Panel A: Yeast two-hybrid assay results showing interactions of RAB27A (wild type and Trp73Arg) with MLPH-SHD. RAB38 is used as a negative control. Panel B: Co-immunoprecipitation assay results showing interactions of FLAG-RAB27A (wild type and Trp73Arg) with T7-MUNC13-4 in COS-7 cells. Immunoblotting is used to detect co-immunoprecipitated T7-MUNC13-4 and immunoprecipitated FLAG-RAB27A. Molecular mass markers in kilodaltons are shown on the left. Panel C: Table outlines the binding activities of RAB27A (wild type, Trp73Arg, and Val143Ala) toward MLPH and MUNC13-4.
RAB27A(Trp73Arg) was unable to bind to RAB effectors that function in melanocytes (MLPH) and CTLs (MUNC13-4). (A) Interactions of RAB27A (WT or Trp73Arg; indicated as W73R) with MLPH-SHD as assessed using yeast two-hybrid assays. RAB38, another melanosomal protein that does not bind to MLPH, was used as a negative control. (B) Interactions of FLAG-RAB27A (WT or Trp73Arg) with T7-MUNC13-4 as assessed by co-immunoprecipitation assays in COS-7 cells. Co-immunoprecipitated T7-MUNC13-4 and immunoprecipitated (IP) FLAG-RAB27A were detected by immunoblotting with the antibodies indicated. The positions of the molecular mass markers (kDa) are shown on the left. (C) Summary of the binding activities of RAB27A (WT, Trp73Arg, and Val143Ala; indicated as V143A) toward MLPH, which functions in melanocytes, and MUNC13-4, which functions in CTLs. Data for RAB27A(Val143Ala) were obtained previously (14). Data in A and B are representative of the data obtained in two and three independent experiments, respectively, and similar results were obtained in each experiment. Source data are available for this figure: SourceData F3.