Figure 1. TRAPPII and TRAPPIII are specific GEFs for Ypt31/32 and Ypt1, respectively.

(A) Subunit composition of TRAPPII and TRAPPIII complexes. (B) Left: Prenylated Rab/GDI substrates and Arf1 used to recruit TRAPPII to membranes. Right: TRAPPII and rTRAPPIII were purified from yeast or E. coli using the indicated affinity tags. (C) Left: Representative traces showing activation of Rab substrates by TRAPPII in the presence of Ni²⁺ TGN liposomes. mock = buffer only control reaction. Right: Rates of TRAPPII-mediated nucleotide exchange calculated from the traces at left. Error bars represent 95% CIs for n ≥ 3 reactions. (D) Left: Representative traces showing activation of Rab substrates by TRAPPII in the presence of TGN liposomes (without Ni²⁺) and Arf1-GTP. Right: Rates of TRAPPII-mediated nucleotide exchange calculated from the traces at left. Error bars represent 95% CIs for n ≥ 3 reactions. n.d., not detectable (the Rab was not activated by the GEF and exponential functions could not be fit to experimental curves). (E) Left: Representative traces showing activation of Rab substrates by TRAPPIII in the presence of TGN liposomes. Right: Rates of TRAPPIII-mediated nucleotide exchange calculated from the traces at left. Error bars represent 95% CIs for n ≥ 3 reactions. (F) Schematic depicting the setup for the GEF-Rab Interaction Test (GRab-IT). (G) Left: TRAPPII localizes largely to Sec7-labeled late Golgi compartments (top) and representative images showing TRAPPII recruitment to mitochondria by nucleotide-free Ypt32, but not Ypt1 (bottom). Right: Quantification of TRAPPII recruitment to mitochondria. Error bars represent 95% CIs for n > 40 cells. See Figure S1 for images of TRAPPII with nucleotide-free Ypt6 and Sec4. (H) Left: TRAPPIII localizes to late Golgi compartments (top) and representative images showing TRAPPIII recruitment to mitochondria by nucleotide-free Ypt1, but not Ypt32 (bottom). Right: Quantification of TRAPPIII recruitment to mitochondria. Error bars represent 95% CIs for n > 40 cells. Scale bars, 2 μm. White arrowheads denote colocalization of TRAPP complexes and Sec7 at late Golgi compartments.

See also Figures S1 and S2.