Characterization of immunoisolated human gastric parietal cells tubulovesicles: identification of regulators of apical recycling.
Gastric parietal cells possess an amplified apical membrane recycling system dedicated to regulated apical recycling of 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 prepared immunoisolated parietal cell H-K-ATPase-containing recycling membranes from human stomachs and analyzed 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, secretory carrier membrane proteins (SCAMPs), and vesicle-associated membrane protein (VAMP)2. In addition, we detected several previously unrecognized proteins, including Rab10, VAMP8, syntaxin 7, and syntaxin 12/13. We also identified the K(+) channel component KCNQ1. Immunostaining of human gastric mucosal sections confirmed the presence of each of these proteins in parietal cells and their colocalization with H-K-ATPase on tubulovesicles. To investigate the role of the identified soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins in apical recycling, we transfected them as DsRed2 fusions into an enhanced green fluorescent protein (EGFP)-Rab11a-expressing Madin-Darby canine kidney (MDCK) cell line. Syntaxin 12/13 and VAMP8 caused a collapse of the EGFP-Rab11a compartment, whereas 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 a 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 proteomic analysis of an immunoisolated vesicle population from native human tissue, demonstrates the diversity of putative regulators of the apical recycling system.
Lapierre
Lynne A
LA
Dept. of Surgery, Vanderbilt Univ. School of Medicine, 4160A MRB III, 465 21st St. S., Nashville, TN 37232-2733, USA.
Avant
Kenya M
KM
Caldwell
Catherine M
CM
Ham
Amy-Joan L
AJ
Hill
Salisha
S
Williams
Janice A
JA
Smolka
Adam J
AJ
Goldenring
James R
JR
D000328:Adult; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D004285:Dogs; D005260:Female; D017506:H(+)-K(+)-Exchanging ATPase / Q000032:analysis / Q000502:physiology*; D006801:Humans; D008297:Male; D008875:Middle Aged; D010295:Parietal Cells, Gastric / Q000502:physiology* / Q000648:ultrastructure; D020543:Proteome / Q000032:analysis; D050600:SNARE Proteins / Q000502:physiology; D015290:Second Messenger Systems; D053719:Tandem Mass Spectrometry; D020691:rab GTP-Binding Proteins / Q000032:analysis; D000099193:rab11 GTP-Binding Proteins
D017506:H(+)-K(+)-Exchanging ATPase; D020543:Proteome; D050600:SNARE Proteins; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins