Supplemental Material

This article contains the following supporting material:

• Materials and Methods -

• Figure 1 - rab-27 Gene Structure
  A. Genomic structure of the rab-27 gene (Y87G2A.4). Map of construct containing the rab-27 promoter driving GFP tagged RAB-27 (NM1112). Map of construct containing rab-27 promoter driving GFP (NM1030) B. Alignment of mouse Rab27B (m), rat Rab27B (r), and human Rab27A/B (h) with C. elegans RAB-27 (Ce). C. elegans RAB-27 shares about 70% identity (boxed) and about 80% homology (shaded) with other vertebrate Rab27. Mutations found in the different aex-6 mutant alleles are labeled. The aex-6(sa699) lesion is within codon 14 (GGC to GAC), which causes a G14 to D14 amino acid substitution. The aex-6(n2172) lesion is within codon 108 (CGA to TGA), which causes an R108 to premature stop codon substitution. The aex-6(sa24) lesion is within codon 180 (CAA to TAA), which causes a Q180 to premature stop codon substitution.

• Figure 2 - AEX-3 Increases the Amount of GTP-Bound RAB-3 and RAB-27, but not RAB-5 in COS-7 cells - Additional Controls
  A. RBD of Rim2 and Rabphilin specifically interact with RAB-3 (left) and RAB-27 (right), respectively. Glutathione-Sepharose beads coupled with GST-Rabphilin-RBD or GST-Rim2-RBD were incubated with COS-7 cell lysates containing RAB-3, RAB-5, RAB-8, RAB-11A, RAB-27, or RAB-37. Proteins bound to the beads were analyzed by 10% SDS-PAGE followed by immunoblotting with anti-FLAG tag antibody (middle panels) and anti-T7 tag antibody (bottom panels). Input means 1/80 volume of the reaction mixture used for immunoprecipitation (top panels). The positions of molecular mass markers (x 10^-3) are shown on the left. B. GST-Rim2-RBD and GST-Rabphilin-RBD specifically trap the GTP-bound form of RAB-3 (left) and RAB-27 (right), respectively, in COS-7 cells. GTP-bound Rab assay was performed as described in Materials and Methods. Note both RAB-3 and RAB-27 did not bind GST-Rabenosyn-5 or GST alone (top panels). C. AEX-3 does not increase the amount of GTP-bound RAB-5 in COS-7 cells. The GTP-bound RAB-5 was pull-downed by GST-Rabenosyn-5 (Nielsen et al. , 2000).