• Characterization of gastric tubulovesicles: implications for vesicle fusion and recycling

      Calhoun, Benjamin Carlisle; School of Graduate Studies (1997-06)
      Gastric parietal cells undergo a massive morphological transformation in response t. to· secretagogues such as histamine and acetylcholine. The surface area of the apical secretory canaliculus increases, and the volume occupied by the intracellular tubulovesicles, which sequester the H/K-ATPase in resting parietal cells, decreases. These morphological changes are thought to correspond to the delivery of the H/K-ATPase from the intracellular tubulovesicles to the cell surface in a regulated vesicle fusion event. The scope of this reversible membrane addition makes the parietal cell an ideal model for the study of the molecular regulation of regulated apical vesicle fusion and recycling. The aim of this study was to determine if candidate regulators of vesicle fusion in other well-:-characterized systems also participate in the fusion of the tubulovesicles with the secretory canaliculus of the parietal cell. Highly purified ·tubulovesicles were prepar(?d by gradient fractionation and immunoisolation on magnetic beads coated with monoclonal antibodies against the ex.subunit_ ofH/K-ATPase. The association of Rab proteins, vesicle-associated membrane protein (V AMP-2), syntaxins, and secretory carrier membrane proteins (SCAMPs) with inimunoisolated tubulovesicles was determined by western blot analysis. Rab 11 and Rab25 are two ras-related small OTP-binding proteins enriched in gastric parietal cells. The vesicle prote!n V AMP-:-2 forms a putative vesicle docking-and fusion complex with the target membrane proteins syntaxin and synaptosome-associated protein (SNAP-25). The SCAMPs are markers for vesicles that recycle from the cell surface. The gastric H/KA TPase, Rabl 1, Rab25, V AMP-2, syntaxin 3 and SCAMPs were recovered in tubulovesicles immunoisolated with the anti-H/K-ATPase antibody. The association of Rabl 1, V AMP-2 and SCAMPs with H/K-ATPase-containing tubulovesicles was confirmed in immunoisolation experiments using monoclonal antibodies specific for V AMP-2 and SCAMPs. In contrast, immunoreactivity for syntaxin lA/B, syntaxin 4 and SNAP-25 was detected in gradient isolated vesicles but not in the immunoisolated tubulovesicles. 'The association of V AMP-2 and two Rab proteins with immunoisolated tubulovesicles sugges~s that tubulovesicles undergo regulated· ve_sicle fusion with the secretory canaliculus. · The observation that syntaxin 3, a putative target membrq11e docking protein, is a component of immunoisolated tubulovesicles suggests that tubulovesicles undergo vesicle-vesicle fusion during the delivery of the H/K.-ATPase to the secretory canaliculus. In addition~ the association of SCAMPs, along ~ith Rabll and Rab25, with immunoisolated tubulovesicles suggests that tubulovesicles likely comprise a compartment I •of apical recycling vesicles. These data are all consistent with the role of tubulovesicles as an expanded apical recycling system that delivers the H/K.-ATPase to the surface of the gastric parietal cell in a regulated vesicle fusion event.