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ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type Igamma.

Krauss M, Kinuta M, Wenk MR, De Camilli P, Takei K, Haucke V - J. Cell Biol. (2003)

Bottom Line: Phosphoinositides have been implicated in nucleating coat assembly by directly binding to several endocytotic proteins including AP-2 and AP180.Here, we show that the stimulatory effect of ATP and GTPgammaS on clathrin coat recruitment is mediated at least in part by increased levels of PIP2.These data suggest a model according to which activation of PIPKIgamma by ARF6-GTP facilitates clathrin-coated pit assembly at the synapse.

View Article: PubMed Central - PubMed

Affiliation: Zentrum für Biochemie und Molekulare Zellbiologie, Dept. of Biochemistry II, University of Göttingen, Humboldtallee 23, Göttingen D-37073, Germany.

ABSTRACT
Clathrin-mediated endocytosis of synaptic vesicle membranes involves the recruitment of clathrin and AP-2 adaptor complexes to the presynaptic plasma membrane. Phosphoinositides have been implicated in nucleating coat assembly by directly binding to several endocytotic proteins including AP-2 and AP180. Here, we show that the stimulatory effect of ATP and GTPgammaS on clathrin coat recruitment is mediated at least in part by increased levels of PIP2. We also provide evidence for a role of ADP-ribosylation factor 6 (ARF6) via direct stimulation of a synaptically enriched phosphatidylinositol 4-phosphate 5-kinase type Igamma (PIPKIgamma), in this effect. These data suggest a model according to which activation of PIPKIgamma by ARF6-GTP facilitates clathrin-coated pit assembly at the synapse.

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ARF6(Q67L) colocalizes with PIPKIγ in transfected cells. Cos7 cells were cotransfected with plasmids encoding ARF6(Q67L)-EGFP (A), ARF6(T27N)-EGFP (B), or PHPLCδ-EGFP (C) and HA-tagged PIPKIγ. 24 h after transfection, the cells were fixed and analyzed by immunofluorescence microscopy. Merged images are shown in A–C (middle panels). Bar, 20 μm.
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fig6: ARF6(Q67L) colocalizes with PIPKIγ in transfected cells. Cos7 cells were cotransfected with plasmids encoding ARF6(Q67L)-EGFP (A), ARF6(T27N)-EGFP (B), or PHPLCδ-EGFP (C) and HA-tagged PIPKIγ. 24 h after transfection, the cells were fixed and analyzed by immunofluorescence microscopy. Merged images are shown in A–C (middle panels). Bar, 20 μm.

Mentions: The biochemical interaction of ARF6 with PIPKIγ was supported by morphological studies of cotransfected cells. Consistent with previous data (Brown et al., 2001), ARF6(Q67L)-EGFP expressed in Cos7 cells was found in peripheral plasma membrane invaginations, vacuolar structures and membrane ruffles. In these structures, it colocalized with cotransfected PIPKIγ-p90 (Fig. 6Figure 6.


ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type Igamma.

Krauss M, Kinuta M, Wenk MR, De Camilli P, Takei K, Haucke V - J. Cell Biol. (2003)

ARF6(Q67L) colocalizes with PIPKIγ in transfected cells. Cos7 cells were cotransfected with plasmids encoding ARF6(Q67L)-EGFP (A), ARF6(T27N)-EGFP (B), or PHPLCδ-EGFP (C) and HA-tagged PIPKIγ. 24 h after transfection, the cells were fixed and analyzed by immunofluorescence microscopy. Merged images are shown in A–C (middle panels). Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172713&req=5

fig6: ARF6(Q67L) colocalizes with PIPKIγ in transfected cells. Cos7 cells were cotransfected with plasmids encoding ARF6(Q67L)-EGFP (A), ARF6(T27N)-EGFP (B), or PHPLCδ-EGFP (C) and HA-tagged PIPKIγ. 24 h after transfection, the cells were fixed and analyzed by immunofluorescence microscopy. Merged images are shown in A–C (middle panels). Bar, 20 μm.
Mentions: The biochemical interaction of ARF6 with PIPKIγ was supported by morphological studies of cotransfected cells. Consistent with previous data (Brown et al., 2001), ARF6(Q67L)-EGFP expressed in Cos7 cells was found in peripheral plasma membrane invaginations, vacuolar structures and membrane ruffles. In these structures, it colocalized with cotransfected PIPKIγ-p90 (Fig. 6Figure 6.

Bottom Line: Phosphoinositides have been implicated in nucleating coat assembly by directly binding to several endocytotic proteins including AP-2 and AP180.Here, we show that the stimulatory effect of ATP and GTPgammaS on clathrin coat recruitment is mediated at least in part by increased levels of PIP2.These data suggest a model according to which activation of PIPKIgamma by ARF6-GTP facilitates clathrin-coated pit assembly at the synapse.

View Article: PubMed Central - PubMed

Affiliation: Zentrum für Biochemie und Molekulare Zellbiologie, Dept. of Biochemistry II, University of Göttingen, Humboldtallee 23, Göttingen D-37073, Germany.

ABSTRACT
Clathrin-mediated endocytosis of synaptic vesicle membranes involves the recruitment of clathrin and AP-2 adaptor complexes to the presynaptic plasma membrane. Phosphoinositides have been implicated in nucleating coat assembly by directly binding to several endocytotic proteins including AP-2 and AP180. Here, we show that the stimulatory effect of ATP and GTPgammaS on clathrin coat recruitment is mediated at least in part by increased levels of PIP2. We also provide evidence for a role of ADP-ribosylation factor 6 (ARF6) via direct stimulation of a synaptically enriched phosphatidylinositol 4-phosphate 5-kinase type Igamma (PIPKIgamma), in this effect. These data suggest a model according to which activation of PIPKIgamma by ARF6-GTP facilitates clathrin-coated pit assembly at the synapse.

Show MeSH
Related in: MedlinePlus