Syntaxin 1A has a specific binding site in the H3 domain that is critical for targeting of H+-ATPase to apical membrane of renal epithelial cells

Abstract

H+ transport in the collecting duct is regulated by exocytic insertion of H+-ATPase-laden vesicles into the apical membrane. The soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptor (SNARE) proteins are critical for exocytosis. Syntaxin 1A contains three main domains, SNARE N, H3, and carboxy-terminal transmembrane domain. Several syntaxin isoforms form SNARE fusion complexes through the H3 domain; only syntaxin 1A, through its H3 domain, also binds H+-ATPase. This raised the possibility that there are separate binding sites within the H3 domain of syntaxin 1A for H+-ATPase and for SNARE proteins. A series of truncations in the H3 domain of syntaxin 1A were made and expressed as glutathione S-transferase (GST) fusion proteins. We determined the amount of H+-ATPase and SNARE proteins in rat kidney homogenate that complexed with GST-syntaxin molecules. Full-length syntaxin isoforms and syntaxin-1AΔC [amino acids (aa) 1–264] formed complexes with H+-ATPase and SNAP23 and vesicle-associated membrane polypeptide (VAMP). A cassette within the H3 portion was found that bound H+-ATPase (aa 235–264) and another that bound SNAP23 and VAMP (aa 190–234) to an equivalent degree as full-length syntaxin. However, the aa 235–264 cassette alone without the SNARE N (aa 1–160) does not bind but requires ligation to the SNARE N to bind H+-ATPase. When this chimerical construct was transected into inner medullary collecting duct cells it inhibited intracellular pH recovery, an index of H+-ATPase mediated secretion. We conclude that within the H3 domain of syntaxin 1A is a unique cassette that participates in the binding of the H+-ATPase to the apical membrane and confers specificity of syntaxin 1A in the process of H+-ATPase exocytosis.