| In the budding yeast Saccharomyces cerevisiae, the organelle responsible for nucleating both cytoplasmic and nuclear microtubules is called the spindle pole body (SPB). Spc110p is an essential component of the SPB whose amino terminus lies at the inner plaque, from which the nuclear microtubules originate. I have done a synthetic lethal screen to identify mutations that enhance the phenotype of spc110-221, a temperature-sensitive mutant of SPC110 containing point mutations in the amino terminus. The screen identified mutations in three genes: SPC97, SPC98, and VIK1. SPC97 and SPC98 encode components of the yeast gamma-tubulin complex. VIK1 encodes an accessory factor that interacts with the kinesin-related motor Kar3p. The discovery of components involved in both microtubule nucleation and in regulation of microtubule dynamics assigns a pivotal role for Spc110p in both functions.; Though both Spc98p and Spc97p co-purify with Spc110p from yeast extracts, genetic and two-hybrid analyses reveal that the interactions of Spc98p and Spc97p with Spc110p are not equivalent. SPC98 is able to suppress the temperature-sensitivity of spc110-221, while SPC97 does not. Two-hybrid assays suggest that Spc98p binds robustly to Spc110p, whereas Spc97p binds only weakly, if at all. Extra copies of SPC98 promote the binding of Spc97p to Spc110p, but extra copies of SPC97 inhibit the binding of Spc98p to Spc110p. The exacerbation of the spc110-221 phenotype by spc98-63 is caused by the failure of Spc98-63p to bind Spc 110-221p. The synthetic lethality of spc97-62 and spc110-221 can be ascribed to the weakened interaction between Spc97-62p and Spc98p.; Genetic interactions between spc110-221 and genes encoding motors indicate that the spc110-221 defect is specifically worsened by defects in VIK1 or KAR3. Moreover, overexpression of Kar3p suppresses the temperature-sensitivity of spc110-221.; Differences in sensitivity to the microtubule-depolymerization agent benomyl and in localization of Kar3p-GFP to the SPB strongly suggest that the vik1 truncation alleles, vik1-233 and vik1-163, are not equivalent to null alleles. In contrast to the effects of spc110-221 alone, the vik1-233-GFP spc110-221 mutant loses viability at the nonpermissive temperature and suffers more severely from chromosome missegregation. |
