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Zhaoxia Zhang, Henry KW Fong; Low-density RGR–bound membranes from suspension of outer segments. Invest. Ophthalmol. Vis. Sci. 2018;59(9):966.
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© ARVO (1962-2015); The Authors (2016-present)
RGR opsin is expressed in cone photoreceptors of diurnal bovine and human retinas. If RGR were concentrated in cone outer segments, the RGR protein may be identified in preparations of outer segment membranes. To test this hypothesis, we analyzed a suspension of outer segment membranes from frozen bovine retinas.
We placed the thawed tissue into PBS without sucrose, and the suspension was gently mixed with a magnetic stirring bar for 1 hr at 4°C. Retinal suspensions were fractionated by differential centrifugation, and RGR was detected by Western immunoblotting. Bovine and human retina sections were analyzed by double-label immunofluorescent staining.
Rod outer segments in the initial suspension were removed substantially from the solution by centrifugation at 3000g. The remaining rod outer segments in the 3000g supernatant were collected by centrifugation at 25000g. Further serial centrifugation of the suspension at 200000g showed no rod outer segments in either the resultant pellet or supernatant. In addition to rod outer segments, a large proportion of RGR was found in the membranes that were collected by centrifugation at 25000g. Significant amounts of RGR opsin remained in the supernatants after serial centrifugation at 3000g, 25000g, and even 200000g, indicating that RGR–bound membranes from the sheared retina have a lower density than the rod outer segments. The results show that a subpopulation of RGR is bound to rhodopsin-free very low-density membranes from the retina. In all the fractions from differential centrifugation, the RGR protein migrated mainly as non-aggregated and non–ubiquitinated monomeric RGR, since protein bands larger that full-length RGR were faint or not detected. Therefore, RGR in the supernatant fractions was not in the form to bind solubilizing proteins such as Bag6 or ubiquitin-dependent p97/Cdc48.
The LWS/MWS and SWS cone photoreceptors in these mammals express a nonvisual opsin of the Go/RGR or tetraopsin group. RGR and the visual pigments are predominantly co-localized in the cone outer segment, which suggests functional interplay among these opsins. We have obtained RGR–bound membranes of very low density relative to rod outer segments and other intact cellular membranes. Isolation of large amounts of these RGR–bound membranes from the retina may provide a new approach to investigate the biochemical interaction of RGR and visual pigment.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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