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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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Activated ARF6 interacts with PIPKIγ in brain. (A) ARF6(Q67L) but not ARF6(T27N) specifically affinity purifies PIPKIγ. Western blot analysis of proteins pulled down by GST and GST–ARF6 fusion proteins (80 μg) from a detergent extract of rat brain. Samples were analyzed by SDS-PAGE and immunoblotting. 5% Std., 5% of the extract used for affinity purification. (B) PIPKIγ specifically interacts with ARF6(Q67L). Immunoblot analysis of PIPKIγ affinity purified with myristoylated His6-tagged ARF6(Q67L), ARF1(Q71L), or arfaptin 2 as described under A. 5% Std., 5% of the extract used for affinity purification. (C) PIPKIγ can be cross-linked to ARF6(Q67L) during recruitment of clathrin/AP-2 to synaptic membranes. LP2-membranes were incubated with brain cytosol, myristoylated His6-tagged ARF6, and nucleotides. DTSP was added where indicated. His6-tagged ARF6 was recovered and cross-linked proteins were analyzed by immunoblotting. Top, immunoblot analysis with antisera against PIPKIγ, AP180, large and medium subunits of the AP-2 complex β1/2-adaptin, α-adaptin, and μ2-adaptin, respectively, and clathrin heavy chain (HC). Bottom, Coomassie-stained gel demonstrating that equal amounts of ARF6 have been recovered in each sample.
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fig5: Activated ARF6 interacts with PIPKIγ in brain. (A) ARF6(Q67L) but not ARF6(T27N) specifically affinity purifies PIPKIγ. Western blot analysis of proteins pulled down by GST and GST–ARF6 fusion proteins (80 μg) from a detergent extract of rat brain. Samples were analyzed by SDS-PAGE and immunoblotting. 5% Std., 5% of the extract used for affinity purification. (B) PIPKIγ specifically interacts with ARF6(Q67L). Immunoblot analysis of PIPKIγ affinity purified with myristoylated His6-tagged ARF6(Q67L), ARF1(Q71L), or arfaptin 2 as described under A. 5% Std., 5% of the extract used for affinity purification. (C) PIPKIγ can be cross-linked to ARF6(Q67L) during recruitment of clathrin/AP-2 to synaptic membranes. LP2-membranes were incubated with brain cytosol, myristoylated His6-tagged ARF6, and nucleotides. DTSP was added where indicated. His6-tagged ARF6 was recovered and cross-linked proteins were analyzed by immunoblotting. Top, immunoblot analysis with antisera against PIPKIγ, AP180, large and medium subunits of the AP-2 complex β1/2-adaptin, α-adaptin, and μ2-adaptin, respectively, and clathrin heavy chain (HC). Bottom, Coomassie-stained gel demonstrating that equal amounts of ARF6 have been recovered in each sample.

Mentions: To obtain possible insights into the mechanism by which ARF6-GTP stimulates clathrin/AP-2 recruitment to synaptic plasma membranes, we used an affinity purification approach. In pull-down experiments, NH2-terminal GST fusion constructs of the constitutively active ARF6(Q67L) or the GTP-binding defective mutant ARF6(T27N) were analyzed for their ability to retain various endocytotic proteins. Considering that ARF1 directly interacts with AP-1 complexes at the trans-Golgi network and AP-3 complexes on endosomal membranes, we initially focused on coat components as putative interactors of ARF6. No association of either ARF6 mutant with clathrin or subunits of AP-2 could be detected. Likewise, we did not detect any interaction of GST-ARF6 with AP180, amphiphysin I, dynamin I, and auxilin, or the SV protein synaptotagmin I (Fig. 5Figure 5.


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)

Activated ARF6 interacts with PIPKIγ in brain. (A) ARF6(Q67L) but not ARF6(T27N) specifically affinity purifies PIPKIγ. Western blot analysis of proteins pulled down by GST and GST–ARF6 fusion proteins (80 μg) from a detergent extract of rat brain. Samples were analyzed by SDS-PAGE and immunoblotting. 5% Std., 5% of the extract used for affinity purification. (B) PIPKIγ specifically interacts with ARF6(Q67L). Immunoblot analysis of PIPKIγ affinity purified with myristoylated His6-tagged ARF6(Q67L), ARF1(Q71L), or arfaptin 2 as described under A. 5% Std., 5% of the extract used for affinity purification. (C) PIPKIγ can be cross-linked to ARF6(Q67L) during recruitment of clathrin/AP-2 to synaptic membranes. LP2-membranes were incubated with brain cytosol, myristoylated His6-tagged ARF6, and nucleotides. DTSP was added where indicated. His6-tagged ARF6 was recovered and cross-linked proteins were analyzed by immunoblotting. Top, immunoblot analysis with antisera against PIPKIγ, AP180, large and medium subunits of the AP-2 complex β1/2-adaptin, α-adaptin, and μ2-adaptin, respectively, and clathrin heavy chain (HC). Bottom, Coomassie-stained gel demonstrating that equal amounts of ARF6 have been recovered in each sample.
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fig5: Activated ARF6 interacts with PIPKIγ in brain. (A) ARF6(Q67L) but not ARF6(T27N) specifically affinity purifies PIPKIγ. Western blot analysis of proteins pulled down by GST and GST–ARF6 fusion proteins (80 μg) from a detergent extract of rat brain. Samples were analyzed by SDS-PAGE and immunoblotting. 5% Std., 5% of the extract used for affinity purification. (B) PIPKIγ specifically interacts with ARF6(Q67L). Immunoblot analysis of PIPKIγ affinity purified with myristoylated His6-tagged ARF6(Q67L), ARF1(Q71L), or arfaptin 2 as described under A. 5% Std., 5% of the extract used for affinity purification. (C) PIPKIγ can be cross-linked to ARF6(Q67L) during recruitment of clathrin/AP-2 to synaptic membranes. LP2-membranes were incubated with brain cytosol, myristoylated His6-tagged ARF6, and nucleotides. DTSP was added where indicated. His6-tagged ARF6 was recovered and cross-linked proteins were analyzed by immunoblotting. Top, immunoblot analysis with antisera against PIPKIγ, AP180, large and medium subunits of the AP-2 complex β1/2-adaptin, α-adaptin, and μ2-adaptin, respectively, and clathrin heavy chain (HC). Bottom, Coomassie-stained gel demonstrating that equal amounts of ARF6 have been recovered in each sample.
Mentions: To obtain possible insights into the mechanism by which ARF6-GTP stimulates clathrin/AP-2 recruitment to synaptic plasma membranes, we used an affinity purification approach. In pull-down experiments, NH2-terminal GST fusion constructs of the constitutively active ARF6(Q67L) or the GTP-binding defective mutant ARF6(T27N) were analyzed for their ability to retain various endocytotic proteins. Considering that ARF1 directly interacts with AP-1 complexes at the trans-Golgi network and AP-3 complexes on endosomal membranes, we initially focused on coat components as putative interactors of ARF6. No association of either ARF6 mutant with clathrin or subunits of AP-2 could be detected. Likewise, we did not detect any interaction of GST-ARF6 with AP180, amphiphysin I, dynamin I, and auxilin, or the SV protein synaptotagmin I (Fig. 5Figure 5.

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