Regulation of synaptic vesicle recycling by complex formation between intersectin 1 and the clathrin adaptor complex AP2

Abstract

Clathrin-mediated synaptic vesicle (SV) recycling involves the spatiotemporally controlled assembly of clathrin coat components at phosphatidylinositiol (4, 5)-bisphosphate [PI(4,5)P 2 ]-enriched membrane sites within the periactive zone. Such spatiotemporal control is needed to coordinate SV cargo sorting with clathrin/AP2 recruitment and to restrain membrane fission and synaptojanin-mediated uncoating until membrane deformation and clathrin coat assembly are completed. The molecular events underlying these control mechanisms are unknown. Here we show that the endocytic SH3 domaincontaining accessory protein intersectin 1 scaffolds the endocytic process by directly associating with the clathrin adaptor AP2. Acute perturbation of the intersectin 1-AP2 interaction in lamprey synapses in situ inhibits the onset of SV recycling. Structurally, complex formation can be attributed to the direct association of hydrophobic peptides within the intersectin 1 SH3A-B linker region with the "side sites" of the AP2 α-and β-appendage domains. AP2 appendage association of the SH3A-B linker region inhibits binding of the inositol phosphatase synaptojanin 1 to intersectin 1. These data identify the intersectin-AP2 complex as an important regulator of clathrinmediated SV recycling in synapses.endocytosis | synapse | scaffolding proteins | appendage | synaptojanin S ynaptic vesicles (SVs), following their activity-dependent exocytic fusion with the presynaptic plasma membrane, are recycled by compensatory endocytosis at the periactive zone (1-3), largely via clathrin-mediated reinternalization of fully fused SV membrane (4). Clathrin-coated pit (CCP) formation (5) proceeds through the assembly of endocytic proteins at phosphatidylinositiol (4, 5)-bisphosphate [PI(4,5)P 2 ]-enriched membrane sites (6, 7). A key factor in the assembly pathway is the heterotetrameric adaptor complex AP2, whose α-and β2-appendage domains act as major recruitment platforms for accessory proteins (6, 7), regulating distinct steps within the pathway. Despite our extensive knowledge regarding the endocytic interactome, we know comparably little about the structural components within the periactive zone that scaffold the endocytic process, thereby allowing the high fidelity of SV recycling. Such spatiotemporal control is needed to coordinate SV cargo protein sorting with coat recruitment (8) and to restrain membrane fission and uncoating until membrane deformation and CCP assembly are completed. Moreover, stabilizing scaffolds may aid coupling of SV exo-and endocytosis (1, 3). The Drosophila multidomain protein Dap160, an ortholog of mammalian intersectin, has been postulated to act as an endocytic scaffold of the periactive zone (9-11), although its precise role in SV recycling in mammalian nerve terminals remains largely unclear (12).Here we show that intersectin 1 scaffolds the endocytic process by directly associating with AP2. Acute perturbation of intersectin-AP2 complex formation blocks the onset of SV recycling. Moreover, association of the SH3A-B l...

Protein scaffolds in the coupling of synaptic exocytosis and endocytosis

“…For example, intersectin undergoes complex formation with early acting factors including FCHos 73 and EPS15, and with the sorting adaptors AP2 (REF. 71) and stonin 2 (FIG. 3c).…”

“…Genetic studies have identified the multidomain endocytic scaffolding proteins intersectin [60][61][62]71 and epidermal growth factor receptor substrate 15 (EPS15) 72 as crucial for synaptic vesicle membrane retrieval and synapse development in Drosophila melanogaster and Caenorhabditis elegans. Expression levels of intersectin and EPS15 are interdependent, and the phenotypes observed on loss of either protein are nearly identical, indicating a close functional relationship between both proteins 72 .…”

SNARE motif-mediated sorting of synaptobrevin by the endocytic adaptors clathrin assembly lymphoid myeloid leukemia (CALM) and AP180 at synapses

“…On 6-8 days in vitro (DIV), neurons were transfected by calcium phosphate transfection (Promega Inc.). On DIV 12-15, neurons expressing synaptopHluorin or vGLUT1-pHluorin were live-imaged using a charge-coupled device camera (AxioCam; Carl Zeiss, Inc.) on an inverted microscope (Axiovert 200M; Carl Zeiss, Inc.) essentially as described previously (36,40 N-labeled synaptobrevin 2 were recorded on a Bruker DRX-600 NMR spectrometer in aqueous solution at 18°C, in complex with DPC at 30°C. Protein concentration was 0.5-1 mM in a buffer consisting of 150 mM NaCl, 5 mM DTT, 1 mM EDTA, 10% D2O, 20 mM Mes at pH 6.0, and optionally 200 mM DPC.…”

An endophilin–dynamin complex promotes budding of clathrin-coated vesicles during synaptic vesicle recycling

“…It has been shown recently that one mechanism of synaptojanin recruitment to the clathrin coat in neurons involves intersectin 1, and the binding of synaptojanin to intersectin is regulated by the adaptor protein complex AP2 (Pechstein et al, 2010). This allows for a possible synaptojanin-endophilin interaction, which is crucial for efficient uncoating of synaptic vesicles, to occur directly after fission, following disassembly of the endophilin-dynamin complex.…”