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

Effect of brefeldin A on clathrin/AP-2 recruitment. Clathrin/AP-2 recruitment was performed as described in Fig. 1 in the presence or absence of ATP/GTPγS and brefeldin A (BFA). Samples were analyzed by quantitative Western blot analysis using antisera against α-adaptin, AP180, synaptotagmin I as a membrane marker, and Hsc70 as a control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172713&req=5

fig2: Effect of brefeldin A on clathrin/AP-2 recruitment. Clathrin/AP-2 recruitment was performed as described in Fig. 1 in the presence or absence of ATP/GTPγS and brefeldin A (BFA). Samples were analyzed by quantitative Western blot analysis using antisera against α-adaptin, AP180, synaptotagmin I as a membrane marker, and Hsc70 as a control.

Mentions: ARF family members have been shown to facilitate vesicle budding by stimulating recruitment of coat components to the membrane (Springer et al., 1999). Therefore, we tested the effect of brefeldin A (BFA) on clathrin/AP-2 recruitment in our assay. BFA inhibits nucleotide exchange factors acting on ARF family members except most of those specific for ARF6 (Randazzo et al., 2000). Consistent with earlier observations on AP-2 association with the plasma membrane of intact cells (Robinson and Kreis, 1992), no effect on the ATP- and GTPγS-dependent recruitment of AP-2 and AP180 to LP2 membranes (Fig. 2)Figure 2.


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)

Effect of brefeldin A on clathrin/AP-2 recruitment. Clathrin/AP-2 recruitment was performed as described in Fig. 1 in the presence or absence of ATP/GTPγS and brefeldin A (BFA). Samples were analyzed by quantitative Western blot analysis using antisera against α-adaptin, AP180, synaptotagmin I as a membrane marker, and Hsc70 as a control.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Effect of brefeldin A on clathrin/AP-2 recruitment. Clathrin/AP-2 recruitment was performed as described in Fig. 1 in the presence or absence of ATP/GTPγS and brefeldin A (BFA). Samples were analyzed by quantitative Western blot analysis using antisera against α-adaptin, AP180, synaptotagmin I as a membrane marker, and Hsc70 as a control.
Mentions: ARF family members have been shown to facilitate vesicle budding by stimulating recruitment of coat components to the membrane (Springer et al., 1999). Therefore, we tested the effect of brefeldin A (BFA) on clathrin/AP-2 recruitment in our assay. BFA inhibits nucleotide exchange factors acting on ARF family members except most of those specific for ARF6 (Randazzo et al., 2000). Consistent with earlier observations on AP-2 association with the plasma membrane of intact cells (Robinson and Kreis, 1992), no effect on the ATP- and GTPγS-dependent recruitment of AP-2 and AP180 to LP2 membranes (Fig. 2)Figure 2.

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