FIGURE 1:
The DENN domain of connecdenn 3 exhibits higher GEF activity toward Rab35 than Rab13. (A) GST, GST-Rab1A, GST-Rab13, and GST-Rab35, maintained in the nucleotide-free state with 5 mM EDTA or preloaded with GTPγS, were used as bait in affinity selection assays with soluble lysates from HEK-293T cells transfected with FLAG-tagged DENN domains of connecdenn (CD) 1–3. Specifically bound proteins were detected by anti-FLAG antibody. Cell lysate (starting material) equivalent to 1/10 that added to the beads was analyzed in parallel. (B) GEF activity of 350 nM purified DENN domains of connecdenn 2 and 3 (CD2/CD3) or basal exchange activity of Rab13 or Rab35 alone measured as the incorporation of [35S]GTPγS onto 1.25 μM GDP-loaded Rab13 or Rab35 as a function of time. The number of picomoles of incorporated GTPγS is plotted over time, and points represent mean (± SE), n = 2. (C) The exchange activity of the connecdenn 2 and 3 DENN domains against Rab13 from B plotted on an expanded scale. (D) Specific amount of GTPγS loaded onto 1.25 μM Rab13 or Rab35 in the presence of connecdenn 2 or connecdenn 3 DENN domains after 20 min was determined by subtracting the amount of GTPγS loaded in the absence of DENN domains.