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Published 3 September 2001. doi:10.1083/jcb.200103107
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© The Rockefeller University Press, 0021-9525/2001/9/1007 $5.00
The Journal of Cell Biology, Volume 154, Number 5, September 3, 2001 1007-1018

Article

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

Fraser D. Brown1, Andrew L. Rozelle3, Helen L. Yin3, Tamás Balla2 and Julie G. Donaldson1

1 Laboratory of Cell Biology, National Heart Lung and Blood Institute
2 Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892
3 Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390

Address correspondence to Julie G. Donaldson, Laboratory of Cell Biology, Bldg. 50, Rm. 2503, Bethesda, MD 20892. Tel.: (301) 402-2907. Fax: (301) 402-1519. E-mail: jdonalds{at}helix.nih.gov

ADP-ribosylation factor (Arf) 6 regulates the movement of membrane between the plasma membrane (PM) and a nonclathrin-derived endosomal compartment and activates phosphatidylinositol 4-phosphate 5-kinase (PIP 5-kinase), an enzyme 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), colocalized with Arf6 at the PM and on tubular endosomal structures. Activation of Arf6 by expression of its exchange factor EFA6 stimulated protrusion formation, the uptake of PM into macropinosomes enriched in PIP2, and recycling of this membrane back to the PM. By contrast, expression of Arf6 Q67L, a GTP hydrolysis-resistant mutant, induced the formation of PIP2-positive actin-coated vacuoles that were unable to recycle membrane back to the PM. PM proteins, such as ß1-integrin, plakoglobin, and major histocompatibility complex class I, that normally traffic through the Arf6 endosomal compartment became trapped in this vacuolar compartment. Overexpression of human PIP 5-kinase {alpha} mimicked the effects seen with Arf6 Q67L. These results demonstrate that PIP 5-kinase activity and PIP2 turnover controlled by activation and inactivation of Arf6 is critical for trafficking through the Arf6 PM-endosomal recycling pathway.

Key Words: Arf6; membrane traffic; phosphatidylinositol 4,5-bisphosphate; PIP2; PIP 5-kinase


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