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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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Model for the roles of PIP 5-kinase and PIP2 on Arf6-regulated membrane traffic. Under normal growth conditions (Constitutive, bottom half), membrane is transported from the tubular endosome and fuses with the plasma membrane in an Arf6-GTP–dependent step that activates PIP 5-kinase. Upon internalization from the plasma membrane, GTP on Arf6 is hydrolyzed, generating Arf6-GDP, allowing PIP2 levels to be lowered. The membrane fuses with the recycling endosome and then returns to the PM completing the cycle. When GEF activity is elevated, as in overexpression of EFA6 (Stimulated, top half) or transiently upon addition of serum to serum-starved cells, protrusions are formed. This leads to the uptake of membrane into dynamic PIP2-positive macropinosomes that can rapidly turn over, since Arf6-GTP can be converted to Arf6-GDP, allowing PIP2 levels to decrease and sorting through the recycling endosome to proceed. However, expression of Arf6Q67L or PIP 5-kinase results in an accumulation of PIP2 endosomes that tether to and fuse with one another, forming large vacuolar membrane structures (dotted arrow), preventing membrane from recycling back to the PM.
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fig8: Model for the roles of PIP 5-kinase and PIP2 on Arf6-regulated membrane traffic. Under normal growth conditions (Constitutive, bottom half), membrane is transported from the tubular endosome and fuses with the plasma membrane in an Arf6-GTP–dependent step that activates PIP 5-kinase. Upon internalization from the plasma membrane, GTP on Arf6 is hydrolyzed, generating Arf6-GDP, allowing PIP2 levels to be lowered. The membrane fuses with the recycling endosome and then returns to the PM completing the cycle. When GEF activity is elevated, as in overexpression of EFA6 (Stimulated, top half) or transiently upon addition of serum to serum-starved cells, protrusions are formed. This leads to the uptake of membrane into dynamic PIP2-positive macropinosomes that can rapidly turn over, since Arf6-GTP can be converted to Arf6-GDP, allowing PIP2 levels to decrease and sorting through the recycling endosome to proceed. However, expression of Arf6Q67L or PIP 5-kinase results in an accumulation of PIP2 endosomes that tether to and fuse with one another, forming large vacuolar membrane structures (dotted arrow), preventing membrane from recycling back to the PM.

Mentions: We next examined the distribution of PH-GFP in cells expressing the constitutively active mutant of Arf6, Q67L. Previous attempts to understand the phenotype of cells expressing Arf6 Q67L have been hampered by the lack of a clear characterization of the onset and nature of the membrane traffic block imposed. These cells are somewhat protrusive, although not particularly F-actin rich (Radhakrishna et al., 1996), and often take on distorted cell morphology. Furthermore, immunofluorescent localization of Arf6 is poorly resolved in these cells (Peters et al., 1995; Radhakrishna et al., 1996). We assessed the distribution of PIP2 in cells expressing Arf6 Q67L after a 44-h transfection and found that PH-GFP clearly labeled distinct membrane structures in these cells (Fig. 3) . The vacuoles, particularly those in large clusters, were coated with actin and were inside the cells as labeling of the cell surface with fluorescent lectin in fixed cells (shown in Fig. 3) or with the lipid dye, DiI, in live cells (unpublished data) did not label these structures. These actin-coated, PH-GFP–labeled structures were also observed in other cell types including BHK (unpublished data) and Cos cells (see Fig. 8) . However, induction of PIP2-positive vacuoles was specific to Arf6 and was not observed with Arf1. Expression of the constitutively active Arf1 mutant, Q71L, which stably associates with the Golgi complex and causes alterations in Golgi morphology (Zhang et al., 1994), did not induce the formation of PIP2-positive vacuoles nor did the PH-GFP construct label the Golgi complex (unpublished data).


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

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

Model for the roles of PIP 5-kinase and PIP2 on Arf6-regulated membrane traffic. Under normal growth conditions (Constitutive, bottom half), membrane is transported from the tubular endosome and fuses with the plasma membrane in an Arf6-GTP–dependent step that activates PIP 5-kinase. Upon internalization from the plasma membrane, GTP on Arf6 is hydrolyzed, generating Arf6-GDP, allowing PIP2 levels to be lowered. The membrane fuses with the recycling endosome and then returns to the PM completing the cycle. When GEF activity is elevated, as in overexpression of EFA6 (Stimulated, top half) or transiently upon addition of serum to serum-starved cells, protrusions are formed. This leads to the uptake of membrane into dynamic PIP2-positive macropinosomes that can rapidly turn over, since Arf6-GTP can be converted to Arf6-GDP, allowing PIP2 levels to decrease and sorting through the recycling endosome to proceed. However, expression of Arf6Q67L or PIP 5-kinase results in an accumulation of PIP2 endosomes that tether to and fuse with one another, forming large vacuolar membrane structures (dotted arrow), preventing membrane from recycling back to the PM.
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Related In: Results  -  Collection

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fig8: Model for the roles of PIP 5-kinase and PIP2 on Arf6-regulated membrane traffic. Under normal growth conditions (Constitutive, bottom half), membrane is transported from the tubular endosome and fuses with the plasma membrane in an Arf6-GTP–dependent step that activates PIP 5-kinase. Upon internalization from the plasma membrane, GTP on Arf6 is hydrolyzed, generating Arf6-GDP, allowing PIP2 levels to be lowered. The membrane fuses with the recycling endosome and then returns to the PM completing the cycle. When GEF activity is elevated, as in overexpression of EFA6 (Stimulated, top half) or transiently upon addition of serum to serum-starved cells, protrusions are formed. This leads to the uptake of membrane into dynamic PIP2-positive macropinosomes that can rapidly turn over, since Arf6-GTP can be converted to Arf6-GDP, allowing PIP2 levels to decrease and sorting through the recycling endosome to proceed. However, expression of Arf6Q67L or PIP 5-kinase results in an accumulation of PIP2 endosomes that tether to and fuse with one another, forming large vacuolar membrane structures (dotted arrow), preventing membrane from recycling back to the PM.
Mentions: We next examined the distribution of PH-GFP in cells expressing the constitutively active mutant of Arf6, Q67L. Previous attempts to understand the phenotype of cells expressing Arf6 Q67L have been hampered by the lack of a clear characterization of the onset and nature of the membrane traffic block imposed. These cells are somewhat protrusive, although not particularly F-actin rich (Radhakrishna et al., 1996), and often take on distorted cell morphology. Furthermore, immunofluorescent localization of Arf6 is poorly resolved in these cells (Peters et al., 1995; Radhakrishna et al., 1996). We assessed the distribution of PIP2 in cells expressing Arf6 Q67L after a 44-h transfection and found that PH-GFP clearly labeled distinct membrane structures in these cells (Fig. 3) . The vacuoles, particularly those in large clusters, were coated with actin and were inside the cells as labeling of the cell surface with fluorescent lectin in fixed cells (shown in Fig. 3) or with the lipid dye, DiI, in live cells (unpublished data) did not label these structures. These actin-coated, PH-GFP–labeled structures were also observed in other cell types including BHK (unpublished data) and Cos cells (see Fig. 8) . However, induction of PIP2-positive vacuoles was specific to Arf6 and was not observed with Arf1. Expression of the constitutively active Arf1 mutant, Q71L, which stably associates with the Golgi complex and causes alterations in Golgi morphology (Zhang et al., 1994), did not induce the formation of PIP2-positive vacuoles nor did the PH-GFP construct label the Golgi complex (unpublished data).

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