SUBCELLULAR-LOCALIZATION OF CDC42P, A SACCHAROMYCES-CEREVISIAE GTP-BINDING PROTEIN INVOLVED IN THE CONTROL OF CELL POLARITY
SUBCELLULAR-LOCALIZATION OF CDC42P, A SACCHAROMYCES-CEREVISIAE GTP-BINDING PROTEIN INVOLVED IN THE CONTROL OF CELL POLARITY
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DOI：
10.1091/mbc.4.12.1307
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发表时间：
1993-12-01
影响因子：
3.3
通讯作者：
JOHNSON, DI
中科院分区：
文献类型：
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作者：
ZIMAN, M;PREUSS, D;JOHNSON, DI
酿酒酵母Cdc42蛋白是低分子量gtp结合蛋白Ras超家族的一员，在酵母细胞周期中参与控制细胞极性。该蛋白具有香叶基修饰的一致序列（CAAX），并且可能与细胞膜相关，至少部分相关。利用细胞分离和免疫定位技术，我们研究了Cdc42p的亚细胞定位。Cdc42p在可溶性和颗粒池中均有发现，其丰度和分布在细胞周期中均不变化。颗粒形式的Cdc42p可以被洗涤剂溶解，但不能被NaCl或尿素溶解，这表明它与膜紧密结合。在限制温度下生长的香叶基香叶基转移酶突变株（cdc43-2(ts)）中，可溶性Cdc42p增加。此外，来自cdc42（C188S）突变株的Cdc42p（在戊酰化共识位点有一个改变）几乎完全在可溶性部分，这表明膜定位依赖于Cys-188上的香叶基修饰。免疫荧光和免疫电镜实验表明，Cdc42p定位于分泌囊泡附近的质膜上，这些囊泡位于芽出部位、芽扩大的尖端和侧面，以及在ct因子阻滞细胞的交配突起（shmoo尖端）内。这些结果表明，Cdc42p在细胞周期的早期定位于芽位，并表明这种定位对芽萌发和细胞极化生长的合适位置的选择至关重要。
The Saccharomyces cerevisiae Cdc42 protein, a member of the Ras superfamily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity during the yeast cell cycle. This protein has a consensus sequence (CAAX) for geranylgeranyl modification and is likely to be associated, at least in part, with cell membranes. Using cell fractionation and immunolocalization techniques, we have investigated the subcellular localization of Cdc42p. Cdc42p was found in both soluble and particulate pools, and neither its abundance nor its distribution varied through the cell cycle. The particulate form of Cdc42p could be solubilized with detergents but not with NaCl or urea, suggesting that it is tightly associated with membranes. An increase in soluble Cdc42p was observed in a geranylgeranyltransferase mutant strain (cdc43-2(ts)) grown at the restrictive temperature. In addition, Cdc42p from a cdc42(C188S) mutant strain (that has an alteration at the prenylation consensus site) was almost exclusively in the soluble fraction, suggesting that membrane localization is dependent on geranylgeranyl modification at Cys-188. Immunofluorescence and immunoelectron microscopy experiments demonstrated that Cdc42p localizes to the plasma membrane in the vicinity of secretory vesicles that were found at the site of bud emergence, at the tips and sides of enlarging buds, and within mating projections (shmoo tips) in ct-factor-arrested cells. These results indicate that Cdc42p is localized to the bud site early in the cell cycle and suggest that this localization is critical for the selection of the proper site for bud emergence and for polarized cell growth.