Michelitch M, Chant J
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Curr Biol. 1996 Apr 1;6(4):446-54. doi: 10.1016/s0960-9822(02)00512-2.
Yeast cells polarize, bud, and divide in either of two genetically programmed patterns: axial or bipolar. The Saccharomyces cerevisiae gene BUD1 (also known as RSR1) encodes a Ras-related GTPase critical for selection of a bud sites in these patterns. To distinguish between possible mechanisms of Bud1p action, we have examined the function and subcellular localization of Bud1p in a variety of mutant situations.
Bud1p has 57% identity to H-ras, except for an 81 amino-acid insertion near the carboxyl terminus. Mutation of the proposed BUD1 effector domain produces a protein which can neither support normal patterns of budding nor interact with CDC24, which encodes a likely Bud1p effector. A version of Bud1p deleted for the 81 amino-acid unique region is essentially wild-type. Immunofluorescence and cell fractionation indicate that Bud1p remains associated with the membrane throughout its GTPase cycle. Both potential effectors of Bud1p, Bem1p and Cdc24p, are also membrane associated even in the absence of Bud1p, suggesting that Bud1p is not required to dock these proteins from the cytosol but may couple these proteins and others within the plane of the plasma membrane.
Based upon observations reported here and elsewhere, we propose a novel mechanism of Bud1p GTPase action. Like Ras, Bud1p GTPase is constitutively associated with the plasma membrane; however, concentrated activities of Bud5p GDP-GTP exchange factor and Bud2p GTPase-activating protein at the future bud site promote rapid cycling of Bud1p between GTP- and GDP-bound conformations in a spatially restricted manner. Local GTPase cycling serves to efficiently nucleate complexes between polarity establishment functions that direct cytoskeletal polarization towards the bud site.
酵母细胞以两种基因编程模式之一进行极化、出芽和分裂：轴向或双极模式。酿酒酵母基因BUD1（也称为RSR1）编码一种Ras相关的GTP酶，对这些模式中芽位点的选择至关重要。为了区分Bud1p作用的可能机制，我们研究了Bud1p在各种突变情况下的功能和亚细胞定位。
Bud1p与H-ras有57%的同源性，除了在羧基末端附近有一个81个氨基酸的插入序列。推测的BUD1效应结构域的突变产生了一种既不能支持正常出芽模式也不能与编码可能的Bud1p效应物的CDC24相互作用的蛋白质。缺失81个氨基酸独特区域的Bud1p版本基本上是野生型的。免疫荧光和细胞分级分离表明，Bud1p在其整个GTP酶循环中都与膜结合。Bud1p的两个潜在效应物Bem1p和Cdc24p即使在没有Bud1p的情况下也与膜相关，这表明Bud1p不需要将这些蛋白质从细胞质中对接过来，但可能在质膜平面内将这些蛋白质与其他蛋白质偶联起来。
基于此处和其他地方报道的观察结果，我们提出了一种Bud1p GTP酶作用的新机制。与Ras一样，Bud1p GTP酶与质膜组成型结合；然而，Bud5p GDP-GTP交换因子和Bud2p GTP酶激活蛋白在未来芽位点的集中活性以空间受限的方式促进Bud1p在GTP结合和GDP结合构象之间的快速循环。局部GTP酶循环有助于有效地使极性建立功能之间形成复合物，这些功能将细胞骨架极化导向芽位点。
