Functional dissection of the γ-tubulin complex by suppressor analysis of gtb1 and alp4 mutations in Schizosaccharomyces pombe

Abstract

In fission yeast, γ-tubulin (encoded by the gtb1+ gene), Alp4 (Spc97/GCP2), and Alp6 (Spc98/GCP3) are essential components of the γ-tubulin complex. We isolated gtb1 mutants as allele-specific suppressors of temperature-sensitive alp4 mutations. Mutation sites in gtb1 mutants and in several alp4 alleles were determined. The majority of substituted amino acids were mapped to a small area on the predicted surface of the γ-tubulin molecule that might directly interact with the Alp4 protein. The cold sensitivity of γ-tubulin mutants was almost completely suppressed by an α-tubulin mutation and partially suppressed by a low concentration of thiabendazole, a microtubule assembly inhibitor. Other gtb1 mutants had increased resistance to this drug. Gel-filtration and immunoprecipitation analyses suggested that the mutant γ-tubulin formed an altered γ-tubulin complex with increased stability compared to wild-type γ-tubulin. In most gtb1 mutants, sexual development was impaired, and aberrant asci that contained an irregular spore shape and number were produced. In contrast, spore formation was not appreciably damaged in some alp4 and alp6 mutants, even at temperatures where vegetative proliferation was substantially defective. These results suggested that the function of the γ-tubulin complex or the requirement of each component of the complex is differentially regulated between the vegetative and sexual phases of the life cycle in fission yeast. In addition, genetic data indicated intimate functional connections of γ-tubulin with several kinesin-like proteins.