(A) A schematic drawing of a mammalian rod photoreceptor, showing the OS, IS, nucleus (N), and synapse (S).
(B) A magnified view of the junction between the distal IS and the proximal OS of mouse rods. Mouse rod OS is ~30 μm in length and ~1.5 μm in diameter; connecting cilium (CC) is ~1.2 μm in length and ~0.2 μm in diameter. The microtubule-based axoneme (Ax) begins at the basal body (BB) in the distal IS and continues through the CC and into the proximal OS axoneme for ~10 μm.
(C) X-gal filter assay results of yeast transformants expressing SARA and Rho39Tr or the vector.
(D) Schematic representation of SARA.
(E) Pull-down assays were carried out by incubating purified His-SARA with either GST- or GST-Rho39-conjugated glutathione Sepharose. Glutathione eluates and input were immunoblotted with anti-SARA and anti-GST Abs, respectively.
(F) Mouse retinal lysates were immunoprecipitated with anti-rhodopsin mAb B6-30 or control mAb. The input (2 μg total protein) and the immunoprecipitates (from 5 μg total protein) were detected by immunoblotting using anti-SARA and anti-rhodopsin Abs. Bound momomeric rhodopsin was shown.
(G) Confocal images showing the triple labeling of endogenous EEA1 (red) and ectopically expressed rhodopsin (green) and Flag-SARA (cyan) in HEK cells. Arrows point to the colocalization of rhodopsin and SARA on early endosomes, which were also EEA1 labeled. SARA+/rhodopsin+/EEA1–vesicles were also occasionally observed (arrowheads). Bar = 20 μm.