A eukaryotic protein, NOP-1, binds retinal to form an archaeal rhodopsin-like photochemically reactive pigment

The nop-1 gene from Neurospora crassa is predicted to encode a seven-helix protein exhibiting conservation with the rhodopsins of the archaeon Halobac terium salinarum. In the work presented here we have expressed this gene he terologously in the yeast Pichia pastoris, obtaining a relatively high yiel d of 2.2 mg of NOP-1 protein/L of cell culture. The expressed protein is me mbrane-associated and forms with all-trans retinal a visible light-absorbin g pigment with a 534 nm absorption maximum and similar to 100 nm half-bandw idth typical of retinylidene protein absorption spectra. Its lambda(max) in dicates a protonated Schiff base linkage of the retinal. Laser flash kineti c spectroscopy demonstrates that the retinal-reconstituted pigment undergoe s a photochemical reaction cycle with a near-UV-absorbing intermediate that is similar to the M intermediates produced by transient Schiff base deprot onation of the chromophore in the photocycles of bacteriorhodopsin and sens ory rhodopsins I and II. The slow photocycle (seconds) and long-lived inter mediates (M and O) are most similar to those of the phototaxis receptor sen sory rhodopsin II. The results demonstrate a photochemically reactive membe r of the archaeal rhodopsin family in a eukaryotic cell.