The UT-A1 urea transporter interacts with snapin, a SNARE-associated protein.
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.
Mistry
Abinash C
AC
Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Mallick
Rickta
R
Fröhlich
Otto
O
Klein
Janet D
JD
Rehm
Armin
A
Chen
Guangping
G
Sands
Jeff M
JM
D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004285:Dogs; D015723:Gene Library; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008954:Models, Biological; D009865:Oocytes / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000502:physiology*; D014981:Xenopus / Q000378:metabolism
D018076:DNA, Complementary; C118519:SNAPIN protein, human; D033921:Vesicular Transport Proteins