Arf GTP-binding proteins and membrane traffic

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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.