Evidence for a Primary Endocytic Vesicle Involved in Synaptic Vesicle Biogenesis

Abstract

The regulated release of neurotransmitters at synapses is mediated by the fusion of neurotransmitterfilled synaptic vesicles with the plasma membrane. Continuous synaptic activity relies on the constant recycling of synaptic vesicle proteins into newly formed synaptic vesicles. At least two different mechanisms are presumed to mediate synaptic vesicle biogenesis at the synapse as follows: direct retrieval of synaptic vesicle proteins and lipids from the plasma membrane, and indirect passage of synaptic vesicle proteins through an endosomal intermediate. We have identified a vesicle population with the characteristics of a primary endocytic vesicle responsible for the recycling of synaptic vesicle proteins through the indirect pathway. We find that synaptic vesicle proteins colocalize in this vesicle with a variety of proteins known to recycle from the plasma membrane through the endocytic pathway, including three different glucose transporters, GLUT1, GLUT3, and GLUT4, and the transferrin receptor. These vesicles differ from "classical" synaptic vesicles in their size and their generic protein content, indicating that they do not discriminate between synaptic vesicle-specific proteins and other recycling proteins. We propose that these vesicles deliver synaptic vesicle proteins that have escaped internalization by the direct pathway to endosomes, where they are sorted from other recycling proteins and packaged into synaptic vesicles.The biogenesis of synaptic vesicles is a complex orchestration of events culminating in a vesicle population responsible for the uptake, storage, and regulated secretion of neurotransmitters. Current models of synaptic vesicle biogenesis suggest that at least two pathways exist for the formation of new synaptic vesicles after exocytosis: directly from the plasma membrane and indirectly via an intermediate endosomal compartment (1, 2). In neurons, both a direct pathway, which is at the active zone, and a more distal indirect pathway have been demonstrated at synapses in the Drosophila shibire ts1 mutant (3). Studies of membrane recycling in hippocampal synapses using the fluorescent dye FM1-43 also suggest that synaptic vesicles formed by endocytosis can fuse with the plasma membrane directly, without passing through an endosomal compartment (4). In the PC12 neuroendocrine cell line, synaptic vesicles are also derived from two different pathways as follows: directly from the plasma membrane (5, 6) as well as from an endosomal intermediate (7)(8)(9)(10). Together these data form an emerging model of multiple pathways to recycle both synaptic vesicle proteins and synaptic vesicle membrane.Although presumably all synaptic vesicle proteins undergo endocytic trafficking, relatively little is known about the intermediate steps in the pathway to mature synaptic vesicles. In order to understand fully the mechanisms underlying synaptic vesicle biogenesis, it is critical that the synaptic vesicle protein trafficking pathways are fully elucidated. In neurons, synaptic vesicle proteins are presen...

Expression of a synapsin IIb site 1 phosphorylation mutant in 3T3-L1 adipocytes inhibits basal intracellular retention of Glut4

“…3), where both TGN markers and the transferrin receptor are localized (Shewan et al, 2003;Zeigerer et al, 2002). Like Glut4, synaptic vesicle proteins are recycled from the cell surface in part through clathrin-mediated endocytosis and recycling back into regulated secretory vesicles from endosomes (Linstedt and Kelly, 1991;Matteoli et al, 1992;Provoda et al, 2000). Synapsins are not found in clathrincoated vesicles with the other synaptic vesicle proteins.…”

In VivoTrafficking and Targeting of N-Cadherin to Nascent Presynaptic Terminals

“…These transport packets have been shown by electron microscopy to be composed primarily of tubulvesicular structures, pleiomorphic vesicles, and large dense core vesicles (Tsukita and Ishikawa, 1980;Ahmari et al, 2000). The protein composition of these organelles is heterogeneous (Okada et al, 1995;Ahmari et al, 2000;Provoda et al, 2000;Zhai et al, 2001), suggesting either the existence of multiple, distinct populations of transport organelles or the sorting of components through multiple rounds of fusion with plasma membrane and recycling, or both. Our data reveal that N-cadherin is localized to at least two distinct forms of transport packet: discrete-punctate (Fig.…”

Internalization of the TGF-β type I receptor into caveolin-1 and EEA1 double-positive early endosomes

“…The EEA1-positive early endosomes were isolated as previously described [28,29]. In brief, the cells were washed with icecold PBS twice, rinsed with osmotic buffer (10 mM Tris/HCl, pH 7.4) for 1 min, and treated with homogenization buffer (10 mM Tris/HCl, 1 mM EGTA, 0.5 mM EDTA, 3 mM Imidazole, 250 mM sucrose, Complete protease inhibitors, pH 7.4).…”

“…By labeling EEA1 and caveolin-1 using different sizes of gold nanoparticles, we found the distribution of caveolin-1 on the membrane of EEA1-positive early endosomes (Supplementary information, Figure S6). (3) We immunoisolated the EEA1-positive early endosomes using a subcellular fractionation protocol [28,29]. The immunoisolated early endosomes were then analyzed by western blotting with antibodies against EEA1, caveolin-1, TGF-β receptor, Golgi protein p115 and mitochondrial protein Tim23.…”