Rab35 binding activity of the centaurin-β2 homologues centaurin-β1 and centaurin-β5.
A, schematic representation of the domain organization of Centβ1/ACAP1, Centβ2/ACAP2, and Centβ5/ACAP3. B, Centβ2-ANKR, but not Centβ1-ANKR or Centβ5-ANKR, specifically recognized the active form of Rab35. Yeast cells containing pAct2 plasmid expressing Centβ1-ANKR, Centβ2-ANKR, or Centβ5-ANKR and pGBD plasmid expressing Rab35(QL) (a constitutively active form) or Rab35(SN) (a constitutively negative form) were streaked on SC-LW (left panels) and SC-AHLW (right panels) and incubated at 30 °C. C, Centβ2-ANKR, but not Centβ1-ANKR or Centβ5-ANKR, interacted with Rab35 in COS-7 cells. Co-immunoprecipitation assays were performed as described in the legend for Fig. 1E. The positions of the molecular mass markers (in kilodaltons) are shown on the left. IP, immunoprecipitation; PH, pleckstrin homology domain; ANK, ankyrin repeat.