Mammalian Cdc42 is a brefeldin A-sensitive component of the Golgi apparatus
Mammalian Cdc42 is a brefeldin A-sensitive component of the Golgi apparatus
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DOI：
10.1074/jbc.271.43.26850
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发表时间：
1996-10-25
影响因子：
4.8
通讯作者：
Cerione, RA
中科院分区：
文献类型：
--
作者：
Erickson, JW;Zhang, CJ;Cerione, RA
在这项研究中，我们运用免疫细胞化学和分级分离方法，对哺乳动物Cdc42蛋白（命名为Cdc42Hs）在体内的定位进行了描述。针对Cdc42Hs的C末端区域制备了一种特异性抗肽抗体。在间接免疫荧光实验中使用这种抗体的亲和纯化制剂，发现Cdc42Hs定位在高尔基体上。与特征明确的非网格蛋白包被蛋白ADP - 核糖基化因子（ARF）和β - COP类似，当细胞暴露于布雷菲德菌素A时，Cdc42Hs的核周聚集会迅速分散，这表明它可能也在细胞内脂质和蛋白质运输过程中发挥作用。利用具有可诱导形式ARF的细胞系，我们在此证明了ARF的核苷酸结合状态与Cdc42Hs的亚细胞定位紧密耦合。具体而言，野生型ARF的表达对Cdc42Hs的布雷菲德菌素A敏感性没有影响，而正如ARF和β - COP的情况一样，一种缺乏GTP酶活性的ARF形式（ArF(Q71L)）的表达使这些定位在高尔基体的蛋白对布雷菲德菌素A处理具有抗性（蒂尔等人，1994年；张等人，1994年）。此外，一种对核苷酸亲和力低的ARF突变形式的诱导表达导致在没有布雷菲德菌素A处理的情况下Cdc42Hs的组成性重新分布。这些结果表明，Cdc42Hs可能通过作用于高尔基体中指导质膜极化生长的靶标，在细胞形态发生中发挥作用。
In this study, we have used immunocytochemical and fractionation approaches to provide a description oft he localization of the mammalian Cdc42 protein (designated Cdc42Hs) in vivo. A specific anti-peptide antibody was generated against the C-terminal region of Cdc42Hs. Using affinity-purified preparations of this antibody in indirect immunofluorescence experiments, Cdc42Hs was found to be localized to the Golgi apparatus, Similar to the well-characterized non-clathrin coat proteins ADP-ribosylation factor (ARF) and beta-COP, the perinuclear clustering of Cdc42Hs is rapidly dispersed upon exposure of the cells to the drug brefeldin A, suggesting that it too may play a role in the processes of intracellular lipid and protein transport, Employing cell lines possessing inducible forms of ARF, we demonstrate here a tight coupling of the nucleotide bound state of ARF and the subcellular localization of Cdc42Hs. Specifically, the expression of wild-type ARF had no effect on the brefeldin A sensitivity of Cdc42Hs while, as is the case for ARF and P-COP, expression of a GTPase-deficient form of ARF (ArF(Q71L)) renders these Golgi-localized proteins resistant to brefeldin A treatment (Teal et al., 1994; Zhang et al,, 1994). Moreover, the induced expression of a mutant form of ARF with a low affinity for nucleotide resulted in constitutive redistribution of Cdc42Hs in the absence of brefeldin A treatment. These results suggest that Cdc42Hs may play a role in cell morphogenesis by acting on targets in the Golgi that direct polarized growth at the plasma membrane.