Rab 3D- and VAMP2-enriched vesicles represent two potentially distinct populations of secretory vesicles in primary rabbit lacrimal acini. Immunoreactivity associated with these secretory vesicle associated proteins, rab 3D and VAMP2, showed different subcellular distributions when examined under confocal microscopy in both resting and carbachol (100 mM) stimulated cells. Furthermore, isolation of subcellular membrane compartments from CCH-stimulated lacrimal acinar cells by centrifugation over sorbitol density gradients and partitioning into aqueous polyethyleneglycol-dextran followed by SDS-PAGE and Western blotting revealed that much of the rab 3D and VAMP2 immunoreactivity was enriched on distinct membranes. To further test whether these populations are distinct, I propose two specific aims: 1) To determine whether VAMP2- and rab3D-enriched vesicles represent discrete vesicle populations rather than different maturation intermediates by comparing secretagogue sensitivity and the spectrum of secretory products released and 2) To determine whether VAMP2- and rab3D-enriched vesicles represent discrete vesicle populations rather than different maturation intermediates by selectively inhibiting different biosynthetic trafficking processes and evaluating the consequences to each vesicle population. ? ?
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