Volker Haucke

ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type Iγ

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 GTPγS 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 Iγ (PIPKIγ), in this effect. These data suggest a model according to which activation of PIPKIγ by ARF6-GTP facilitates clathrin-coated pit assembly at the synapse.

Clathrin-mediated endocytosis: membrane factors pull the trigger

Clathrin-mediated endocytosis is a vesicular transport event involved in the internalization and recycling of receptors participating in signal transduction events and nutrient import as well as in the reformation of synaptic vesicles. Recent studies in vitro and in living cells have provided a number of new insights into the initial steps of clathrin-coated vesicle formation and the membrane factors involved in this process. The unexpected complexity of these interactions at the cytosol-membrane interface suggests that clathrin-coated vesicle assembly is a highly cooperative process occurring under tight regulatory control. In this review, we focus on the role of membrane proteins and lipids in the nucleation of clathrin-coated pits and provide a hypothetical model for the early steps in clathrin-mediated endocytosis.

A phosphatidylinositol (4,5)-bisphosphate binding site within μ2-adaptin regulates clathrin-mediated endocytosis

The clathrin adaptor complex AP-2 serves to coordinate clathrin-coated pit assembly with the sorting of transmembrane cargo proteins at the plasmalemma. How precisely AP-2 assembly and cargo protein recognition at sites of endocytosis are regulated has remained unclear, but recent evidence implicates phosphoinositides, in particular phosphatidylinositol (4,5)-bisphosphate (PI[4,5]P2), in these processes. Here we have identified and functionally characterized a conserved binding site for PI(4,5)P2 within μ2-adaptin, the medium chain of the clathrin adaptor complex AP-2. Mutant μ2 lacking a cluster of conserved lysine residues fails to bind PI(4,5)P2 and to compete the recruitment of native clathrin/AP-2 to PI(4,5)P2-containing liposomes or to presynaptic membranes. Moreover, we show that expression of mutant μ2 inhibits receptor-mediated endocytosis in living cells. We suggest that PI(4,5)P2 binding to μ2-adaptin regulates clathrin-mediated endocytosis and thereby may contribute to structurally linking cargo recognition to coat formation.

Dual interaction of synaptotagmin with μ2‐ and α‐adaptin facilitates clathrin‐coated pit nucleation

The synaptic vesicle protein synaptotagmin was proposed to act as a major docking site for the recruitment of clathrin coats implicated in endocytosis, including the recycling of synaptic vesicles. We show here that the C2B domain of synaptotagmin binds μ2‐ and α‐adaptin, two of the four subunits of the endocytic adaptor complex AP‐2. μ2 represents the major interacting subunit of AP‐2 within this complex. Its binding to synaptotagmin is mediated by a site in subdomain B that is distinct from the binding site for tyrosine‐based sorting motifs located in subdomain A. The presence of the C2B domain of synaptotagmin at the surface of liposomes enhances the recruitment of AP‐2 and clathrin. Conversely, perturbation of the interaction between synaptotagmin and AP‐2 by synprint, the cytoplasmic synaptotagmin‐binding domain of N‐type calcium channels, inhibits transferrin internalization in living cells. We conclude that a dual interaction of synaptotagmin with the clathrin adaptor AP‐2 plays a key physiological role in the nucleation of endocytic clathrin‐coated pits.

Human stoned B interacts with AP-2 and synaptotagmin and facilitates clathrin-coated vesicle uncoating

Synaptic vesicle biogenesis involves the recycling of synaptic vesicle components by clathrin‐mediated endocytosis from the presynaptic membrane. stoned B, a protein encoded by the stoned locus in Drosophila melanogaster has been shown to regulate vesicle recycling by interacting with synaptotagmin. We report here the identification and characterization of a human homolog of stoned B (hStnB). Human stoned B is a brain‐specific protein which co‐enriches with other endocytic proteins such as AP‐2 in a crude synaptic vesicle fraction and at nerve terminals. A domain with homology to the medium chain of adaptor complexes binds directly to both AP‐2 and synaptotagmin and competes with AP‐2 for the same binding site within synaptotagmin. Finally we show that the μ2 homology domain of hStnB stimulates the uncoating of both clathrin and AP‐2 adaptors from clathrin‐coated vesicles. We hypothesize that hStnB regulates synaptic vesicle recycling by facilitating vesicle uncoating.

Vesicle budding: a coat for the COPs

Although vesicular transport in eukaryotic cells involves a number of different carriers, one common feature is that most of them use small GTPases to direct coat assembly at the donor membrane. COPII coated vesicles bud from the endoplasmic reticulum to selectively export secretory cargo en route to the Golgi complex. Vesicle formation involves the stepwise recruitment of the small GTPase Sar1 and two large heterodimeric complexes Sec23-Sec24 and Sec13-Sec31 to the membrane. A new structural study now provides breathtaking molecular insights into the formation of the Sec23-Sec24-Sar1 pre-budding complex and into COPII coat assembly.

Tyrosine-based endocytic motifs stimulate oligomerization of AP-2 adaptor complexes

The clathrin adaptor complex AP-2 functions in the assembly of clathrin-coated vesicles at the plasma membrane where it serves to couple endocytic vesicle formation to the selection of membrane cargo proteins. Recent evidence suggests that binding of tyrosine-based endocytic sorting motifs may induce a conformational change within the AP-2 adaptor complex that could enhance its interaction with other cargo molecules and with the membrane. We report here that soluble tyrosine-based endocytic sorting motif peptides facilitate clathrin/AP-2 recruitment to liposomal membranes and induce adaptor oligomerization even in the absence of a lipid bilayer. These effects are specific for endocytic motifs of the type Yxxphi whereas peptides corresponding to NPxY- or di-leucine-containing sorting signals are ineffective. Our data may help to explain how the highly cooperative assembly of clathrin and adaptors could be linked to the selection of membrane cargo proteins.

Dissecting the ins and outs of excitement: glutamate receptors on the move.

AMPA, NMDA or insulin can cause endocytosis of AMPA receptors. Two papers now show that these stimuli act via distinct signaling pathways, some of which also induce LTD.