Phosphatidylinositol 4,5-bisphosphate and Arf6-regulated membrane traffic

ADP ribosylation factor (Arf) 6 regulates the movement of membrane between the plasma membrane (PM) and a nonclathrin-derived endosomal compartment an d activates phosphatidylinositol 4-phosphate 5-kinase (PIP 5-kinase), an en zyme that generates phosphatidylinositol 4,5-bisphosphate (PIP2). Here, we show that PIP2 visualized by expressing a fusion protein of the pleckstrin homology domain from PLC delta and green fluorescent protein (PH-GFP), colo calized with Arf6 at the PM and on tubular endosomal structures. Activation of Arf6 by expression of its exchange factor EFA6 stimulated protrusion fo rmation, the uptake of PM into macropinosomes enriched in PIP2, and recycli ng of this membrane back to the PM. By contrast, expression of Arf6 Q67L, a GTP hydrolysis-resistant mutant, induced the formation of PIP2 positive ac tin-coated vacuoles that were unable to recycle membrane back to the PM. PM proteins, such as beta1-integrin, plakoglobin, and major histocompatibilit y complex class I, that normally traffic through the Arf6 endosomal compart ment became trapped in this vacuolar compartment. Overexpression of human P IP 5-kinase a mimicked the effects seen with Arf6 Q67L. These results demon strate that PIP 5-kinase activity and PIP2 turnover controlled by activatio n and inactivation of Arf6 is critical for trafficking through the Arf6 PM- endosomal recycling pathway.