Nguyen T, Vinh D B, Crawford D K, Davis T N
Molecular and Cellular Biology Program, University of Washington, Seattle, Washington 98195, USA.
Mol Biol Cell. 1998 Aug;9(8):2201-16. doi: 10.1091/mbc.9.8.2201.
The spindle pole body (SPB) in Saccharomyces cerevisiae functions as the microtubule-organizing center. Spc110p is an essential structural component of the SPB and spans between the central and inner plaques of this multilamellar organelle. The amino terminus of Spc110p faces the inner plaque, the substructure from which spindle microtubules radiate. We have undertaken a synthetic lethal screen to identify mutations that enhance the phenotype of the temperature-sensitive spc110-221 allele, which encodes mutations in the amino terminus. The screen identified mutations in SPC97 and SPC98, two genes encoding components of the Tub4p complex in yeast. The spc98-63 allele is synthetic lethal only with spc110 alleles that encode mutations in the N terminus of Spc110p. In contrast, the spc97 alleles are synthetic lethal with spc110 alleles that encode mutations in either the N terminus or the C terminus. Using the two-hybrid assay, we show that the interactions of Spc110p with Spc97p and Spc98p are not equivalent. The N terminus of Spc110p displays a robust interaction with Spc98p in two different two-hybrid assays, while the interaction between Spc97p and Spc110p is not detectable in one strain and gives a weak signal in the other. Extra copies of SPC98 enhance the interaction between Spc97p and Spc110p, while extra copies of SPC97 interfere with the interaction between Spc98p and Spc110p. By testing the interactions between mutant proteins, we show that the lethal phenotype in spc98-63 spc110-221 cells is caused by the failure of Spc98-63p to interact with Spc110-221p. In contrast, the lethal phenotype in spc97-62 spc110-221 cells can be attributed to a decreased interaction between Spc97-62p and Spc98p. Together, these studies provide evidence that Spc110p directly links the Tub4p complex to the SPB. Moreover, an interaction between Spc98p and the amino-terminal region of Spc110p is a critical component of the linkage, whereas the interaction between Spc97p and Spc110p is dependent on Spc98p.
酿酒酵母中的纺锤体极体（SPB）作为微管组织中心发挥作用。Spc110p是SPB的一个必需结构成分，横跨这个多层细胞器的中央片层和内片层。Spc110p的氨基末端面向内片层，纺锤体微管从该亚结构辐射而出。我们进行了一项合成致死筛选，以鉴定增强温度敏感型spc110 - 221等位基因表型的突变，该等位基因编码氨基末端的突变。筛选鉴定出SPC97和SPC98中的突变，这两个基因编码酵母中Tub4p复合体的成分。spc98 - 63等位基因仅与编码Spc110p氨基末端突变的spc110等位基因合成致死。相比之下，spc97等位基因与编码Spc110p氨基末端或羧基末端突变的spc110等位基因合成致死。使用双杂交试验，我们表明Spc110p与Spc97p和Spc98p的相互作用并不等同。在两种不同的双杂交试验中，Spc110p的氨基末端与Spc98p表现出强烈的相互作用，而在一种菌株中Spc97p与Spc110p之间的相互作用无法检测到，在另一种菌株中则给出微弱信号。SPC98的额外拷贝增强了Spc97p与Spc110p之间的相互作用，而SPC97的额外拷贝则干扰了Spc98p与Spc110p之间的相互作用。通过测试突变蛋白之间的相互作用，我们表明spc98 - 63 spc110 - 221细胞中的致死表型是由Spc98 - 63p无法与Spc110 - 221p相互作用导致的。相比之下，spc97 - 62 spc110 - 221细胞中的致死表型可归因于Spc97 - 62p与Spc98p之间相互作用的减弱。总之，这些研究提供了证据表明Spc110p直接将Tub4p复合体与SPB连接起来。此外，Spc98p与Spc110p氨基末端区域之间的相互作用是这种连接的关键组成部分，而Spc97p与Spc110p之间的相互作用则依赖于Spc98p。
