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 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 PLCdelta 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 beta1-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.
Brown
F D
FD
Laboratory of Cell Biology, National Heart Lung and Blood Institute, National Institute of Health, Bethesda, MD 20892, USA.
Rozelle
A L
AL
Yin
H L
HL
Balla
T
T
Donaldson
J G
JG
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D017930:Genes, Reporter; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009419:Nerve Tissue Proteins; D010445:Peptide Elongation Factors / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D054803:Phospholipase C delta; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology*; D015375:Receptors, Interleukin-2 / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism
D000089982:ADP-Ribosylation Factor 6; D020662:Guanine Nucleotide Exchange Factors; D007527:Isoenzymes; D009419:Nerve Tissue Proteins; C109970:PSD protein, human; D010445:Peptide Elongation Factors; D019269:Phosphatidylinositol 4,5-Diphosphate; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins; D010738:Type C Phospholipases; D054803:Phospholipase C delta; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human