The ADP-ribosylation factors (Arfs) are a family of GTP binding proteins that regulate membrane traffic and organelle structure in the cell. We have been studying the cellular function of Arf6, an Arf that affects membrane movement between the plasma membrane and an endosomal compartment and the actin cytoskeleton. In many cells this pathway is the route followed by membrane proteins that lack clathrin localization sequences and hence are endocytosed into cells by non-clathrin mechanisms. Recycling of these membrane proteins back to the plasma membrane requires Arf6 activation whereas entry of these proteins into the endosomal recycling system requires hydrolysis of GTP-bound Arf6. While this pathway has been shown to influence plasma membrane remodeling events such as phagocytosis, and cell spreading and migration, the molecules involved in mediating Arf6 responses remain to be identified. Membrane phosphoinositides, such as phosphatidylinositol-4,5-bisphosphate (PIP2), have been implicated in multiple processes such as membrane trafficking and actin remodeling. We have investigated the subcellular distribution of PIP2 and its role in Arf6 function. We find that Arf6 regulates the localization of PIP2 within the cell. There is also evidence that Arf6, when activated, may stimulate the activity of an enzyme, phosphatidylinositol 4 phosphate 5-kinase, responsible for generating PIP2 on membranes. Indeed, we find that overexpression of this enzyme in cells recreates a phenotype similar to that observed when activated Arf6 mutants are expressed in cells. Notably, these cells accumulate massive amounts of PIP2-enriched plasma membrane in vacuolar-like structures that are coated with actin. Ordinarily, the inactivation of Arf6, would prevent such aberrant vacuole accumulations from forming. In a collaboration with Paul Randazzo at the NCI, we have identified two new Arf GAP molecules ACAP1 and ACAP2, that function to inactivate Arf6. Thus, Arf6 activation and inactivation may mediate the regulation of PIP2 synthesis and/or PIP2 trafficking at defined membrane locations. Another project in the lab concerns the fate of proteins that move through the Arf6 endosome. We find that proteins that traverse this pathway get degraded in lysosomes and gain access to the lysosomes via the Arf6 endosome. This alternative route to the lysosome contrasts to the route followed by proteins that are endocytosed into cells by clathrin-mediated mechanisms. Both pathways do converge, however, in a late endosomal compartment. We are in the process of further characterizing this alternative route to the lysosome since many important molecules, including MHC class I, integrins, src, and catenins, have been observed to traffic through this pathway. This route to degradation may be utilized or subverted by viruses or pathogenic bacteria known to influence lysosomal targeting mechanisms.
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