Arf GTP-binding proteins and membrane traffic

ARF GTP酶调节细胞器的膜运输和结构。Arf1和Arf6是研究最多的ARF，分别在高尔基复合体和质膜上发挥功能。Arf1和Arf6都可以通过激活磷脂酰肌醇4-磷酸-5-激酶(PIP-5-K)和磷脂酶D(PLD)作用于细胞膜来改变脂质组成。由此产生的脂类，磷脂酰肌醇4，5-二磷酸(PIP2)和磷脂酸(PA)可以将特定的蛋白质募集到膜上，改变膜的行为，或转化为其他脂类。我们一直在研究Arf6在调控不依赖于笼蛋白的内体内化和再循环途径中的作用，以前报道过Arf6激活PIP5-激酶和产生PIP2的能力与Arf6作用的许多方面有关。我们想要研究Arf6对PLD的激活是否对Arf6功能的任何方面都很重要。为此，我们表达了Arf6、N48R和N48I的效应域突变体，这些突变体以前被证明在激活PLD的能力方面受到了损害，同时保持了激活PIP 5-激酶的能力。我们发现，Arf6N48R或N48I的表达导致含有内吞货物和PIP2的内膜堆积(Jovanovic等人，2006年)。这些突变导致内细胞膜再循环受阻，表现为显性负表型。这表明，内吞循环回到质膜需要产生PA，可能还需要下一代二酰甘油(DAG)的生成。结合我们所做的其他观察，这表明内细胞膜回到质膜的循环需要Rab22、Rab11的活性，特别是Arf6激活PLD的能力。研究正在进行中，以确定PA是否是囊泡形成、分裂或融合所必需的。

Arf GTPases regulate membrane traffic and the structure of organelles. Arf1 and Arf6 are the most studied Arfs with functions at the Golgi complex and plasma membrane, respectively. Both Arf1 and Arf6 can act at membranes to alter lipid composition by activating phosphatidylinositol 4-phosphate 5-kinase (PIP 5-kinase) and phospholipase D (PLD). The resultant generated lipids, phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA) can recruit specific proteins to the membranes, alter the behavior of the membrane, or be converted to other lipid species. We have been studying the role of Arf6 in regulating a clathrin-independent endosomal internalization and recycling pathway and previously reported that the ability of Arf6 to activate PIP 5-kinase and generate PIP2 was responsible for many aspects of Arf6 action. We wanted to examine whether Arf6 activation of PLD was important for any aspect of Arf6 function. To do this, we expressed effector domain mutants of Arf6, N48R and N48I, that were previously shown to be impaired in their ability to activate PLD while maintaining their ability to activate PIP 5-kinase. We found that expression of Arf6N48R or N48I resulted in an accumulation of internal endosomal membranes that contained endocytic cargo and PIP2 (Jovanovic et al 2006). These mutants caused a dominant negative phenotype in that endocytic membrane recycling was blocked. This indicates that endocytic recycling back to the plasma membrane requires the generation of PA and possibly the subsequent generation diacylglycerol (DAG). Together with other observations that we have made, this indicates that recycling of endocytic membrane back to the plasma membrane requires the activity of Rab22, Rab11and specifically the ability of Arf6 to activate PLD. Studies are ongoing to ascertain whether the PA is required for vesicle formation, fission or fusion.