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Vol. 14, Issue 12, 4909-4919, December 2003
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* Department of Neuroscience, San Raffaele Scientific Institute and "Vita-Salute" University, 20132 Milano, Italy;
Department of Experimental Medicine, Section of Human Physiology, University of Genova, 16132 Genova, Italy
Submitted June 10, 2003;
Revised July 24, 2003;
Accepted July 30, 2003
Monitoring Editor: Lawrence Goldstein
Synaptic vesicle (SV) proteins are synthesized at the level of the cell body and transported down the axon in membrane precursors of SVs. To investigate the mechanisms underlying sorting of proteins to SVs, fluorescent chimeras of vesicle-associated membrane protein (VAMP) 2, its highly homologous isoform VAMP1 and synaptotagmin I (SytI) were expressed in hippocampal neurons in culture. Interestingly, the proteins displayed a diffuse component of distribution along the axon. In addition, VAMP2 was found to travel in vesicles that constitutively fuse with the plasma membrane. Coexpression of VAMP2 with synaptophysin I (SypI), a major resident of SVs, restored the correct sorting of VAMP2 to SVs. The effect of SypI on VAMP2 sorting was dose dependent, being reversed by increasing VAMP2 expression levels, and highly specific, because the sorting of the SV proteins VAMP1 and SytI was not affected by SypI. The cytoplasmic domain of VAMP2 was found to be necessary for both the formation of VAMP2-SypI hetero-dimers and for VAMP2 sorting to SVs. These data support a role for SypI in directing the correct sorting of VAMP2 in neurons and demonstrate that a direct interaction between the two proteins is required for SypI in order to exert its effect.
Abbreviations used: 
-Ltx, -latrotoxin; DIV, days in vitro; DSS, disuccinimidyl suberate; ECFP, enhanced cyan fluorescent protein; ER, endoplasmic reticulum; EYFP, enhanced yellow fluorescent protein; FP, fluorescent protein; GFP, green fluorescent protein; GSDB, goat serum dilution buffer; KRH, Krebs-Ringer's solution; LDL-R, low-density lipoprotein receptor; MAP2, microtubule-associated protein 2; PBS, phosphate-buffered saline; PEI 25, 25-kDa polyethylenimine; SLMV, synaptic-like microvesicle; SV, synaptic vesicle; SV2, synaptic vesicle protein 2; SypI, synaptophysin I; SytI, synaptotagmin I; TfR, transferrin receptor; TGN, trans-Golgi network; VAMP, vesicle-associated membrane protein.
These authors contributed equally to this work.
Corresponding author. E-mail address: valtorta.flavia{at}hsr.it.
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