Figure 6.

SipA acts as a cognate R-SNARE to promote fusion of SCVs with EEs. (A) To determine the role of Syn8, in vitro fusion was performed between SCVs containing biotinylated Salmonella:WT or Salmonella:sipAKO and EEs containing avidin-HRP in the presence of ATP-regenerating fusion buffer supplemented with gel-filtered cytosol for 1 h at 37°C. Fusion was measured as described in Materials and methods. (B) To determine the role of Syn8 and Rab5, a similar fusion assay was performed between SCV:WT and EE in the presence of indicated antibodies. (C) To determine the role of identified SipA cognate SNAREs, fusion was performed between SCV:WT and EE in presence of the cytoplasmic domain of indicated SNARE proteins. Fusion obtained with SCV:WT with EE was chosen as 1 U in all experiments, and the results are expressed as relative fusion of three independent experiments ± SEM. 1 U corresponds with ∼30 ng, 34 ng, or 36 ng HRP activity per mg protein in the fusion assay reported in A–C, respectively. Levels of significance are indicated by P values.