Mutagenesis of β-tubulin cysteine residues in Saccharomyces cerevisiae:: Mutation of cysteine 354 results in cold-stable microtubules
Mutagenesis of β-tubulin cysteine residues in Saccharomyces cerevisiae:: Mutation of cysteine 354 results in cold-stable microtubules
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DOI：
10.1002/cm.1021
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发表时间：
2001-06-01
影响因子：
--
通讯作者：
Himes, RH
中科院分区：
文献类型：
--
作者：
Gupta, ML;Bode, CJ;Himes, RH
半胱氨酸残基在微管蛋白功能调控中起着重要作用。为了确定β-微管蛋白中六个半胱氨酸残基中的哪一个是微管蛋白功能的关键，我们将酿酒酵母β-微管蛋白中的半胱氨酸分别突变为丙氨酸和丝氨酸残基。在12个突变中，只有3个突变产生了显著影响：C12S、C354A和C354S。C12S突变在单倍体中是致死的，但C12A突变没有明显的表型。基于微管蛋白电子晶体结构的相互作用观点，我们认为该位置的丝氨酸取代半胱氨酸对微管蛋白与可交换的GTP的相互作用有不稳定的影响。这两个C354突变虽然不致命，但对微管和需要微管的细胞过程产生了戏剧性的影响。C354突变细胞的生长速度减慢，有丝分裂减慢，对苯菌灵的抵抗力增强，核迁移和纺锤体组装受损。C354A突变比C354S突变产生了更严重的表型：单倍体细胞有染色体分离缺陷，培养物中只有50%的细胞存活，而且有相当大比例的细胞畸形。C354S和C354A细胞的细胞质微管较对照株长，纺锤体结构变短变厚。两个C354突变体的细胞质微管和纺锤体微管对低温都非常稳定。在4℃24小时后，微管仍然存在，实际上已经形成了很长很厚的微管聚合物。有证据表明，哺乳动物微管蛋白中的C354残基位于秋水仙素结合部位附近，微管蛋白的电子晶体结构将残基放置在α和β亚基之间的界面上。巯基位于极性环境中，这可能解释了为什么丙氨酸突变比丝氨酸突变更严重。当在一个二倍体菌株中进行C12S和两个C354突变时，突变的微管蛋白被整合到微管中，得到的杂合子的表型介于突变的单倍体和野生型菌株之间。结果表明，C12和C354残基在微管蛋白的结构和功能中起着重要作用。细胞运动。细胞骨架49：67-77,2001。(C)2001年Wiley-Liss，Inc.
Cysteine residues play important roles in the control of tubulin function. To determine which of the six cysteine residues in beta -tubulin are critical to tubulin function, we mutated the cysteines in Saccharomyces cerevisiae beta -tubulin individually to alanine and serine residues. Of the twelve mutations, only three produced significant effects: C12S, C354A, and C354S. The C12S mutation was lethal in the haploid, but the C12A mutation had no observable phenotype. Based on interactive views of the electron crystallographic structure of tubulin, we suggest that substitution of serine for cysteine at this position has a destabilizing effect on the interaction of tubulin with the exchangeable GTP. The two C354 mutations, although not lethal, produced dramatic effects on microtubules and cellular processes that require microtubules. The C354 mutant cells had decreased growth rates, a slowed mitosis, increased resistance to benomyl, and impaired nuclear migration and spindle assembly. The C354A mutation produced a more severe phenotype than the C354S mutation: the haploid cells had chromosome segregation defects, only 50% of cells in a culture were viable, and a significant percentage of the cells were misshapened. Cytoplasmic microtubules in the C354S and C354A cells were longer than in the control strain and spindle structures appeared shorter and thicker. Both cytoplasmic and spindle microtubules in the two C354 mutants were extremely stable to cold temperature. After 24 h at 4 degreesC, the microtubules were still present and, in fact, very long and thick tubulin polymers had formed. Evidence exists to indicate that the C354 residue in mammalian tubulin is near the colchicine binding site and the electron crystal structure of tubulin places the residue at the interface between the alpha- and beta -subunits. The sulfhydryl group is situated in a polar environment, which may explain why the alanine mutation is more severe than the serine mutation. When the C12S and the two C354 mutations were made in a diploid strain, the mutated tubulin was incorporated into microtubules and the resulting heterozygotes had phenotypes that were intermediate between those of the mutated haploids and the wild-type strains. The results suggest that the C12 and C354 residues play important roles in the structure and function of tubulin. Cell Motil. Cytoskeleton 49:67-77, 2001. (C) 2001 Wiley-Liss, Inc.