Growth site localization of Rho1 small GTP-binding protein and its involvement in bud formation in Saccharomyces cerevisiae.
Growth site localization of Rho1 small GTP-binding protein and its involvement in bud formation in Saccharomyces cerevisiae.
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DOI：
10.1083/jcb.125.5.1077
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发表时间：
1994-06
期刊：
影响因子：
--
通讯作者：
Takai Y
中科院分区：
文献类型：
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作者：
Yamochi W;Tanaka K;Nonaka H;Maeda A;Musha T;Takai Y
Rho 小 GTP 结合蛋白家族调节各种肌动球蛋白依赖性细胞功能，例如细胞形态、运动、胞质分裂、膜皱褶和平滑肌收缩。在酿酒酵母中，有哺乳动物 RhoA 的同源物 RHO1，它对于酵母细胞的营养生长至关重要。为了探索 RHO1 基因的功能，我们分离了 RHO1 的隐性温度敏感突变体 rho1-104。 rho1-104 突变导致 Rho1p 的 Asp 72 氨基酸替换为 Asn，Cys 164 替换为 Tyr。携带 rho1-104 突变的菌株积累了微小或小芽细胞，其中皮质肌动蛋白斑块在限制温度下聚集成芽。 rho1-104 突变体也观察到细胞裂解和细胞死亡。间接免疫荧光显微镜研究表明，Rho1p集中在皮质肌动蛋白斑块聚集的细胞外围，包括芽出现的部位、生长芽的尖端以及胞质分裂前细胞的母芽颈区域。对过表达 RHO1 的细胞进行的间接免疫荧光研究表明，Rho1p 结合位点是可饱和的。脂质修饰位点发生氨基酸取代的突变 Rho1p 保留在细胞质中。这些结果表明，Rho1 小 GTP 结合蛋白与细胞生长区域的特定位点结合，Rho1p 在该位点发挥控制细胞生长的功能。
The Rho small GTP-binding protein family regulates various actomyosin- dependent cell functions, such as cell morphology, locomotion, cytokinesis, membrane ruffling, and smooth muscle contraction. In the yeast Saccharomyces cerevisiae, there is a homologue of mammalian RhoA, RHO1, which is essential for vegetative growth of yeast cells. To explore the function of the RHO1 gene, we isolated a recessive temperature-sensitive mutation of RHO1, rho1-104. The rho1-104 mutation caused amino acid substitutions of Asp 72 to Asn and Cys 164 to Tyr of Rho1p. Strains bearing the rho1-104 mutation accumulated tiny- or small- budded cells in which cortical actin patches were clustered to buds at the restrictive temperature. Cell lysis and cell death were also seen with the rho1-104 mutant. Indirect immunofluorescence microscopic study demonstrated that Rho1p was concentrated to the periphery of the cells where cortical actin patches were clustered, including the site of bud emergence, the tip of the growing buds, and the mother-bud neck region of cells prior to cytokinesis. Indirect immunofluorescence study with cells overexpressing RHO1 suggested that the Rho1p-binding site was saturable. A mutant Rho1p with an amino acid substitution at the lipid modification site remained in the cytoplasm. These results suggest that Rho1 small GTP-binding protein binds to a specific site at the growth region of cells, where Rho1p exerts its function in controlling cell growth.