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The UT-A1 urea transporter interacts with snapin, a SNARE-associated protein

Item Type:Article
Title:The UT-A1 urea transporter interacts with snapin, a SNARE-associated protein
Creators Name:Mistry, A.C. and Mallick, R. and Froehlich, O. and Klein, J.D. and Rehm, A. and Chen, G. and Sands, J.M.
Abstract:The UT-A1 urea transporter mediates rapid transepithelial urea transport across the inner medullary collecting duct and plays a major role in the urinary concentrating mechanism. To transport urea, UT-A1 must be present in the plasma membrane. The purpose of this study was to screen for UT-A1-interacting proteins and to study the interactions of one of the identified potential binding partners with UT-A1. Using a yeast two-hybrid screen of a human kidney cDNA library with the UT-A1 intracellular loop (residues 409-594) as bait, we identified snapin, a ubiquitously expressed SNARE-associated protein, as a novel UT-A1 binding partner. Deletion analysis indicated that the C-terminal coiled-coil domain (H2) of snapin is required for UT-A1 interaction. Snapin binds to the intracellular loop of UT-A1 but not to the N- or C-terminal fragments. Glutathione S-transferase pulldown experiments and co-immunoprecipitation studies verified that snapin interacts with native UT-A1, SNAP23, and syntaxin-4 (t-SNARE partners), indicating that UT-A1 participates with the SNARE machinery in rat kidney inner medulla. Confocal microscopic analysis of immunofluorescent UT-A1 and snapin showed co-localization in both the cytoplasm and in the plasma membrane. When we co-injected UT-A1 with snapin cRNA in Xenopus oocytes, urea influx was significantly increased. In the absence of snapin, the influx was decreased when UT-A1 was combined with t-SNARE components syntaxin-4 and SNAP23. We conclude that UT-A1 may be linked to the SNARE machinery via snapin and that this interaction may be functionally and physiologically important for urea transport.
Keywords:Biological Models, Biological Transport, Cell Membrane, Complementary DNA, Gene Library, Kidney, Oocytes, Tertiary Protein Structure, Two-Hybrid System Techniques, Vesicular Transport Proteins, Animals, Dogs, Rats, Xenopus
Source:Journal of Biological Chemistry
Publisher:American Society for Biochemistry and Molecular Biology
Page Range:30097-30106
Date:12 October 2007
Official Publication:https://doi.org/10.1074/jbc.M705866200
PubMed:View item in PubMed

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