TARGETING OF THE SYNAPTIC VESICLE PROTEIN SYNAPTOBREVIN IN THE AXON OF CULTURED HIPPOCAMPAL-NEURONS - EVIDENCE FOR 2 DISTINCT SORTING STEPS

Synaptic vesicles are concentrated in the distal axon, far from the si te of protein synthesis. Integral membrane proteins destined for this organelle must therefore make complex targeting decisions. Short amino acid sequences have been shown to act as targeting signals directing proteins to a variety of intracellular locations. To identify synaptic vesicle targeting sequences and to follow the path that proteins trav el en route to the synaptic vesicle, we have used a defective herpes v irus amplicon expression system to study the targeting of a synaptobre vin-transferrin receptor (SB-TfR) chimera in cultured hippocampal neur ons. Addition of the cytoplasmic domain of synaptobrevin onto human tr ansferrin receptor was sufficient to retarget the transferrin receptor from the dendrites to presynaptic sites in the axon. At the synapse, the SB-TfR chimera did not localize to synaptic vesicles, but was inst ead found in an organelle with biochemical and functional characterist ics of an endosome. The chimera recycled in parallel with synaptic ves icle proteins demonstrating that the nerve terminal efficiently sorts transmembrane proteins into different pathways. The synaptobrevin sequ ence that controls targeting to the presynaptic endosome was not local ized to a single, 10-amino acid region of the molecule, indicating tha t this targeting signal may be encoded by a more distributed structura l conformation. However, the chimera could be shifted to synaptic vesi cles by deletion of amino acids 61-70 in synaptobrevin, suggesting tha t separate signals encode the localization of synaptobrevin to the syn apse and to the synaptic vesicle.