Limits...
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.

Show MeSH

Related in: MedlinePlus

Overexpression of PIP 5-kinase induces the accumulation of PIP2-positive actin-coated vacuoles. HeLa cells were cotransfected with PIP 5-kinase and PH-GFP for 44 h, fixed, and probed with myc antibody to detect PIP 5-kinase (top), with phalloidin (middle), and the surface was stained using Alexa 594–conjugated concanavalin A (bottom). Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196179&req=5

fig5: Overexpression of PIP 5-kinase induces the accumulation of PIP2-positive actin-coated vacuoles. HeLa cells were cotransfected with PIP 5-kinase and PH-GFP for 44 h, fixed, and probed with myc antibody to detect PIP 5-kinase (top), with phalloidin (middle), and the surface was stained using Alexa 594–conjugated concanavalin A (bottom). Bars, 10 μm.

Mentions: The PH-GFP labeling of the vacuoles that accumulate in cells expressing Arf6 Q67L suggests the involvement of PIP2 and therefore a type I PIP 5-kinase in Arf6 function. Overexpression of human PIP 5-kinase α (homologue of the mouse PIP 5-kinase β) alone resulted in cells containing vacuolar structures that were indistinguishable from those formed in cells expressing Arf6 Q67L. The vacuoles labeled with PH-GFP were actin coated and within the cell (Fig. 5) . These vacuoles were also formed in Cos cells expressing PIP 5-kinase (see Fig. 7). Kinase activity was necessary for the formation of vacuoles, since expression of a PIP 5-kinase mutant, D270A, which lacks kinase activity (Mejillano et al., 2001), did not induce their formation (unpublished data). Vacuoles induced in cells expressing human PIP 5-kinase labeled with antibodies to MHC I, integrins, and plakoglobin but not with antibodies to the transferrin receptor (unpublished data) similar to that observed for the expression of Arf6 Q67L (Fig. 4). Induction of vacuoles by PIP 5-kinase appeared to occur downstream of Arf6 activation, since expression of dominant negative Arf6 did not block vacuole formation (unpublished data). Furthermore, at low expression levels barely detected by antibody PIP 5-kinase had no effect on cell morphology but could synergize with wild-type Arf6 to form protrusive structures (unpublished data). In contrast to the results with human PIP 5-kinase α, expression of the mouse α isoform used by Honda et al. (1999), a homologue of human PIP 5-kinase β, was less effective at inducing the vacuolar structures, although it synergized with Arf6 to form protrusions (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)

Overexpression of PIP 5-kinase induces the accumulation of PIP2-positive actin-coated vacuoles. HeLa cells were cotransfected with PIP 5-kinase and PH-GFP for 44 h, fixed, and probed with myc antibody to detect PIP 5-kinase (top), with phalloidin (middle), and the surface was stained using Alexa 594–conjugated concanavalin A (bottom). Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Overexpression of PIP 5-kinase induces the accumulation of PIP2-positive actin-coated vacuoles. HeLa cells were cotransfected with PIP 5-kinase and PH-GFP for 44 h, fixed, and probed with myc antibody to detect PIP 5-kinase (top), with phalloidin (middle), and the surface was stained using Alexa 594–conjugated concanavalin A (bottom). Bars, 10 μm.
Mentions: The PH-GFP labeling of the vacuoles that accumulate in cells expressing Arf6 Q67L suggests the involvement of PIP2 and therefore a type I PIP 5-kinase in Arf6 function. Overexpression of human PIP 5-kinase α (homologue of the mouse PIP 5-kinase β) alone resulted in cells containing vacuolar structures that were indistinguishable from those formed in cells expressing Arf6 Q67L. The vacuoles labeled with PH-GFP were actin coated and within the cell (Fig. 5) . These vacuoles were also formed in Cos cells expressing PIP 5-kinase (see Fig. 7). Kinase activity was necessary for the formation of vacuoles, since expression of a PIP 5-kinase mutant, D270A, which lacks kinase activity (Mejillano et al., 2001), did not induce their formation (unpublished data). Vacuoles induced in cells expressing human PIP 5-kinase labeled with antibodies to MHC I, integrins, and plakoglobin but not with antibodies to the transferrin receptor (unpublished data) similar to that observed for the expression of Arf6 Q67L (Fig. 4). Induction of vacuoles by PIP 5-kinase appeared to occur downstream of Arf6 activation, since expression of dominant negative Arf6 did not block vacuole formation (unpublished data). Furthermore, at low expression levels barely detected by antibody PIP 5-kinase had no effect on cell morphology but could synergize with wild-type Arf6 to form protrusive structures (unpublished data). In contrast to the results with human PIP 5-kinase α, expression of the mouse α isoform used by Honda et al. (1999), a homologue of human PIP 5-kinase β, was less effective at inducing the vacuolar structures, although it synergized with Arf6 to form protrusions (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