Limits...
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.

Show MeSH

Related in: MedlinePlus

ARF6 is enriched in synaptic plasma membrane fractions. (A) Polyclonal anti-ARF6 antibodies specifically recognize ARF6, but not ARF1. Top, Ponceau-stained nitrocellulose membrane. 3 μg ARF protein was loaded per lane. Bottom, immunoblot analysis using anti-ARF6 antibodies. (B) Subcellular fractionation of pig brain homogenate according to Maycox et al. (1992). 15 μg protein was loaded per lane and analyzed by immunoblotting against clathrin heavy chain (HC), μ2-adaptin, dynamin I, synaptophysin, and ARF6. H, brain homogenate; P2, crude synaptosomes; P2′, washed synaptosomes; S3, cytosol; LP1, 20,000-g pellet after lysis of synaptosomes; LP2, 55,000-g pellet; CCV, purified clathrin-coated vesicles. (C) Localization of ARF6(Q67L) in transfected cortical neurons. Neurons at 11 days in vitro were fixed and analyzed for the distribution of HA-tagged ARF6(Q67L) and the presynaptic marker protein synaptophysin using a Leica confocal laser microscope. Bar, 10 μm. Note that synaptophysin-positive synapses devoid of ARF6(Q67L) may originate from nontransfected neurons.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172713&req=5

fig3: ARF6 is enriched in synaptic plasma membrane fractions. (A) Polyclonal anti-ARF6 antibodies specifically recognize ARF6, but not ARF1. Top, Ponceau-stained nitrocellulose membrane. 3 μg ARF protein was loaded per lane. Bottom, immunoblot analysis using anti-ARF6 antibodies. (B) Subcellular fractionation of pig brain homogenate according to Maycox et al. (1992). 15 μg protein was loaded per lane and analyzed by immunoblotting against clathrin heavy chain (HC), μ2-adaptin, dynamin I, synaptophysin, and ARF6. H, brain homogenate; P2, crude synaptosomes; P2′, washed synaptosomes; S3, cytosol; LP1, 20,000-g pellet after lysis of synaptosomes; LP2, 55,000-g pellet; CCV, purified clathrin-coated vesicles. (C) Localization of ARF6(Q67L) in transfected cortical neurons. Neurons at 11 days in vitro were fixed and analyzed for the distribution of HA-tagged ARF6(Q67L) and the presynaptic marker protein synaptophysin using a Leica confocal laser microscope. Bar, 10 μm. Note that synaptophysin-positive synapses devoid of ARF6(Q67L) may originate from nontransfected neurons.

Mentions: A role of ARF6 in presynaptic clathrin-mediated vesicle recycling is plausible given the enrichment of the ARF6-specific nucleotide exchange factor mSec7 in nerve terminals (Ashery et al., 1999). pAbs that recognize ARF6 but not ARF1 (Fig. 3Figure 3.


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 is enriched in synaptic plasma membrane fractions. (A) Polyclonal anti-ARF6 antibodies specifically recognize ARF6, but not ARF1. Top, Ponceau-stained nitrocellulose membrane. 3 μg ARF protein was loaded per lane. Bottom, immunoblot analysis using anti-ARF6 antibodies. (B) Subcellular fractionation of pig brain homogenate according to Maycox et al. (1992). 15 μg protein was loaded per lane and analyzed by immunoblotting against clathrin heavy chain (HC), μ2-adaptin, dynamin I, synaptophysin, and ARF6. H, brain homogenate; P2, crude synaptosomes; P2′, washed synaptosomes; S3, cytosol; LP1, 20,000-g pellet after lysis of synaptosomes; LP2, 55,000-g pellet; CCV, purified clathrin-coated vesicles. (C) Localization of ARF6(Q67L) in transfected cortical neurons. Neurons at 11 days in vitro were fixed and analyzed for the distribution of HA-tagged ARF6(Q67L) and the presynaptic marker protein synaptophysin using a Leica confocal laser microscope. Bar, 10 μm. Note that synaptophysin-positive synapses devoid of ARF6(Q67L) may originate from nontransfected neurons.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: ARF6 is enriched in synaptic plasma membrane fractions. (A) Polyclonal anti-ARF6 antibodies specifically recognize ARF6, but not ARF1. Top, Ponceau-stained nitrocellulose membrane. 3 μg ARF protein was loaded per lane. Bottom, immunoblot analysis using anti-ARF6 antibodies. (B) Subcellular fractionation of pig brain homogenate according to Maycox et al. (1992). 15 μg protein was loaded per lane and analyzed by immunoblotting against clathrin heavy chain (HC), μ2-adaptin, dynamin I, synaptophysin, and ARF6. H, brain homogenate; P2, crude synaptosomes; P2′, washed synaptosomes; S3, cytosol; LP1, 20,000-g pellet after lysis of synaptosomes; LP2, 55,000-g pellet; CCV, purified clathrin-coated vesicles. (C) Localization of ARF6(Q67L) in transfected cortical neurons. Neurons at 11 days in vitro were fixed and analyzed for the distribution of HA-tagged ARF6(Q67L) and the presynaptic marker protein synaptophysin using a Leica confocal laser microscope. Bar, 10 μm. Note that synaptophysin-positive synapses devoid of ARF6(Q67L) may originate from nontransfected neurons.
Mentions: A role of ARF6 in presynaptic clathrin-mediated vesicle recycling is plausible given the enrichment of the ARF6-specific nucleotide exchange factor mSec7 in nerve terminals (Ashery et al., 1999). pAbs that recognize ARF6 but not ARF1 (Fig. 3Figure 3.

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