Rab27b localizes to the tubulovesicle membranes of gastric parietal cells and regulates acid secretion.
OBJECTIVE
Rabs are monomeric guanosine triphosphatases that regulate membrane trafficking and acid secretion in gastric parietal cells. Using a proteomics approach, we identified a new Rab, Rab27b, in tubulovesicle membranes and determined its role in parietal cell activation.


METHODS
We used mass spectrometry (MS) to identify Rab27b in purified tubulovesicular membrane fractions and used immunoblot and immunofluorescence analyses to study its expression. Wild-type, constitutively active (Rab27bQ78L), and dominant negative (Rab27bN133I) forms of Rab27b were tagged with yellow fluorescent protein (YFP) and expressed in parietal cells using adenoviral constructs to study localization and function. Localization was visualized by fluorescence microscopy in resting and stimulated cells. Acid secretion in primary cell cultures was measured by aminopyrine accumulation.


RESULTS
A tandem MS peptide mass fingerprint was matched to 7 peptides of Rab27b. Rab27b localized to tubulovesicle membranes, based on immunoblot and immunocytochemical analyses. Endogenous Rab27b, YFP/wild-type Rab27b, Rab27bQ78L, and Rab27bN133I all distributed throughout the cytoplasm of resting parietal cells. After stimulation, wild-type Rab27b and YFP-Rab27bQ78L translocated to the apical membrane, but YFPR-ab27bN133I did not. Expression of wild-type YFP-Rab27b or YFP-Rab27bQ78L did not affect acid secretion, whereas expression of Rab27bN133I almost completely inhibited acid secretion.


CONCLUSIONS
Rab27b is associated with tubulovesicle membranes in the parietal cell and Rab27b may play a role in stimulation-associated membrane recruitment and gastric acid secretion.
Suda
Jo
J
Division of Gastrointestinal & Liver Diseases, University of Southern California Keck School of Medicine, Los Angeles, California 90033, USA.
Zhu
Lixin
L
Okamoto
Curtis T
CT
Karvar
Serhan
S
D000632:Aminopyrine / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D005744:Gastric Acid / Q000378:metabolism*; D017506:H(+)-K(+)-Exchanging ATPase / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009154:Mutation; D010295:Parietal Cells, Gastric / Q000378:metabolism*; D010449:Peptide Mapping; D021381:Protein Transport; D040901:Proteomics / Q000379:methods; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014162:Transfection; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D000632:Aminopyrine; C512960:Rab27B protein, human; D017506:H(+)-K(+)-Exchanging ATPase; D020691:rab GTP-Binding Proteins