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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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Effects of PIP2 masking or degradation on clathrin/AP-2–coated pit assembly. (A) Recombinant human PHPLCδ1 inhibits ATP/GTPγS-induced membrane recruitment of clathrin/AP-2 coat components. Clathrin/AP-2 recruitment onto presynaptic membranes was performed as described in Fig. 1 in the presence or absence of ATP/GTPγS, purified PHPLCδ1, or BSA. Samples were analyzed by quantitative Western blotting using antisera against clathrin heavy chain (HC), α-adaptin, synaptotagmin I as a membrane marker, and Hsc70 as a control. (B and C) Overexpression of membrane-targeted HA-tagged inositol 5-phosphate phosphatase domain of synaptojanin 1 (HA-IPP1-CAAX) mislocalizes clathrin and AP-2. Cos7 cells expressing HA-IPP1-CAAX were fixed 24 h after transfection and analyzed for the distribution of AP-2 (B) or clathrin (C) by immunofluorescence microscopy. Bar, 20 μm.
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fig8: Effects of PIP2 masking or degradation on clathrin/AP-2–coated pit assembly. (A) Recombinant human PHPLCδ1 inhibits ATP/GTPγS-induced membrane recruitment of clathrin/AP-2 coat components. Clathrin/AP-2 recruitment onto presynaptic membranes was performed as described in Fig. 1 in the presence or absence of ATP/GTPγS, purified PHPLCδ1, or BSA. Samples were analyzed by quantitative Western blotting using antisera against clathrin heavy chain (HC), α-adaptin, synaptotagmin I as a membrane marker, and Hsc70 as a control. (B and C) Overexpression of membrane-targeted HA-tagged inositol 5-phosphate phosphatase domain of synaptojanin 1 (HA-IPP1-CAAX) mislocalizes clathrin and AP-2. Cos7 cells expressing HA-IPP1-CAAX were fixed 24 h after transfection and analyzed for the distribution of AP-2 (B) or clathrin (C) by immunofluorescence microscopy. Bar, 20 μm.

Mentions: Finally, if the ARF6-mediated stimulation of PIPKIγ played a major role in mediating the effect of ATP/GTPγS on clathrin/AP-2 recruitment, one would expect that masking PIP2 with a PIP2-binding module or degradation of PIP2 by an inositol phosphatase would inhibit such recruitment to synaptic membranes or in living cells. We performed recruitment experiments in the presence of the recombinant PH domain of human PLCδ1 (PHPLCδ1), a specific PI(4,5)P2-binding protein. Addition of recombinant PHPLCδ1 to synaptic membranes inhibited the ATP/GTPγS-induced binding of clathrin and AP-2 in a concentration-dependent manner, whereas addition of GST had no effect (Fig. 8Figure 8.


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)

Effects of PIP2 masking or degradation on clathrin/AP-2–coated pit assembly. (A) Recombinant human PHPLCδ1 inhibits ATP/GTPγS-induced membrane recruitment of clathrin/AP-2 coat components. Clathrin/AP-2 recruitment onto presynaptic membranes was performed as described in Fig. 1 in the presence or absence of ATP/GTPγS, purified PHPLCδ1, or BSA. Samples were analyzed by quantitative Western blotting using antisera against clathrin heavy chain (HC), α-adaptin, synaptotagmin I as a membrane marker, and Hsc70 as a control. (B and C) Overexpression of membrane-targeted HA-tagged inositol 5-phosphate phosphatase domain of synaptojanin 1 (HA-IPP1-CAAX) mislocalizes clathrin and AP-2. Cos7 cells expressing HA-IPP1-CAAX were fixed 24 h after transfection and analyzed for the distribution of AP-2 (B) or clathrin (C) by immunofluorescence microscopy. Bar, 20 μm.
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Related In: Results  -  Collection

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fig8: Effects of PIP2 masking or degradation on clathrin/AP-2–coated pit assembly. (A) Recombinant human PHPLCδ1 inhibits ATP/GTPγS-induced membrane recruitment of clathrin/AP-2 coat components. Clathrin/AP-2 recruitment onto presynaptic membranes was performed as described in Fig. 1 in the presence or absence of ATP/GTPγS, purified PHPLCδ1, or BSA. Samples were analyzed by quantitative Western blotting using antisera against clathrin heavy chain (HC), α-adaptin, synaptotagmin I as a membrane marker, and Hsc70 as a control. (B and C) Overexpression of membrane-targeted HA-tagged inositol 5-phosphate phosphatase domain of synaptojanin 1 (HA-IPP1-CAAX) mislocalizes clathrin and AP-2. Cos7 cells expressing HA-IPP1-CAAX were fixed 24 h after transfection and analyzed for the distribution of AP-2 (B) or clathrin (C) by immunofluorescence microscopy. Bar, 20 μm.
Mentions: Finally, if the ARF6-mediated stimulation of PIPKIγ played a major role in mediating the effect of ATP/GTPγS on clathrin/AP-2 recruitment, one would expect that masking PIP2 with a PIP2-binding module or degradation of PIP2 by an inositol phosphatase would inhibit such recruitment to synaptic membranes or in living cells. We performed recruitment experiments in the presence of the recombinant PH domain of human PLCδ1 (PHPLCδ1), a specific PI(4,5)P2-binding protein. Addition of recombinant PHPLCδ1 to synaptic membranes inhibited the ATP/GTPγS-induced binding of clathrin and AP-2 in a concentration-dependent manner, whereas addition of GST had no effect (Fig. 8Figure 8.

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