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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-GTP stimulates clathrin/AP-2 recruitment to synaptic membranes. (A) Coat recruitment to LP2 membranes was performed and analyzed as described in the legend to Fig. 1. Samples containing 1-μM ARF6 mutants were incubated in the presence of 200 μM GTP and analyzed by quantitative immunoblotting against clathrin heavy chain (HC), AP180, α-adaptin, Hsc70, and synaptotagmin I. (B) Dose dependence of the stimulatory effect of ARF6(Q67L) on clathrin recruitment to membranes as shown in A. Values were normalized to the amount of clathrin bound in the presence of ATP and GTPγS (100%). (C) Dose dependence of the inhibitory effect of ARF6(T27N) on clathrin recruitment to membranes as shown in A. Values were normalized to the amount of clathrin bound in the presence of ATP and GTPγS (100%).
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fig4: ARF6-GTP stimulates clathrin/AP-2 recruitment to synaptic membranes. (A) Coat recruitment to LP2 membranes was performed and analyzed as described in the legend to Fig. 1. Samples containing 1-μM ARF6 mutants were incubated in the presence of 200 μM GTP and analyzed by quantitative immunoblotting against clathrin heavy chain (HC), AP180, α-adaptin, Hsc70, and synaptotagmin I. (B) Dose dependence of the stimulatory effect of ARF6(Q67L) on clathrin recruitment to membranes as shown in A. Values were normalized to the amount of clathrin bound in the presence of ATP and GTPγS (100%). (C) Dose dependence of the inhibitory effect of ARF6(T27N) on clathrin recruitment to membranes as shown in A. Values were normalized to the amount of clathrin bound in the presence of ATP and GTPγS (100%).

Mentions: To investigate directly whether ARF6 can trigger AP-2/clathrin coat formation, we tested recombinant myristoylated ARF6 mutants for their ability to facilitate clathrin/AP-2 recruitment to carbonate-washed LP2 membranes devoid of endogenous membrane-bound ARF6 (Fig. 1 A). Reactions were performed in the presence of 200 μM GTP instead of GTPγS to prevent generalized activation of all endogenous GTPases. Addition of 1 μM recombinant ARF6(Q67L), an ARF6 mutant locked in the GTP-bound state due to defective GTP hydrolysis, was sufficient to promote recruitment of clathrin, AP-2, and AP180 as efficiently as addition of GTPγS in the absence of exogenous ARF (Fig. 4Figure 4.


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-GTP stimulates clathrin/AP-2 recruitment to synaptic membranes. (A) Coat recruitment to LP2 membranes was performed and analyzed as described in the legend to Fig. 1. Samples containing 1-μM ARF6 mutants were incubated in the presence of 200 μM GTP and analyzed by quantitative immunoblotting against clathrin heavy chain (HC), AP180, α-adaptin, Hsc70, and synaptotagmin I. (B) Dose dependence of the stimulatory effect of ARF6(Q67L) on clathrin recruitment to membranes as shown in A. Values were normalized to the amount of clathrin bound in the presence of ATP and GTPγS (100%). (C) Dose dependence of the inhibitory effect of ARF6(T27N) on clathrin recruitment to membranes as shown in A. Values were normalized to the amount of clathrin bound in the presence of ATP and GTPγS (100%).
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Related In: Results  -  Collection

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fig4: ARF6-GTP stimulates clathrin/AP-2 recruitment to synaptic membranes. (A) Coat recruitment to LP2 membranes was performed and analyzed as described in the legend to Fig. 1. Samples containing 1-μM ARF6 mutants were incubated in the presence of 200 μM GTP and analyzed by quantitative immunoblotting against clathrin heavy chain (HC), AP180, α-adaptin, Hsc70, and synaptotagmin I. (B) Dose dependence of the stimulatory effect of ARF6(Q67L) on clathrin recruitment to membranes as shown in A. Values were normalized to the amount of clathrin bound in the presence of ATP and GTPγS (100%). (C) Dose dependence of the inhibitory effect of ARF6(T27N) on clathrin recruitment to membranes as shown in A. Values were normalized to the amount of clathrin bound in the presence of ATP and GTPγS (100%).
Mentions: To investigate directly whether ARF6 can trigger AP-2/clathrin coat formation, we tested recombinant myristoylated ARF6 mutants for their ability to facilitate clathrin/AP-2 recruitment to carbonate-washed LP2 membranes devoid of endogenous membrane-bound ARF6 (Fig. 1 A). Reactions were performed in the presence of 200 μM GTP instead of GTPγS to prevent generalized activation of all endogenous GTPases. Addition of 1 μM recombinant ARF6(Q67L), an ARF6 mutant locked in the GTP-bound state due to defective GTP hydrolysis, was sufficient to promote recruitment of clathrin, AP-2, and AP180 as efficiently as addition of GTPγS in the absence of exogenous ARF (Fig. 4Figure 4.

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