Identification of the retinal-binding protein in halorhodopsin

Cell envelope vesicles prepared from a retinal-deficient strain of Halobacterium halobium contained the apoprotein of halorhodopsin, but not the apoprotein of bacteriorhodopsin. Halorhodopsin was reconstituted in these membranes with tritium-labeled retinal and the preparation was reduced with sodium cyanoborohydride. The product was a bleached pigment in which the retinal-protein bond was resistant to hydroxylamine cleavage. Fluorography of sodium dodecyl sulfate/urea-polyacrylamide gels showed that one protein was radioactively labeled, almost exclusively. This protein migrated with an apparent molecular weight somewhat lower than that of bacteriorhodopsin, which was reconstituted in membranes from another strain and radioactively labeled under the same conditions. Thus, the retinal-binding component of halorhodopsin is a small protein, with an apparent molecular weight of approximately 25,000.