Syntaxin isoform specificity in the regulation of renal H+-ATPase exocytosis.
Intercalated and inner medullary collecting duct (IMCD) cells of the kidney mediate the transport of H+ by a plasma membrane H+-ATPase. The rate of H+ transport in these cells is regulated by exocytic insertion of H+-ATPase-laden vesicles into the apical membrane. We have shown that the exocytic insertion of proton pumps (H+-ATPase) into the apical membrane of rat IMCD cells, in culture, involves SNARE proteins (syntaxin (synt), SNAP-23, and VAMP). The membrane fusion complex observed in IMCD cells with the induction of proton pump exocytosis not only included these SNAREs but also the H+-ATPase. Based on these observations, we suggested that the targeting of these vesicles to the apical membrane is mediated by an interaction between the H+-ATPase and a specific t-SNARE. To evaluate this hypothesis, we utilized a "pull-down" assay in which we identified, by Western analysis, the proteins in a rat kidney medullary homogenate that complexed with glutathione S-transferase (GST) fusion syntaxin isoforms attached to Sepharose 4B-glutathione beads. The syntaxin isoforms employed were 1A, 1B, 2, 4, 5, and also 1A that was truncated to exclude the H3 SNARE binding domain (synt-1ADeltaH3). All full-length syntaxin isoforms formed complexes with SNAP-23 and VAMP. Neither GST nor synt-1ADeltaH3 formed complexes with these SNAREs. H+-ATPase (subunits E, a, and c) bound to syntaxin-1A and to a lesser extent to synt-1B but not to synt-1ADeltaH3 or synt-2, -4, and -5. In cultured IMCD cells transfected to express syntaxin truncated for the membrane binding domain (synt-DeltaC), expression of synt-1ADeltaC, but not synt-4DeltaC, inhibited H+-ATPase exocytosis. In conclusion, because all full-length syntaxins examined bound VAMP-2 and SNAP-23, but only non-H3-truncated syntaxin-1 bound H+-ATPase, and synt-1ADeltaC expression by intact IMCD cells inhibited H+-ATPase exocytosis, it is likely that the H+-ATPase binds directly to the H3 domain of syntaxin-1 and not through VAMP-2 or SNAP-23. Interaction between the syntaxin-1A and H+-ATPase is important in the targeted exocytosis of the proton pump to the apical membrane of intercalated cells.
Li
Guangmu
G
Renal Section, Boston University Medical Center and Department of Medicine, Boston University School of Medicine, Massachusetts 02118-2908, USA.
Alexander
Edward A
EA
Schwartz
John H
JH
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D005089:Exocytosis; D007679:Kidney Medulla / Q000201:enzymology*; D008565:Membrane Proteins / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D050765:Qa-SNARE Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050827:Syntaxin 1
D017931:DNA Primers; D008565:Membrane Proteins; D020033:Protein Isoforms; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins; C494213:Stx1a protein, rat; D050827:Syntaxin 1; D006180:Proton-Translocating ATPases