The carboxyl terminus of the membrane-binding domain of cytochrome b5 spans the bilayer of the endoplasmic reticulum.
Preliminary studies (Vergères, G., and Waskell, L. (1992) J. Biol. Chem. 267, 12583-12591) have suggested that the carboxyl-terminal membrane-binding domain of cytochrome b5 traverses the membrane and that the carboxyl terminus is in the lumen of the endoplasmic reticulum. In order to confirm and extend these studies, additional experiments were conducted. The gene coding for rat cytochrome b5 was transcribed and the resulting mRNA was translated in vitro in a rabbit reticulocyte lysate in the presence of microsomes. The binding and topology of cytochrome b5 were investigated by treating microsomes containing the newly incorporated cytochrome b5 with carboxypeptidase Y and trypsin. Our studies indicate that cytochrome b5 is inserted both co- and post-translationally into microsomes in a topology in which the membrane-binding domain spans the bilayer with its COOH terminus in the lumen. Cytochrome b5 is also incorporated into microsomes pretreated with trypsin in a topology indistinguishable from the one resulting from the insertion of the protein into untreated microsomes, reconfirming that cytochrome b5 does not use the signal recognition particle-dependent translocation machinery. Our results do not allow a distinction to be made between a spontaneous insertion mode or some other trypsin-resistant receptor-mediated mechanism. A role for Pro115 in the middle of the membrane-binding domain of cytochrome b5 was also examined by mutating it to an alanine and subsequently characterizing the ability of the mutant protein to be incorporated into membranes. The mutant protein inserted more slowly in vitro into microsomes as well as into pure lipid bilayers by a factor of 2 to 3.
Department of Anesthesia, University of California, San Francisco.
G
G
Vergères
J
J
Ramsden
L
L
Waskell
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002268:Carboxypeptidases; D043402:Cathepsin A; D002474:Cell-Free System; D015786:Cytochromes b5 / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure; D007700:Kinetics; D008051:Lipid Bilayers; D008099:Liver / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D010179:Pancreas / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D015183:Restriction Mapping; D012156:Reticulocytes / Q000378:metabolism; D014357:Trypsin
D008051:Lipid Bilayers; D010446:Peptide Fragments; D012333:RNA, Messenger; D015786:Cytochromes b5; D002268:Carboxypeptidases; D043402:Cathepsin A; D014357:Trypsin