Sec15 and Rab11 homologues interact in T. brucei. (A) A 799-amino-acid fragment encompassing an N-terminal deletion of TbSec15 was expressed downstream of the activation domain (AD) in a yeast two-hybrid system. A mating yeast assay of TbSec15 against Rab11QL or Rab11SN (GTP and GDP locked configurations), fused to the binding domain (BD) in the yeast two-hybrid system, indicates a specific interaction of TbSec15 with the QL dominant-active GTP form of Rab11. (B) Procyclic-form parasites expressing GFP-tagged TbSec15 were fixed and prepared for immunofluorescence analysis using anti-GFP antibody. Left, merge of TbSec15-GFP (green) and DAPI fluorescence (blue); right, fluorescence channels merged with the transmitted light image. TbSec15-GFP distributes next to the flagellar pocket, the only site for exocytosis in T. brucei. Note the presence of a “C”-shaped profile for TbSec15, which is observed in the majority of stained cells. Bar, 2 μm. (C) Location of YFP-TbSec15 in bloodstream-form parasites compared to the locations of endosomes by immunostaining for Rab11 and Rab5. YFP-TbSec15 is visualized in green (white arrows), and rabbit polyclonal antibodies against Rab11 (left) and Rab5 (right) are in red. DNA was visualized with DAPI (blue, right panels, merge). Bar, 2 μm.