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

Brown FD, Rozelle AL, Yin HL, Balla T, Donaldson JG - J. Cell Biol. (2001)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, National Heart Lung and Blood Institute, National Institute of Health, Bethesda, MD 20892, USA.

ABSTRACT
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.

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Overexpression of Arf6 Q67L traps proteins that traffic through the Arf6 compartment but not the transferrin receptor into PIP2-positive vacuoles. HeLa cells were transfected with Arf6 Q67L and PH-GFP and probed with antibodies against MHC I, β1-integrin, plakoglobin, and the transferrin receptor as shown. Bars, 10 μm.
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fig4: Overexpression of Arf6 Q67L traps proteins that traffic through the Arf6 compartment but not the transferrin receptor into PIP2-positive vacuoles. HeLa cells were transfected with Arf6 Q67L and PH-GFP and probed with antibodies against MHC I, β1-integrin, plakoglobin, and the transferrin receptor as shown. Bars, 10 μm.

Mentions: The vacuolar membranes accumulated integral and peripheral membrane proteins that traffic through the Arf6 endosome in untransfected cells. As can be seen in Fig. 4 , MHC I, β1-integrin, and plakoglobin (γ-catenin) were all enriched in the PH-GFP–labeled structures induced by expression of Arf6 Q67L. The normal distribution of these proteins in cells not expressing Q67L can also be observed (Fig. 4). In contrast, transferrin receptor distribution in cells expressing Q67L was similar to that in untransfected cells, and transferrin receptor was absent from these vacuolar structures, consistent with the distinction between the clathrin-coated and Arf6-regulated endocytic recycling systems in HeLa cells as observed previously (Radhakrishna and Donaldson, 1997).


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

Brown FD, Rozelle AL, Yin HL, Balla T, Donaldson JG - J. Cell Biol. (2001)

Overexpression of Arf6 Q67L traps proteins that traffic through the Arf6 compartment but not the transferrin receptor into PIP2-positive vacuoles. HeLa cells were transfected with Arf6 Q67L and PH-GFP and probed with antibodies against MHC I, β1-integrin, plakoglobin, and the transferrin receptor as shown. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196179&req=5

fig4: Overexpression of Arf6 Q67L traps proteins that traffic through the Arf6 compartment but not the transferrin receptor into PIP2-positive vacuoles. HeLa cells were transfected with Arf6 Q67L and PH-GFP and probed with antibodies against MHC I, β1-integrin, plakoglobin, and the transferrin receptor as shown. Bars, 10 μm.
Mentions: The vacuolar membranes accumulated integral and peripheral membrane proteins that traffic through the Arf6 endosome in untransfected cells. As can be seen in Fig. 4 , MHC I, β1-integrin, and plakoglobin (γ-catenin) were all enriched in the PH-GFP–labeled structures induced by expression of Arf6 Q67L. The normal distribution of these proteins in cells not expressing Q67L can also be observed (Fig. 4). In contrast, transferrin receptor distribution in cells expressing Q67L was similar to that in untransfected cells, and transferrin receptor was absent from these vacuolar structures, consistent with the distinction between the clathrin-coated and Arf6-regulated endocytic recycling systems in HeLa cells as observed previously (Radhakrishna and Donaldson, 1997).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, National Heart Lung and Blood Institute, National Institute of Health, Bethesda, MD 20892, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus