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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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ATP/GTPγS triggers clathrin/AP-2 coat recruitment to synaptic membranes in vitro. (A) Immunoblot analysis of untreated, carbonate-washed, or LP2 membranes (6 μg) incubated with 120 μg cytosol plus ATP/GTPγS for clathrin, AP180, α-adaptin, synaptotagmin I, and ARF6. (B–E) Washed 4-μg LP2 membranes were incubated with 80 μg rat brain cytosol in the presence or absence of the indicated nucleotides. Membranes were recovered by centrifugation and analyzed by immunoblotting against clathrin heavy chain (HC), α-adaptin, AP180, Hsc70, tubulin, endophilin I, PIPKIγ, ARF6, and synaptotagmin I as a membrane marker. (B) Clathrin/AP-2 coat components associate with synaptic membranes in a nucleotide-dependent manner. (C) Quantification of the results shown in A from two independent experiments (mean ± SD). Data were normalized with respect to the amount of protein recruited in the absence of nucleotides (fold stimulation). (D) Protein recruitment in the absence of nucleotides or in the presence of ATP plus GTPγS. (E) Protein recruitment in the presence of the indicated nucleotides and with or without the broad-specificity kinase inhibitor A3.
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fig1: ATP/GTPγS triggers clathrin/AP-2 coat recruitment to synaptic membranes in vitro. (A) Immunoblot analysis of untreated, carbonate-washed, or LP2 membranes (6 μg) incubated with 120 μg cytosol plus ATP/GTPγS for clathrin, AP180, α-adaptin, synaptotagmin I, and ARF6. (B–E) Washed 4-μg LP2 membranes were incubated with 80 μg rat brain cytosol in the presence or absence of the indicated nucleotides. Membranes were recovered by centrifugation and analyzed by immunoblotting against clathrin heavy chain (HC), α-adaptin, AP180, Hsc70, tubulin, endophilin I, PIPKIγ, ARF6, and synaptotagmin I as a membrane marker. (B) Clathrin/AP-2 coat components associate with synaptic membranes in a nucleotide-dependent manner. (C) Quantification of the results shown in A from two independent experiments (mean ± SD). Data were normalized with respect to the amount of protein recruited in the absence of nucleotides (fold stimulation). (D) Protein recruitment in the absence of nucleotides or in the presence of ATP plus GTPγS. (E) Protein recruitment in the presence of the indicated nucleotides and with or without the broad-specificity kinase inhibitor A3.

Mentions: Assembly of clathrin- and AP-2–coated endocytotic intermediates was previously reconstituted from lysed nerve terminal lysed synaptosomal membrane fraction (LP2) membranes incubated with brain cytosol and nucleotides (Takei et al., 1996). LP2 membranes mainly consist of synaptic vesicles (SV) and endosomal membranes. Carbonate treatment of LP2 efficiently removed associated clathrin, AP180, and AP-2 (Fig. 1Figure 1.


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)

ATP/GTPγS triggers clathrin/AP-2 coat recruitment to synaptic membranes in vitro. (A) Immunoblot analysis of untreated, carbonate-washed, or LP2 membranes (6 μg) incubated with 120 μg cytosol plus ATP/GTPγS for clathrin, AP180, α-adaptin, synaptotagmin I, and ARF6. (B–E) Washed 4-μg LP2 membranes were incubated with 80 μg rat brain cytosol in the presence or absence of the indicated nucleotides. Membranes were recovered by centrifugation and analyzed by immunoblotting against clathrin heavy chain (HC), α-adaptin, AP180, Hsc70, tubulin, endophilin I, PIPKIγ, ARF6, and synaptotagmin I as a membrane marker. (B) Clathrin/AP-2 coat components associate with synaptic membranes in a nucleotide-dependent manner. (C) Quantification of the results shown in A from two independent experiments (mean ± SD). Data were normalized with respect to the amount of protein recruited in the absence of nucleotides (fold stimulation). (D) Protein recruitment in the absence of nucleotides or in the presence of ATP plus GTPγS. (E) Protein recruitment in the presence of the indicated nucleotides and with or without the broad-specificity kinase inhibitor A3.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: ATP/GTPγS triggers clathrin/AP-2 coat recruitment to synaptic membranes in vitro. (A) Immunoblot analysis of untreated, carbonate-washed, or LP2 membranes (6 μg) incubated with 120 μg cytosol plus ATP/GTPγS for clathrin, AP180, α-adaptin, synaptotagmin I, and ARF6. (B–E) Washed 4-μg LP2 membranes were incubated with 80 μg rat brain cytosol in the presence or absence of the indicated nucleotides. Membranes were recovered by centrifugation and analyzed by immunoblotting against clathrin heavy chain (HC), α-adaptin, AP180, Hsc70, tubulin, endophilin I, PIPKIγ, ARF6, and synaptotagmin I as a membrane marker. (B) Clathrin/AP-2 coat components associate with synaptic membranes in a nucleotide-dependent manner. (C) Quantification of the results shown in A from two independent experiments (mean ± SD). Data were normalized with respect to the amount of protein recruited in the absence of nucleotides (fold stimulation). (D) Protein recruitment in the absence of nucleotides or in the presence of ATP plus GTPγS. (E) Protein recruitment in the presence of the indicated nucleotides and with or without the broad-specificity kinase inhibitor A3.
Mentions: Assembly of clathrin- and AP-2–coated endocytotic intermediates was previously reconstituted from lysed nerve terminal lysed synaptosomal membrane fraction (LP2) membranes incubated with brain cytosol and nucleotides (Takei et al., 1996). LP2 membranes mainly consist of synaptic vesicles (SV) and endosomal membranes. Carbonate treatment of LP2 efficiently removed associated clathrin, AP180, and AP-2 (Fig. 1Figure 1.

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