Covalent modification of rhodopsin with imidoesters: evidence for transmembrane arrangement of rhodopsin in rod outer segment disk membranes

Abstract

The transmembrane disposition of the visual pigment rhodopsin was studied by the covalent labeling of protein amino groups with membrane-permeable and -impermeable imidoesters. A new, highly reactive permeable reagent, 2-(methylsulfonyl)ethyl acetimidate (SAI) was developed. The permeabilities of this compound and the impermeable reagent isethionyl acetimidate (IAI) across the rod outer segment disc membrane were directly measured. Rhodopsin apparently contains 3 classes of amino groups. One class (35-55% of the total) reacts rapidly with the membrane-impermeable reagent and is presumably exposed on the outside surface of the membrane. A 2nd class (35-55% of the total) is located on the internal surface of the disc since its rate of reaction is dependent on the relative permeabilities of the labeling reagents. The remaining 10% of the rhodopsin amino groups are inaccessible to either type of imidate and are largely accounted for by the single lysine residue which specificially binds the chromophore retinal. Freeze-fracture EM and the above properties imply rhodopsin is a transmembrane protein.