XendoU plays a role in vesicle fusion and controls local RNA degradation on membranes. (A) Light membranes were washed one time in buffer containing 200 mM KCl (wlm 200) and incubated in the absence of ATP and GTP (no fusion) or in the presence of ATP and GTP (vesicle fusion). Alternatively, wlms were incubated for 30 min at RT with 5 µM control (IgG) or XendoU antibody followed by the addition of ATP and GTP. Aliquots were mixed with octadecyl rhodamine at 15 min, incubated for an additional 15 min at RT, and imaged live. Representative images of each condition are shown. (B) RNA was isolated from membrane pellets from the end points of reactions containing IgG or XendoU antibodies in A, 5′ end-labeled with [³²P-γ]ATP as described, and run on a denaturing gel. (C) Membranes were pelleted from standard vesicle fusion reactions (+ATP +GTP) or control reactions (−ATP –GTP), and RNA was isolated from the supernatant, run on a denaturing gel, and imaged with SYBR Green II. (D) Western blots of XendoU, ribosomal protein S6 (RibS6), ribosomal protein L7a (RibL7a), dynein, and TRAPα on membranes after vesicle fusion (+ATP +GTP) or in the absence of fusion (−ATP −GTP) in the presence of IgG or XendoU antibody. (E) Wlms were mock-treated or RNaseA treated (0.01 ng/µl, 0.1 ng/µl, or 1 ng/µl), then washed once more, and RNA was isolated and imaged as in C. (F) RNaseA-treated vesicles from E were incubated and imaged as in A at 20 min and 50 min after addition of ATP and GTP. Bars, 10 µm.