A RAB PROTEIN REGULATES THE LOCALIZATION OF SECRETORY GRANULES IN ATT-20 CELLS

Low molecular weight (LMW) GTP-binding proteins are hypothesized to pl ay a role in the vectorial transport of intracellular vesicles. Mutati onal studies in yeast and subcellular localization in mammalian cells suggest that a family of LMW GTP-binding proteins, termed rab, target intracellular vesicles to their appropriate acceptor compartment. In t his report, we demonstrate that an elasmobranch homologue of rab3A, o- rab3, plays a significant role in the sequestration of regulated secre tory vesicles. When transfected into the murine endocrine cell line At T-20, the wild-type o-rab3 protein is localized exclusively to the tip s of the processes, a region of the cell known to accumulate proteins associated with regulated secretory vesicles. Two mutations, Gln81 to Leu (Q81L) and Asn135 Ile (N135I), which alter GTP binding or rate of hydrolysis, blocked the localization of the o-rab3 protein to the tips of cell processes. These mutations also hindered the sequestration of ACTH-containing secretory vesicles to the process tips but did not af fect the basal or stimulated release of ACTH. Moreover, the sequestrat ion of the protein VAMP to the process tip was also hindered by the mu tation. The results demonstrate a role for the rab3 proteins in locali zation, sequestration, and storage of secretory vesicles near their re lease site.