partial rescue of the spindle defect. iii) Inactivation of the spindle assembly checkpoint by Mad2 depletion also leads to only a partial rescue of the spindle defect. iv) A considerable number of cells separate their spindle pole bodies in presence of active Esp1, but nevertheless display a spindle defect. v) Most spindle defects occur at spindle pole distances that are characteristic of metaphase, when spindle stability is independent of the presence and activity of Esp1. • The ame1-2 kinetochore defect is more severe (particularly the kinetochore localization of the Mtw1 complex) when the mutation is induced prior to an establishment of bipolar attachment than after. This differentially compromised kinetochore structure in ame1-2 is apparently reflective of derived kinetochore functions. Similar kinetochore defects (monopolar segregation, failure in Cdc14 release) are observed, no matter if the mutation is induced before or after bipolar attachment. However, only the latter situation allows for the assembly of wild type metaphase and anaphase spindles. Thus, a certain kinetochore structure (including the Mtw1 complex) may be involved in generating a spindle stabilizing factor. 3. The present structural model of the S. cerevisiae kinetochore has been refined in the current work by the following findings: • A direct protein interaction network between the Okp1 / Mtw1 / Spc105 / Ndc80 kinetochore complexes could be established by in vitro binding assays performed with isolated protein complexes. • These data together with those derived from ChIP analyses of the ame1-2 mutant show a clear dependency of the centromeric association of all other central and outer kinetochore complexes on the Okp1 complex and are thus placing this complex in close proximity to the DNA binding CBF3 complex. • However, when bipolar attachments are achieved prior to the induction of ame1-2, the localization of the Mtw1 complex becomes independent of the presence of the Okp1 complex. The functional characterization of Ame1 in combination with the biochemical mapping of intra-kinetochore interactions allowed for a structural refinement of the present-state kinetochore model. Moreover, a direct influence of the kinetochore on spindle stability has been uncovered, which may be attributed to the presence of the Mtw1 complex.