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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON

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

¹Renal Section, Boston University Medical Center, and Departments of ²Medicine, ³Physiology, and ⁴Pathology, Boston University School of Medicine, Boston, Massachusetts; and ⁵Department of Nephrology, First Affiliated Hospital, Zhongshan University, Guangzhou, China

Submitted 2 February 2005 ; accepted in final form 27 April 2005

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-1AC [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.

soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor proteins; exocytosis; H⁺⁺ transport

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