We propose to develop a complete picture for the vectorial orientation and sites of surface exposure of rhodopsin in the rod outer segment disc membrane. This should firmly establish whether rhodopsin is a transmembrane protein and lead to a determination of its sidedness in the membrane. Localization of various important sites in rhodopsin should assist in construction of theories to explain the mode of action of rhodopsin in visual transduction. Osmotically intact disc membranes of native sidedness will be prepared and characterized. They will be labeled with various enzymes and chemical reagents. Transglutaminase will be used to label rhodopsin with DNP-cadaverine, and the site(s) of attachment will be determined. The highly reactive nonpenetrating nitrene precursr N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate will be used to determine the surface exposure of additional peptide regions of rhodopsin. Antibodies to rhodopsin's carboxyl-terminal region 1'-34' will be prepared. Antibody conjugates will be used to label rhodopsin in the plasma membrane and disc membranes in order to determine the surface localization of rhodopsin's carboxyl terminal. The region of rhodopsin which exists in the membrane-lipid environment will be labeled using the photoreactive reagent 1-azidopyrene. Labeled peptides from rhodopsin's hydrophobic surface will be isolated.
