Two Rab proteins, vesicle-associated membrane protein 2 (VAMP-2) and secretory carrier membrane proteins (SCAMPs), are present on immunoisolated parietal cell tubulovesicles.
The tubulovesicles of gastric parietal cells sequester H+/K+-ATPase molecules within resting parietal cells. Stimulation of parietal cell secretion elicits delivery of intracellular H+/K+-ATPase to the apically oriented secretory canaliculus. Previous investigations have suggested that this process requires the regulated fusion of intracellular tubulovesicles with the canalicular target membrane. We have sought to investigate the presence of critical putative regulators of vesicle fusion on immunoisolated gastric parietal cell tubulovesicles. Highly purified tubulovesicles were prepared by gradient fractionation and immunoisolation on magnetic beads coated with monoclonal antibodies against the alpha subunit of H+/K+-ATPase. Western blot analysis revealed the presence of Rab11, Rab25, vesicle-associated membrane protein 2 (VAMP-2) and secretory carrier membrane proteins (SCAMPs) on immunoisolated vesicles. The same cohort of proteins was recovered on vesicles immunoisolated with monoclonal antibodies against SCAMPs and VAMP-2. In contrast, whereas immunoreactivities for syntaxin 1A/1B and synaptosome-associated protein (SNAP-25) were present in gradient-isolated vesicles, none of the immunoreactivity was associated with immunoisolated vesicles. The observation of VAMP-2 and two Rab proteins on immunoisolated H+/K+-ATPase-containing tubulovesicles supports the role for tubulovesicles in a regulated vesicle fusion process. In addition, the presence of SCAMPs along with Rab11 and Rab25 implicates the tubulovesicles as a critical apical recycling vesicle population.
Calhoun
B C
BC
Institute for Molecular Medicine and Genetics, Department of Medicine, Medical College of Georgia, 1120 Fifteenth Street, Augusta, GA 30912-3175, USA.
Goldenring
J R
JR
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000032:analysis*; D017506:H(+)-K(+)-Exchanging ATPase / Q000378:metabolism; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D008565:Membrane Proteins / Q000032:analysis*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000032:analysis; D010295:Parietal Cells, Gastric / Q000737:chemistry* / Q000201:enzymology / Q000648:ultrastructure; D050683:R-SNARE Proteins; D011817:Rabbits; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins
D002352:Carrier Proteins; D019204:GTP-Binding Proteins; D017506:H(+)-K(+)-Exchanging ATPase; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C082888:Rab25 protein, Oryctolagus cuniculus