A Eukaryotic Protein, NOP-1, Binds Retinal To Form an Archaeal Rhodopsin-like Photochemically Reactive Pigment

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Abstract
The nop-1 gene from Neurospora crassa is predicted to encode a seven-helix protein exhibiting conservation with the rhodopsins of the archaeon Halobacteriumsalinarum. In the work presented here we have expressed this gene heterologously in the yeast Pichia pastoris, obtaining a relatively high yield of 2.2 mg of NOP-1 protein/L of cell culture. The expressed protein is membrane-associated and forms with all-trans retinal a visible light-absorbing pigment with a 534 nm absorption maximum and ∼100 nm half-bandwidth typical of retinylidene protein absorption spectra. Its λmax indicates a protonated Schiff base linkage of the retinal. Laser flash kinetic spectroscopy demonstrates that the retinal-reconstituted pigment undergoes a photochemical reaction cycle with a near-UV-absorbing intermediate that is similar to the M intermediates produced by transient Schiff base deprotonation of the chromophore in the photocycles of bacteriorhodopsin and sensory rhodopsins I and II. The slow photocycle (seconds) and long-lived intermediates (M and O) are most similar to those of the phototaxis receptor sensory rhodopsin II. The results demonstrate a photochemically reactive member of the archaeal rhodopsin family in a eukaryotic cell.