Proteomics of Photoreceptor Outer Segments Identifies a Subset of SNARE and Rab Proteins Implicated in Membrane Vesicle Trafficking and Fusion

The outer segment is a specialized compartment of vertebrate rod and cone photoreceptor cells where phototransduction takes place. In rod cells it consists of an organized stack of disks enclosed by a separate plasma membrane. Although most proteins involved in phototransduction have been identified and characterized, little is known about the proteins that are responsible for outer segment structure and renewal. In this study we used a tandem mass spectrometry-based proteomics approach to identify proteins in rod outer segment preparations as an initial step in defining their roles in photoreceptor structure, function, renewal, and degeneration. Five hundred and sixteen proteins were identified including 41 proteins that function in rod and cone phototransduction and the visual cycle and most proteins previously shown to be involved in outer segment structure and metabolic pathways. In addition, numerous proteins were detected that have not been previously reported to be present in outer segments including a subset of Rab and SNARE proteins implicated in vesicle trafficking and membrane fusion. Western blotting and immunofluorescence microscopy confirmed the presence of Rab 11b, Rab 18, Rab 1b, and Rab GDP dissociation inhibitor in outer segments. The SNARE proteins, VAMP2/3, syntaxin 3, N-ethylmaleimide-sensitive factor, and Munc 18 detected in outer segment preparations by mass spectrometry and Western blotting were also observed in outer segments by immunofluorescence microscopy. Syntaxin 3 and N-ethylmaleimide- sensitive factor had a restricted localization at the base of the outer segments, whereas VAMP2/3 and Munc 18 were distributed throughout the outer segments. These results suggest that Rab and SNARE proteins play a role in vesicle trafficking and membrane fusion as part of the outer segment renewal process. The data set generated in this study is a valuable resource for further analysis of photoreceptor outer segment structure and function. The outer segment is a specialized compartment of vertebrate rod and cone photoreceptor cells where phototransduction takes place. In rod cells it consists of an organized stack of disks enclosed by a separate plasma membrane. Although most proteins involved in phototransduction have been identified and characterized, little is known about the proteins that are responsible for outer segment structure and renewal. In this study we used a tandem mass spectrometry-based proteomics approach to identify proteins in rod outer segment preparations as an initial step in defining their roles in photoreceptor structure, function, renewal, and degeneration. Five hundred and sixteen proteins were identified including 41 proteins that function in rod and cone phototransduction and the visual cycle and most proteins previously shown to be involved in outer segment structure and metabolic pathways. In addition, numerous proteins were detected that have not been previously reported to be present in outer segments including a subset of Rab and SNARE proteins implicated in vesicle trafficking and membrane fusion. Western blotting and immunofluorescence microscopy confirmed the presence of Rab 11b, Rab 18, Rab 1b, and Rab GDP dissociation inhibitor in outer segments. The SNARE proteins, VAMP2/3, syntaxin 3, N-ethylmaleimide-sensitive factor, and Munc 18 detected in outer segment preparations by mass spectrometry and Western blotting were also observed in outer segments by immunofluorescence microscopy. Syntaxin 3 and N-ethylmaleimide- sensitive factor had a restricted localization at the base of the outer segments, whereas VAMP2/3 and Munc 18 were distributed throughout the outer segments. These results suggest that Rab and SNARE proteins play a role in vesicle trafficking and membrane fusion as part of the outer segment renewal process. The data set generated in this study is a valuable resource for further analysis of photoreceptor outer segment structure and function. Vertebrate photoreceptor cells are highly specialized, photosensitive neurons that function in the transduction of light into an electrical signal and the transmission of this signal to other neurons in the retina as the initial steps in vision. Rod and cone photoreceptor cells consist of five principle regions: the outer segment where the process of phototransduction takes place, a thin connecting cilium that joins the outer segment to the inner segment and allows for the passage of proteins and other molecules between the inner and outer segments, the inner segment that contains the biosynthetic and metabolic machinery of the cell, the cell body harboring the nucleus, and the synaptic region containing the synaptic vesicles and the ribbon synapse for transmission of electrical signals to secondary neurons of the retina. The rod outer segment (ROS) 1The abbreviations used are: ROS, rod outer segment; COS, cone outer segment; RPE, retinal pigment epithelium, pAb, polyclonal antibody; mAb, monoclonal antibody, ER, endoplasmic reticulum, NSF, N-ethylmaleimide-sensitive factor; VAMP, vesicle-associated membrane protein; SNARE, soluble NSF attachment protein receptor; GDI, GDP dissociation inhibitor; ABC, ATP-binding cassette; IPI, International Protein Index; APEX, absolute protein expression measurement(s); DAPI, 4′,6-diamidino-2-phenylindole; LTQ, linear trapping quadrupole. consists of an ordered stack of over 1000 closed disks surrounded by a separate plasma membrane. Cone outer segments (COSs) have a similar stacked membrane organization, although the disk membranes are continuous with the plasma membrane. Outer segments undergo a continual renewal process in which new disk membrane is added at the base of the outer segment while packets of aged disks are shed from the distal end and removed by a phagocytic process mediated by adjacent retinal pigment epithelial (RPE) cells (1Young R.W. Bok D. Participation of the retinal pigment epithelium in the rod outer segment renewal process.J. Cell R.W. 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