Rho1p-Bni1p-Spa2p interactions:: Implication in localization of Bni1p at the bud site and regulation of the actin cytoskeleton in Saccharomyces cerevisiae
Rho1p-Bni1p-Spa2p interactions:: Implication in localization of Bni1p at the bud site and regulation of the actin cytoskeleton in Saccharomyces cerevisiae
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DOI：
10.1091/mbc.9.5.1221
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发表时间：
1998-05-01
影响因子：
3.3
通讯作者：
Takai, Y
中科院分区：
文献类型：
--
作者：
Fujiwara, T;Tanaka, K;Takai, Y
Rho 1 p是哺乳动物RhoA小GTP结合蛋白的酵母同源物。Rho 1 p定位于生长部位，是芽形成所必需的。我们最近发现，Bni 1 p是Rho 1 p的一个潜在的目标，Bni 1 p通过与profilin（一种肌动蛋白单体结合蛋白）的相互作用调节肌动蛋白细胞骨架的重组。利用酵母双杂交筛选系统，我们克隆了一个编码与Bni 1 p相互作用的蛋白的基因。已知这种蛋白Spa 2 p定位于芽尖，并与细胞极性的建立有关。Spa 2 p（1213-1466）的C端254个氨基酸区域直接结合Bni 1 p（826-987）的162个氨基酸区域。遗传分析表明，bni 1和spa 2突变显示出与Pkc 1 p-促分裂原活化蛋白激酶途径的基因编码组分中的突变的合成致死相互作用，其中Pkc 1 p是Rho 1 p的另一个靶点。免疫荧光显微镜分析表明，Bni 1 p定位在芽尖在野生型细胞。然而，在spa 2突变体中，Bni 1 p不定位在芽尖，而是弥漫定位在细胞质中。突变体Bni 1 p，缺乏Rho 1 p结合区，也未能定位在芽尖。这些结果表明，Rho 1 p和Spa 2 p都参与了Bni 1 p在生长位点的定位，Rho 1 p通过Bni 1 p调节肌动蛋白细胞骨架的重组。
Rho1p is a yeast homolog of mammalian RhoA small GTP-binding protein. Rho1p is localized at the growth sites and required for bud formation. We have recently shown that Bni1p is a potential target of Rho1p and that Bni1p regulates reorganization of the actin cytoskeleton through interactions with profilin, an actin monomer-binding protein. Using the yeast two-hybrid screening system, we cloned a gene encoding a protein that interacted with Bni1p. This protein, Spa2p, was known to be localized at the bud tip and to be implicated in the establishment of cell polarity. The C-terminal 254 amino acid region of Spa2p, Spa2p(1213-1466), directly bound to a 162-amino acid region of Bni1p, Bni1p(826-987). Genetic analyses revealed that both the bni1 and spa2 mutations showed synthetic lethal interactions with mutations in the genes encoding components of the Pkc1p-mitogen-activated protein kinase pathway, in which Pkc1p is another target of Rho1p. Immunofluorescence microscopic analysis showed that Bni1p was localized at the bud tip in wild-type cells. However, in the spa2 mutant, Bni1p was not localized at the bud tip and instead localized diffusely in the cytoplasm. A mutant Bni1p, which lacked the Rho1p-binding region, also failed to be localized at the bud tip. These results indicate that both Rho1p and Spa2p are involved in the localization of Bni1p at the growth sites where Rho1p regulates reorganization of the actin cytoskeleton through Bni1p.