Intracellular distribution of Arf proteins in mammalian cells - Arf6 is uniquely localized to the plasma membrane
Intracellular distribution of Arf proteins in mammalian cells - Arf6 is uniquely localized to the plasma membrane
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DOI：
10.1074/jbc.271.36.21767
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发表时间：
1996-09-06
影响因子：
4.8
通讯作者：
Kahn, RA
中科院分区：
文献类型：
--
作者：
Cavenagh, MM;Whitney, JA;Kahn, RA
使用一组亚型特异性多克隆抗体和一组泛Arf单克隆抗体检测了5种人Arf蛋白的亚细胞分布。对培养的哺乳动物细胞进行亚细胞分离，证实Arf6只存在于中国仓鼠卵巢细胞的质膜上，其质膜分布不受GTP γ S（鸟苷5′- o -（3-硫）三磷酸）和brefeldin A （Arf活性的激活剂和抑制剂）的影响。相比之下，Arf蛋白1、3、4和5主要存在于细胞质中，但可以被募集到各种细胞膜上，但不能被募集到质膜上。在GTP γ S的存在下孵卵，GTP γ S促进的胞质Arf蛋白与细胞膜的结合被brefeldin A阻断。Arf6与质膜的稳定结合以及其定位对GTP γ S或brefeldin A的不敏感，揭示了Arf6与其他Arf亚型之间的明显区别。Arf6在质膜上的定位表明，该异构体在质膜上具有独特的细胞作用，但未能在胞内结构（包括网格蛋白包被的囊泡）上发现内源性Arf6，这似乎与Arf6在转染成纤维细胞的外壳结构或核内体组装中的作用不一致（1,2）。
Subcellular distributions of the five human Arf proteins were examined, using a set of isoform-specific polyclonal and a pan-Arf monoclonal antibodies. Subcellular fractionation of cultured mammalian cells allowed the demonstration that Arf6 is uniquely localized to the plasma membranes of Chinese hamster ovary cells, The plasma membrane distrubution was unaffected by either GTP gamma S (guanosine 5'-O-(3-thio)triphosphate) or brefeldin A, an activator and inhibitor of Arf activities, respectively. In contrast, Arf proteins 1, 3, 4, and 5 were predominantly cytosolic but could be recruited to a variety of intracellular membranes, but not plasma membranes, upon incubation in the presence of GTP gamma S. The GTP gamma S-promoted binding of the cytosolic Arf proteins to membranes was blocked by brefeldin A. The stable association of Arf6 with plasma membranes and the insensitivity of its localization to either GTP gamma S or brefeldin A revealed a clear distinction between Arf6 and the other Arf isoforms. Localization of Arf6 to the plasma membrane suggests a unique cellular role for this isoform at the plasma membrane, but failure to find endogenous Arf6 on endocytic structures, including clathrin-coated vesicles, appears inconsistent with the proposed role of Arf6 in assembly of coat structures or endosomes in transfected fibroblasts (1, 2).