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The initial events of vision at low light take place in vertebrate retinal rods. The rod outer segment consists of a stack of flattened disks surrounded by the plasma membrane. A list of the proteins that reside in disks has not been achieved yet. We present the first comprehensive proteomic analysis of purified rod disks, obtained by combining the results of two-dimensional gel electrophoresis separation of disk proteins to MALDI-TOF or nLC-ESI-MS/MS mass spectrometry techniques. Intact disks were isolated from bovine retinal rod outer segments by a method that minimizes contamination from inner segment. Out of a total of 187 excised spots, 148 proteins were unambiguously identified. An additional set of 61 proteins (partially overlapping with the previous ones) was generated by one-dimensional (1D) gel nLC-ESI-MS/MS method. Proteins involved in vision as well as in aerobic metabolism were found, among which are the five complexes of oxidative phosphorylation. Results from biochemical, Western blot, and confocal laser scanning microscopy immunochemistry experiments suggest that F 1F o-ATP synthase is located and catalytically active in ROS disk membranes. This study represents a step toward a global physiological characterization of the disk proteome and provides information necessary for future studies on energy supply for phototransduction.

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The vertebrate retina is an array of "narrow-capture" photoreceptive elements of diverse cellular types that allow the fine spatial resolution characteristic of vision. Imaging of photoreceptors and of the whole retina has been previously reported; however, both were achieved exclusively after fixation. We report our development of a new technique for imaging live bovine retinas ex vivo. Using this technique, we conducted fluorescence confocal laser scanning microscopic imaging of bovine retinas. Eyecups were incubated with conventional fluorescent mitochondrial probes (MitoTracker and JC-1). Unexpectedly, we found that, besides the retinal mitochondria, the rod outer segments that are devoid of mitochondria were also stained. No other neuron was stained. Both protonophores, which decrease mitochondrial membrane potential, or inhibit electron transport strongly inhibited the selective association of dyes with both retinal rod outer segments and mitochondria. This is the first time that living rod outer segments were visualized by this technique. This finding may shed light on previous reports of the existence of a proton potential across the disk membranes and on the mechanism of the adenosine tri-phosphate (ATP) supply for phototransduction, which still requires investigation.