Fig. 3.

Rinl preferentially interacts with nucleotide free Rab5a and acts as GEF for Rab5a. (A) Yeast was transformed with the indicated Rab5a bait constructs and Rinl full-length and deletion prey constructs. Interaction was screened by growth on –HIS. (B) Table showing the activation of the reporter genes HIS and lacZ. (C) Rinl GST-deletion constructs were purified from bacteria and used for a pulldown of GFP-Rab5a (wt, S34N, Q79L) expressed in COS7 cells. Proteins were detected by immunoblotting (IB) using anti-GFP antibodies. 5% of the total Rab5a lysates are shown as input. (D) Extracts of transiently transfected HEK 293T cells were used to immunoprecipitate Rinl with anti-Rinl antibodies. Co-immunoprecipitated Rab5a was detected by immunoblotting with anti-GFP. No Rab7a co-immunoprecipitation is detected. 10% of the input is shown (total lysate). IP, immunoprecipitation. (E) 200 nM Rab5a or Rab7a loaded with mGDP were incubated with increasing amounts of Rinl in the presence of 20 μM unlabeled GDP. The exchange of mGDP for GDP was measured as decay in fluorescence signal. In case of Rab7a proper nucleotide loading was demonstrated by the addition of EDTA at the indicated time point, which induces the release of nucleotides and a rapid decay in the fluorescence signal.