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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 directly stimulates the activity of endogenous or recombinant PIPKIγ. (A) Liposomes containing phosphatidylinositol 4-phosphate (6%, wt/wt) as a substrate were incubated for 10 min at 37°C with brain cytosol and recombinant myristoylated ARF6 mutants (200 nM) as indicated in the presence of neomycin, GTP, and γ[32P]ATP. Lipids were extracted and separated by HPTLC. Data represent mean values (± SD) from four independent experiments. Values were normalized to the amount of PIP2 generated in the absence of ARF6 proteins (fold stimulation). (B) 4 ng recombinant PIPKIγ was pre-incubated for 20 min at 4°C with recombinant myristoylated ARF6 proteins, GTP, and total brain liposomes. Reactions were started by addition of γ[32P]ATP, and after a 7-min incubation (37°C), lipid products were extracted, separated by TLC as described previously (Wenk et al., 2001), analyzed by autoradiography (see bottom for a typical experiment) and quantified (Phosphor- Imager). The top shows the quantification of three independent experiments (mean ± SD); the data presented are normalized to the activity of PIPKIγ in the absence of exogenously added ARF6 proteins (fold stimulation). (C) 100 μg rat brain cytosol that had either been mock-depleted (ctrl) or depleted of PIPKIγ was pre-incubated (20 min at 4°C) with myristoylated ARF6 proteins, GTP, and total brain liposomes. The reaction (7 min at 37°C) was started by addition of γ[32P]ATP. Samples were analyzed as described above. Formation of PIP2 and PIP is depicted as mean ± SD (n = 3). (D) Brain cytosol was pre-incubated (2 min at 37°C) with GTP, γ[32P]ATP, neomycin, and recombinant ARF6 protein as indicated. Reactions (15 min at 37°C) were started by addition of LP2 membranes. Samples were analyzed as described under A. Data are depicted as mean (± SD) from four independent experiments.
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fig7: ARF6 directly stimulates the activity of endogenous or recombinant PIPKIγ. (A) Liposomes containing phosphatidylinositol 4-phosphate (6%, wt/wt) as a substrate were incubated for 10 min at 37°C with brain cytosol and recombinant myristoylated ARF6 mutants (200 nM) as indicated in the presence of neomycin, GTP, and γ[32P]ATP. Lipids were extracted and separated by HPTLC. Data represent mean values (± SD) from four independent experiments. Values were normalized to the amount of PIP2 generated in the absence of ARF6 proteins (fold stimulation). (B) 4 ng recombinant PIPKIγ was pre-incubated for 20 min at 4°C with recombinant myristoylated ARF6 proteins, GTP, and total brain liposomes. Reactions were started by addition of γ[32P]ATP, and after a 7-min incubation (37°C), lipid products were extracted, separated by TLC as described previously (Wenk et al., 2001), analyzed by autoradiography (see bottom for a typical experiment) and quantified (Phosphor- Imager). The top shows the quantification of three independent experiments (mean ± SD); the data presented are normalized to the activity of PIPKIγ in the absence of exogenously added ARF6 proteins (fold stimulation). (C) 100 μg rat brain cytosol that had either been mock-depleted (ctrl) or depleted of PIPKIγ was pre-incubated (20 min at 4°C) with myristoylated ARF6 proteins, GTP, and total brain liposomes. The reaction (7 min at 37°C) was started by addition of γ[32P]ATP. Samples were analyzed as described above. Formation of PIP2 and PIP is depicted as mean ± SD (n = 3). (D) Brain cytosol was pre-incubated (2 min at 37°C) with GTP, γ[32P]ATP, neomycin, and recombinant ARF6 protein as indicated. Reactions (15 min at 37°C) were started by addition of LP2 membranes. Samples were analyzed as described under A. Data are depicted as mean (± SD) from four independent experiments.

Mentions: Given the interaction of ARF6-GTP with PIPKIγ, we asked whether ARF6 might regulate phosphatidylinositol 4-phosphate 5-kinase activity in bovine brain cytosol. To this end, cytosol was incubated with γ[32P]ATP, phosphatidylinositol 4-phosphate (PI4P)–containing liposomes, and neomycin to prevent PIP2 degradation. Lipid products were analyzed by TLC, and the amount of incorporated radioactivity was quantified. Addition of 1 μM recombinant myristoylated ARF6(Q67L)-GTP greatly stimulated PI4P 5-kinase activity compared with ARF6(T27N). A slightly less pronounced increase in PI4P kinase activity was seen if wild-type ARF6-GTP was used instead (Fig. 7Figure 7.


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 directly stimulates the activity of endogenous or recombinant PIPKIγ. (A) Liposomes containing phosphatidylinositol 4-phosphate (6%, wt/wt) as a substrate were incubated for 10 min at 37°C with brain cytosol and recombinant myristoylated ARF6 mutants (200 nM) as indicated in the presence of neomycin, GTP, and γ[32P]ATP. Lipids were extracted and separated by HPTLC. Data represent mean values (± SD) from four independent experiments. Values were normalized to the amount of PIP2 generated in the absence of ARF6 proteins (fold stimulation). (B) 4 ng recombinant PIPKIγ was pre-incubated for 20 min at 4°C with recombinant myristoylated ARF6 proteins, GTP, and total brain liposomes. Reactions were started by addition of γ[32P]ATP, and after a 7-min incubation (37°C), lipid products were extracted, separated by TLC as described previously (Wenk et al., 2001), analyzed by autoradiography (see bottom for a typical experiment) and quantified (Phosphor- Imager). The top shows the quantification of three independent experiments (mean ± SD); the data presented are normalized to the activity of PIPKIγ in the absence of exogenously added ARF6 proteins (fold stimulation). (C) 100 μg rat brain cytosol that had either been mock-depleted (ctrl) or depleted of PIPKIγ was pre-incubated (20 min at 4°C) with myristoylated ARF6 proteins, GTP, and total brain liposomes. The reaction (7 min at 37°C) was started by addition of γ[32P]ATP. Samples were analyzed as described above. Formation of PIP2 and PIP is depicted as mean ± SD (n = 3). (D) Brain cytosol was pre-incubated (2 min at 37°C) with GTP, γ[32P]ATP, neomycin, and recombinant ARF6 protein as indicated. Reactions (15 min at 37°C) were started by addition of LP2 membranes. Samples were analyzed as described under A. Data are depicted as mean (± SD) from four independent experiments.
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fig7: ARF6 directly stimulates the activity of endogenous or recombinant PIPKIγ. (A) Liposomes containing phosphatidylinositol 4-phosphate (6%, wt/wt) as a substrate were incubated for 10 min at 37°C with brain cytosol and recombinant myristoylated ARF6 mutants (200 nM) as indicated in the presence of neomycin, GTP, and γ[32P]ATP. Lipids were extracted and separated by HPTLC. Data represent mean values (± SD) from four independent experiments. Values were normalized to the amount of PIP2 generated in the absence of ARF6 proteins (fold stimulation). (B) 4 ng recombinant PIPKIγ was pre-incubated for 20 min at 4°C with recombinant myristoylated ARF6 proteins, GTP, and total brain liposomes. Reactions were started by addition of γ[32P]ATP, and after a 7-min incubation (37°C), lipid products were extracted, separated by TLC as described previously (Wenk et al., 2001), analyzed by autoradiography (see bottom for a typical experiment) and quantified (Phosphor- Imager). The top shows the quantification of three independent experiments (mean ± SD); the data presented are normalized to the activity of PIPKIγ in the absence of exogenously added ARF6 proteins (fold stimulation). (C) 100 μg rat brain cytosol that had either been mock-depleted (ctrl) or depleted of PIPKIγ was pre-incubated (20 min at 4°C) with myristoylated ARF6 proteins, GTP, and total brain liposomes. The reaction (7 min at 37°C) was started by addition of γ[32P]ATP. Samples were analyzed as described above. Formation of PIP2 and PIP is depicted as mean ± SD (n = 3). (D) Brain cytosol was pre-incubated (2 min at 37°C) with GTP, γ[32P]ATP, neomycin, and recombinant ARF6 protein as indicated. Reactions (15 min at 37°C) were started by addition of LP2 membranes. Samples were analyzed as described under A. Data are depicted as mean (± SD) from four independent experiments.
Mentions: Given the interaction of ARF6-GTP with PIPKIγ, we asked whether ARF6 might regulate phosphatidylinositol 4-phosphate 5-kinase activity in bovine brain cytosol. To this end, cytosol was incubated with γ[32P]ATP, phosphatidylinositol 4-phosphate (PI4P)–containing liposomes, and neomycin to prevent PIP2 degradation. Lipid products were analyzed by TLC, and the amount of incorporated radioactivity was quantified. Addition of 1 μM recombinant myristoylated ARF6(Q67L)-GTP greatly stimulated PI4P 5-kinase activity compared with ARF6(T27N). A slightly less pronounced increase in PI4P kinase activity was seen if wild-type ARF6-GTP was used instead (Fig. 7Figure 7.

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