Fig. 6: SPIRE1/2 interact with active Rab27a via their membrane-binding C-termini.
a A schematic representation of the domain structure of SPIRE1/2, Mlph and truncations used in interaction studies (b, c, e, f). Interaction of SPIRE1/2 and Rab27a was investigated using GST pull-down (b, c, e, f) and BiFC (d) assays (see Experimental procedures). b, c, e Western blots and Ponceau S stained filters showing the results of pull-down assays measuring the interaction of GST-Rab27a (active Q78L and inactive T23N mutants) with SPIRE1/2 and the indicated truncations (Myc-tagged (b, c) and GFP-tagged (e) in vitro). c is a contrast enhanced version of the section of b showing interaction of SPIRE2 with Rab27a-Q78L and Rab27a-T23N. d Fluorescence images and a bee swarm plot (upper and lower panels) showing the results of the BiFC assay, reporting the interaction of Rab27a with SPIRE1 and SPIRE2 in HEK293a cells (see Experimental procedures). Images of mCherry indicate transfection efficiency and vYFP indicates BiFC, i.e., interaction. The bee swarm plot shows the BiFC signal for populations of cells expressing SPIRE1/2 with and without active and inactive Rab27a mutants. Data shown are from three independent experiments. ****, *** and ** indicate significant differences of p = <0.0001, p = <0.001 and p = <0.01 between the adjacent dataset and Rab27a wild-type/SPIRE1/2 expressing cells as determined by one-way ANOVA of data for SPIRE1 and SPIRE2 with different Rab27a proteins. No other significant differences were observed. Two-way ANOVA comparison of BiFC signal for the Rab27a proteins with different SPIREs revealed no significant differences. Bars indicate the mean and 25th and 75th percentile of data. Scale bar = 250 μm. f Line plots showing the extent of binding of GFP-SPIRE1-MSFH (n = 4) and GFP-Mlph-RBD (n = 3) as a function of increasing GST-Rab27a-Q78L concentrations. Data are presented as mean values ± SEM and the equilibrium dissociation constants (Kd) are provided. K KIND, W WH2, M GTBM globular tail domain binding motif, S SB SPIRE box, F FYVE-type zinc finger, H C-terminal flanking sequences similar to H2 of Slp/Slac-proteins, WB western blotting. Source data b–f are provided in the Supplementary Source data file.
