An effector domain mutant of Arf6 implicates phospholipase D in endosomal membrane recycling
An effector domain mutant of Arf6 implicates phospholipase D in endosomal membrane recycling
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DOI：
10.1091/mbc.e05-06-0523
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发表时间：
2006-01-01
影响因子：
3.3
通讯作者：
Donaldson, JG
中科院分区：
文献类型：
--
作者：
Jovanovic, OA;Brown, FD;Donaldson, JG
在这项研究中，我们研究了磷脂酶D（PLD）在介导Arf 6功能的细胞中的作用。在激活PLD方面有缺陷的Arf 6突变体Arf 6 N48 R和Arf 6 N481的表达抑制了膜向质膜（PM）的再循环，导致管状内体膜的积累。此外，与野生型Arf 6不同，在氟化铝的存在下，Arf 6突变体都不能产生突起或将Arf 6 GTdR激活蛋白（GAP）ACAP 1募集到内体上。值得注意的是，所有这些表型，包括积累的管状内体，阻断再循环，以及未能有效地制造突起和招募ACAP，可以在未转染的细胞或表达野生型Arf 6的细胞中通过用1-丁醇处理以抑制磷脂酸（PA）（PLD的产物）的形成来重建。此外，表达Arf 6 N48 R或N481的细胞中存在的大多数缺陷可以通过用预期提高细胞中PA水平的试剂处理来逆转。总之，这些观察结果提供了令人信服的证据，即Arf 6刺激PLD是内体膜再循环和GAP募集所必需的。
In this study, we investigated the role of phospholipase D (PLD) in mediating Arf6 function in cells. Expression of Arf6 mutants that are defective in activating PLD, Arf6N48R and Arf6N481, inhibited membrane recycling to the plasma membrane (PM), resulting in an accumulation of tubular endosomal membranes. Additionally, unlike wild-type Arf6, neither Arf6 mutant could generate protrusions or recruit the Arf6 GTPase activating protein (GAP) ACAP1 onto the endosome in the presence of aluminum fluoride. Remarkably, all of these phenotypes, including accumulated tubular endosomes, blocked recycling, and failure to make protrusions and recruit ACAP effectively, could be recreated in either untransfected cells or cells expressing wild-type Arf6 by treatment with 1-butanol to inhibit the formation of phosphatidic acid (PA), the product of PLD. Moreover, most of the defects present in cells expressing Arf6N48R or N481 could be reversed by treatment with agents expected to elevate PA levels in cells. Together, these observations provide compelling evidence that Arf6 stimulation of PLD is required for endosomal membrane recycling and GAP recruitment.