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1 Surgery/Epithelial Biology Program, Vanderbilt University School of Medicine, Nashville, Tennessee, United States
2 Surgery, Vanderbilt University School of Medicine, Nashville, Tennessee, United States
3 Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, United States
4 Dept. of Medicine/CSB 921E, Medical University of South Carolina, Charleston, South Carolina, United States
* To whom correspondence should be addressed. E-mail: jim.goldenring{at}vanderbilt.edu.
Gastric parietal cells possess an amplified apical membrane recycling system dedicated to regulated apical recycling of the H/K-ATPase. While amplified in parietal cells, apical recycling is critical to polarized secretory processes in most epithelial cells. To clarify putative regulators of apical recycling, we have prepared immunoisolated parietal cell H/K-ATPase-containing recycling membranes from human stomachs and analyzed the protein contents by tryptic digestion and mass spectrometry. We identified and validated by western blots many of the proteins previously identified on immunoisolated rabbit tubulovesicles including Rab11, Rab25, syntaxin 3, SCAMPs and VAMP2. In addition, we detected several previously unrecognized proteins including Rab10, VAMP8, syntaxin 7 and syntaxin 12/13. We also identified the potassium channel component, KCNQ1. Immunostaining of human gastric mucosal sections confirmed the presence of each of these proteins in parietal cells and their co-localization with H/K-ATPase on the tubulovesicles. To investigate the role of the identified SNARE proteins in apical recycling, we transfected them as DsRed2 fusions into an EGFP-Rab11a expressing MDCK cell line. Syntaxin 12/13 and VAMP8 caused a collapse of the EGFP-Rab11a compartment, while a less dramatic effect was observed in cells transfected with syntaxin 3, syntaxin 7 or VAMP2. The five DsRed2-SNARE chimeras were also transfected into an MDCK cell line overexpressing Rab11-FIP2(129-512). All five of the chimeras were drawn into the collapsed apical recycling system. This study, which represents the first proteomics analysis of an immunoisolated vesicle population from native human tissue, demonstrates the diversity of putative regulators of the apical recycling system.
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