SYNAPTOTAGMIN - A MEMBRANE CONSTITUENT OF NEUROPEPTIDE-CONTAINING LARGE DENSE-CORE VESICLES

Synaptotagmin is known to be a major membrane protein of synaptic vesi cles (SVs) in neurons. We have now used an immunoisolation procedure t o demonstrate that synaptotagmin is also present in the membranes of p eptide containing large dense-core vesicles (LDCVs) of rat hypothalamu s and bovine posterior pituitary. Synaptotagmin bead-immunoisolated or ganelles from these tissues primarily consisted of SVs but contained o ccasionally larger structures reminiscent of LDCVs that were absent fr om vesicle populations immunoisolated with a synaptophysin antibody. F urthermore, the vesicles immunoisolated with synaptotagmin beads conta ined significant amounts of neuropeptide Y (NPY). In contrast, vesicle s immunoisolated with synaptophysin beads did not contain detectable l evels of NPY. Sucrose density gradient fractionation of postnuclear su pernatants obtained from the bovine posterior pituitary resulted in a bimodal distribution of synaptotagmin, corresponding to the positions of both SVs and neurosecretory granules. A similar distribution was fo und for cytochrome b561 and the 116 kDa subunit of the vacuolar proton pump. In contrast, the SV proteins synaptophysin, SV2, and p29 were r estricted to the SV-containing fractions. Immunoisolation of small and large vesicles from the sucrose gradient confirmed the differential d istribution of synaptotagmin and synaptophysin in the two types of sec retory vesicles in nerve endings of the posterior pituitary. We conclu de that synaptotagmin is a constituent of both SVs and peptide-contain ing secretory vesicles in the nervous system. Since both types of orga nelles undergo Ca2+-dependent exocytosis, these findings support a gen eral role of synaptotagmin as an exocytotic Ca2+ receptor.