SNARE Proteins Regulate H+-ATPase Redistribution to the Apical Membrane in Rat Renal Inner Medullary Collecting Duct Cells

Abhijit Banerjee,Theodora Shih,Edward A Alexander,John H Schwartz

doi:10.1074/jbc.274.37.26518

Abstract

The interaction of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins provides the necessary steps for vesicle docking fusion. In inner medullary collecting duct (IMCD) cells, acid secretion is regulated in part by exocytotic insertion and endocytotic retrieval of an H(+)-ATPase to and from the apical membrane. We previously suggested a role for SNARE proteins in exocytotic insertion of proton pumps in IMCD cells. The purpose of the present study was to determine whether SNARE proteins are associated with the 31-kDa subunit of H(+)-ATPase in IMCD cells during exocytosis and to determine the effects of clostridial toxins on SNARE-mediated trafficking of H(+)-ATPase. Cell acidification induced a marked increment of H(+)-ATPase in the apical membrane. However, pretreating cells with clostridial toxins blocked the cellular translocation of the 31-kDa subunit. Immunoprecipitation of IMCD cell homogenate, using antibodies against either the 31-kDa subunit of H(+)-ATPase or vesicle-associated membrane protein-2, co-immunoprecipitated N-ethylmaleimide-sensitive factor, alpha-soluble NSF attachment protein (alpha-SNAP), synaptosome-associated protein-23, syntaxin, and vesicle-associated membrane protein-2. Pretreatment with clostridial toxin resulted in reduced co-immunoprecipitation of H(+)-ATPase and syntaxin. These experiments document, for the first time, a putative docking fusion complex in IMCD cells and a physical association of the H(+)-ATPase with the complex. The sensitivity to the action of clostridial toxin indicates the docking-fusion complex is a part of the exocytotic mechanism of the proton pump.

Highlights

The vacuolar Hϩ-ATPase is a ubiquitous multisubunit enzyme that participates in a wide variety of cellular functions [1]
To characterize the mechanisms involved in inner medullary collecting duct (IMCD) trafficking of Hϩ-ATPase and to characterize the role of sensitive factor attachment protein receptor (SNARE) in this process, we determined the effect of clostridial toxins on cell acidification-induced translocation of both Hϩ-ATPase and SNAREs to the apical membrane, identified the v-SNARE proteins that participate in the translocation of Hϩ-ATPase from vesicle to apical membrane following an acid load, and isolated a complex of proteins similar to the docking complex (20 S complex) that is required for docking and fusion of the vesicles
Hϩ-ATPase Translocation to Apical Membrane and Toxin Action—After acute cell acidification, a maneuver that enhances the rate of Hϩ transport by IMCD cells [2], the mass of the immunodetectable 31-kDa subunit of Hϩ-ATPase increased in apical membrane by a factor of 2.5, compared with that present in apical membrane of control cells (Fig. 1, A and B)

Summary

Introduction

The vacuolar Hϩ-ATPase is a ubiquitous multisubunit enzyme that participates in a wide variety of cellular functions [1]. To date, no studies have described the exocytotic mechanisms regulating Hϩ-ATPase insertion into the apical plasma membrane or provided direct evidence for the participation of the SNARE proteins in this process. To characterize the mechanisms involved in IMCD trafficking of Hϩ-ATPase and to characterize the role of SNAREs in this process, we determined the effect of clostridial toxins on cell acidification-induced translocation of both Hϩ-ATPase and SNAREs to the apical membrane, identified the v-SNARE proteins that participate in the translocation of Hϩ-ATPase from vesicle to apical membrane following an acid load, and isolated a complex of proteins similar to the docking complex (20 S complex) that is required for docking and fusion of the vesicles. The present study, utilizing previously characterized cultured IMCD cells [15], provides direct evidence for the participation of SNARE proteins in Hϩ-ATPase vesicle trafficking and elucidates an oligomeric protein complex that is comprised of the v- and t-SNAREs, NSF, SNAP, and Hϩ-ATPase and that resembles the previously described 20 S docking complex in neuronal cells

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