Orientation of bacteriorhodopsin in Halobacterium halobium as studied by selective proteolysis

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Abstract
The orientation of bacteriorhodopsin in the purple membrane of H. halobium was studied by proteolytic degradation of purple membrane sheets, reconstituted vesicles and whole cells, with the following results: bacteriorhodopsin in purple membrane sheets is cleaved at a single site by Pronase or trypsin; a polypeptide segment of about 15 amino acids is lost from the carboxyl end. Carboxypeptidase A sequentially releases amino acids from the carboxyl end; the tetrapeptide sequence -Ala-Ala-Thr-Ser(COOH) was tentatively deduced for this terminus. The apomembrane, which lacks retinal, undergoes a 2nd cleavage with trypsin releasing a fragment of .apprx. 6300 MW from the amino terminus. Vesicles reconstituted from the purple membrane sheets and synthetic lecithins, in which the direction of proton pumping is opposite to that in the whole cells, have the carboxyl terminus of bacteriorhodopsin accessible to proteolysis. In envelope vesicles, which largely pump protons in the same direction as the whole cells, the carboxyl terminus is largely protected against proteolysis. Treatment of whole cells with proteinase K hydrolyzes the cell wall proteins but has no effect on bacteriorhodopsin. The same treatment after lysis of the cells results in degradation of the hydrophilic region at the carboxyl terminus. The carboxyl terminus and the additional cleavage site near the amino terminus observed in apomembrane are on the cytoplasmic side of the purple membrane.