Horio T, Oakley B R
Department of Molecular Genetics, Ohio State University, Columbus 43210.
J Cell Biol. 1994 Sep;126(6):1465-73. doi: 10.1083/jcb.126.6.1465.
gamma-Tubulin is a phylogenetically conserved component of microtubule-organizing centers that is essential for viability and microtubule function. To examine the functional conservation of gamma-tubulin, we have tested the ability of human gamma-tubulin to function in the fission yeast Schizosaccharomyces pombe. We have found that expression of a human gamma-tubulin cDNA restores viability and a near-normal growth rate to cells of S. pombe lacking endogenous gamma-tubulin. Immunofluorescence microscopy showed that these cells contained normal mitotic spindles and interphase microtubule arrays, and that human gamma-tubulin, like S. pombe gamma-tubulin, localized to spindle pole bodies, the fungal microtubule-organizing centers. These results demonstrate that human gamma-tubulin functions in fission yeast, and they suggest that in spite of the great morphological differences between the microtubule-organizing centers of humans and fission yeasts, gamma-tubulin is likely to perform the same tasks in both. They suggest, moreover, that the proteins that interact with gamma-tubulin, including, most obviously, microtubule-organizing center proteins, must also be conserved. We have also found that a fivefold overexpression of S. pombe gamma-tubulin causes no reduction in growth rates or alteration of microtubule organization. We hypothesize that the excess gamma-tubulin is maintained in the cytoplasm in a form incapable of nucleating microtubule assembly. Finally, we have found that expression of human gamma-tubulin or overexpression of S. pombe gamma-tubulin causes no significant alteration of resistance to the antimicrotubule agents benomyl, thiabendazole and nocodazole.
γ-微管蛋白是微管组织中心在系统发育上保守的组成部分,对细胞存活和微管功能至关重要。为了研究γ-微管蛋白的功能保守性,我们测试了人类γ-微管蛋白在裂殖酵母粟酒裂殖酵母中发挥功能的能力。我们发现,人类γ-微管蛋白cDNA的表达可恢复缺乏内源性γ-微管蛋白的粟酒裂殖酵母细胞的活力和接近正常的生长速率。免疫荧光显微镜检查显示,这些细胞含有正常的有丝分裂纺锤体和间期微管阵列,并且人类γ-微管蛋白与粟酒裂殖酵母γ-微管蛋白一样,定位于纺锤极体,即真菌的微管组织中心。这些结果表明人类γ-微管蛋白在裂殖酵母中发挥功能,并且表明尽管人类和裂殖酵母的微管组织中心在形态上存在很大差异,但γ-微管蛋白在两者中可能执行相同的任务。此外,这表明与γ-微管蛋白相互作用的蛋白质,最明显的包括微管组织中心蛋白,也必定是保守的。我们还发现,粟酒裂殖酵母γ-微管蛋白的五倍过表达不会导致生长速率降低或微管组织改变。我们推测过量的γ-微管蛋白以无法启动微管组装的形式保留在细胞质中。最后,我们发现人类γ-微管蛋白的表达或粟酒裂殖酵母γ-微管蛋白的过表达不会导致对抗微管药物苯菌灵、噻苯达唑和诺考达唑的抗性发生显著改变。