Genetic and biochemical resolution of the chromophoric polypeptide of halorhodopsin

Retinal-binding proteins in bacteriorhodopsin-deficient membranes of Halobacterium halobium have been separated on polyacrylamide gels after labeling with (3H)retinal. Four labeled polypeptides are observed: one at an Mr of 19,000, two distinct bands near 25,000, and one at 94,000. Comparing halorhodopsin-containing and halorhodopsin-deficient mutants and using ion translocation measurements and flash spectroscopy to test for the photoactive retinal pigments, we show that the lower of the two radiolabeled bands at 25,000 is the retinal-binding polypeptide of halorhodopsin. The identities of the other three polypeptides are not known, but one of them, probably the upper of the two 25,000 bands, is likely to be s-rhodopsin.