Cytosolic RAB3D is associated with RAB escort protein (REP), not RAB-GDP dissociation inhibitor (GDI), in dispersed chief cells from guinea pig stomach

Rab3D, a low‐molecular‐weight GTP‐binding protein believed to be involved with regulated exocytosis, is associated with secretory granules in gastric chief cells. Although Rab3D is predominantly membrane associated, a significant fraction is cytosolic. Rab proteins are geranylgeranylated on their C‐terminal cysteine motifs by geranylgeranyltransferase (GGTase). Rab escort protein (REP) is required to present Rab proteins to GGTase and may accompany newly modified Rab proteins to their target membrane. In most tissues, cytosolic Rab proteins are complexed with rab‐GDP dissociation inhibitor (rab‐GDI). In the present study, we examined the interactions of Rab3D with cytosolic proteins in dispersed chief cells. Two REP isoforms and at least two GDI isoforms are present in chief cell and brain cytosol. When chief cell cytosol was fractionated by gel filtration chromatography, Rab3D eluted with REP at >150 kDa, whereas rab‐GDI eluted as a separate 65‐kDa peak, suggesting that Rab3D exists as a complex with REP, but not with rab‐GDI. In addition, a small fraction of Rab3D eluted as a monomer at 29 kDa. As has been demonstrated previously, in brain cytosol, Rab3 proteins co‐elute with rab‐GDI at approx. 90 kDa, suggesting that Rab3 proteins undergo active cycling between membrane and cytosolic compartments in this tissue. In vitro experiments revealed that Rab3D remains associated with REP after geranylgeranylation. Our findings suggest that, in gastric chief cells, Rab3D remains associated with REP after geranylgeranylation until it is presented to its target membrane. J. Cell. Biochem. 72:540–548, 1999. © 1999 Wiley‐Liss, Inc.