FIG. 1.
Interaction of TRPV5 and TRPV6 with Rab11a. (A) The carboxyl terminus of TRPV6 or γENaC and full-length Rab11a were cotransformed into the Y153 yeast strain and grown on media without tryptophan and leucine. β-Galactosidase activity was determined for TRPV6- and Rab11a-cotransformed yeast, and γENaC- and Rab11a-cotransformed yeast was used as a negative control. Two representative colonies are depicted. (B) [35S]methionine-labeled TRPV5 (left) or TRPV6 (right) was incubated with GST-Rab11a WT, GTP-locked mutant Rab11a (S20V), GDP-locked Rab11a (S25N), or GST alone immobilized on glutathione-Sepharose 4B beads. (C) Coomassie staining of the gels shows equal expression of GST, GST-fused Rab11a WT S20V, or S25N. (D) [35S]methionine-labeled Rab11a S20V or Rab11a S25N was incubated with GST-TRPV5, GST-TRPV6, or GST immobilized on glutathione-Sepharose 4B beads. Both TRPV5 and TRPV6 strongly interacted with Rab11a S25N, whereas no binding to Rab11a S20V was observed. (E) Coomassie staining of the gels demonstrating similar amounts of GST, GST fused to the carboxyl terminus of TRPV5, or GST fused to the carboxyl terminus of TRPV6. (F and G) Xenopus laevis oocytes were injected with Rab11a S25N or coinjected with HA-tagged TRPV5 and Rab11a S25N cRNAs. Oocyte lysates were subjected to immunoprecipitation using monoclonal anti-HA antibodies. The precipitated sample was immunoblotted for the presence of TRPV5 (F) or coprecipitated Rab11a (G).