Investigation of Protein - Protein Interactions in Clathrin-Mediated Membrane Transport

Abstract

Ever since it was reported that synaptic vesicle (SV) retrieval follows two kinetic phases, the molecular mechanisms corresponding to these kinetic modes have been controversially discussed. This work aimed at elucidating the molecular details of SV recycling in small central synapses. We have generated tools to inhibit clathrin-mediated endocytosis (CME) in primary hippocampal neurons and analyzed their effects on SV retrieval using the optical tracers FM1-43 and synaptopHluorin. We found that the majority of SVs was recycled via an AP-2/clathrin- and dynamin-dependent pathway. Fast and slow components of SV retrieval were equally affected by CME inhibition. While CME as such is generally well understood, the question how select SV proteins are targeted for endocytosis has remained elusive. Genetic and biochemical studies have implicated the SV membrane protein synaptotagmin 1 (syt1) in connecting the exo- and endocytic limbs of the SV cycle. We have identified stonin 2 as syt1-specific endocytic sorting adaptor, which serves as linker between syt1 and the clathrin machinery by directly interacting with syt1 and AP-2 in vitro and in vivo. The interaction between stonin 2 and syt1, and the endocytic function of stonin 2 are directly dependent on residues KYE783-785 within the stonin 2 µ-homology domain (µHD). Our data indicate a synergistic effect between the syt1 C2 domains with respect to stonin 2 interaction, however, when C2 domains are offered separately, stonin 2 associates primarily with the C2A domain. We found that the C2B domain as well as the direct association of syt1 and AP-2 are dispensable for AP-2/stonin 2-dependent syt1 internalization in fibroblasts. We hypothesize that stonin 2 may serve as a linker between syt1 and AP-2/clathrin by directly recognizing syt1 C2 domains.In addition, we were able to identify the G protein-coupled receptor kinase interacting protein GIT1 as novel stonin 2 binding partner. Stonin 2 directly associates with the ADP-ribosylation factor GTPase activating protein (ARF-GAP) domain of GIT1, indicating a potential regulatory function of stonin 2 for the enzymatic activity of GIT1. GIT1 is involved in various processes such as cell motility by cytoskeletal rearrangements, trafficking between plasma membrane and endosomal compartments, or synapse formation. The functional relevance of the stonin 2 GIT1 interaction for one or more of these processes remains to be unraveled.