Orientation of bacteriorhodopsin in Halobacterium halobium as studied by selective proteolysis.
The orientation of bacteriorhodopsin in the purple membrane of Halobacterium halobium has been studied by proteolytic degradation of purple membrane sheets, reconstituted vesicles, and whole cells, with the following results: (i) 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. (ii) The apomembrane, which lacks retinal, undergoes a second cleavage with trypsin releasing a fragment of approximately 6300 molecular weight from the amino terminus. (iii) 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. (iv) In envelope vesicles, which largely pump protons in the same direction as the whole cells, the carboxyl terminus is largely protected against proteolysis. (v) Treatment of whole cells with proteinase K hydrolyzes the cell wall proteins but has no effect on acteriorhodopsin. However, the same treatment after lysis of the cells results in degradation of the hydrophilic region at the carboxyl terminus. The results show that the carboxyl terminus as well as the additional cleavage site near the amino terminus observed in apomembrane are on the cytoplasmic side of the purple membrane.
D001059:Apoproteins; D001426:Bacterial Proteins; D001436:Bacteriorhodopsins; D002338:Carotenoids; D002462:Cell Membrane / Q000648:ultrastructure; D006217:Halobacterium / Q000648:ultrastructure; D008565:Membrane Proteins; D008567:Membranes, Artificial; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism; D013499:Surface Properties
D001059:Apoproteins; D001426:Bacterial Proteins; D008565:Membrane Proteins; D008567:Membranes, Artificial; D002338:Carotenoids; D001436:Bacteriorhodopsins; D010447:Peptide Hydrolases
J Biol Chem. 1972 May 25;247(10):3242-51
4112808
Nat New Biol. 1971 Sep 29;233(39):149-52
4940442
Biochemistry. 1971 Jun 22;10(13):2606-17
4326772
Biochem Biophys Res Commun. 1971 Apr 2;43(1):88-93
4325496
J Biol Chem. 1974 Jan 25;249(2):662-3
4272126
Biochemistry. 1975 Jul;14(13):2882-9
50859
Biochim Biophys Acta. 1976 Apr 5;426(4):703-10
4105
Biochem Biophys Res Commun. 1975 Jan 2;64(3):1054-61
238520
Proc Natl Acad Sci U S A. 1973 Oct;70(10):2853-7
4517939
Proc Natl Acad Sci U S A. 1974 Apr;71(4):1234-8
4524635
FEBS Lett. 1974 Aug 30;44(3):257-61
4414481
Angew Chem Int Ed Engl. 1976 Jan;15(1):17-24
814845
Proc Natl Acad Sci U S A. 1976 Aug;73(8):2687-91
1066681
Proc Natl Acad Sci U S A. 1976 Apr;73(4):1159-63
772680
Proc Natl Acad Sci U S A. 1975 Aug;72(8):2964-8
1059087
Biochemistry. 1976 Feb 24;15(4):792-8
1247534
Nature. 1975 Sep 4;257(5521):28-32
1161000
Can J Biochem. 1975 Mar;53(3):284-92
1125815
Nature. 1970 Aug 15;227(5259):680-5
5432063
Proc Natl Acad Sci U S A. 1977 Jul;74(7):2830-4
331317
Biochim Biophys Acta. 1977 Apr 18;466(2):315-24
857886
Fed Proc. 1977 May;36(6):1799-804
856634