Jovanovic Olivera A, Brown Fraser D, Donaldson Julie G
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Mol Biol Cell. 2006 Jan;17(1):327-35. doi: 10.1091/mbc.e05-06-0523. Epub 2005 Nov 9.
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 Arf6N48I, 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 N48I 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.
在本研究中，我们调查了磷脂酶D（PLD）在介导细胞中Arf6功能方面的作用。激活PLD有缺陷的Arf6突变体Arf6N48R和Arf6N48I的表达，抑制了向质膜（PM）的膜回收，导致管状内体膜的积累。此外，与野生型Arf6不同，在存在氟化铝的情况下，两种Arf6突变体都不能在内体上产生突起或招募Arf6 GTP酶激活蛋白（GAP）ACAP1。值得注意的是，所有这些表型，包括积累的管状内体、受阻的回收以及无法产生突起和有效招募ACAP，在用1-丁醇处理以抑制PLD产物磷脂酸（PA）形成时，可在未转染细胞或表达野生型Arf6的细胞中重现。此外，表达Arf6N48R或N48I的细胞中存在的大多数缺陷，可通过用预期能提高细胞内PA水平的试剂处理来逆转。这些观察结果共同提供了令人信服的证据，表明内体膜回收和GAP招募需要Arf6对PLD的刺激。
