Expression of cytochrome b5 in yeast and characterization of mutants of the membrane-anchoring domain.
The soluble and membrane-bound forms of the synthetic rat cytochrome b5 gene have been expressed in Saccharomyces cerevisiae. In order to examine the topology and function of the COOH-terminal membrane binding domain of cytochrome b5, mutants have been constructed, expressed, purified, and partially characterized. Pro-115 is located in the middle of the putative alpha-helical membrane-anchoring domain of cytochrome b5 and has been hypothesized to give rise to either a hairpin-like loop or approximately equal to 26 degrees kink in the helix, depending on whether it exists, respectively, in the cis or trans configuration. The Pro-115----Ala mutant, which is expected to have a straight transmembrane helix, inserted normally into the endoplasmic reticulum and exhibited wild type levels of activity in yeast microsomes and in vitro in the cytochrome P-450 mixed function oxidation system. Since a hairpin structure does not appear to be essential, it is likely that the membrane binding domain of cytochrome b5 spans the membrane. Characterization of the truncated cytochrome b5 molecule, Pro-115----Stop, lacking 19 amino acids at the COOH terminus indicates that the distal part of the membrane binding domain of cytochrome b5 is necessary for in vivo binding to the endoplasmic reticulum and for functioning with its membrane-associated electron transfer partners. Replacement of Ser-104 to Met-125, the putative membrane-anchoring domain of cytochrome b5, with 22 leucine residues results in a protein which targets to the endoplasmic reticulum but the extent of its reduction is only 50% of that of the wild type in yeast microsomes. In vitro, the polyleucine mutant is unable to support substrate oxidation by cytochrome P-450. The mutation of Ala-131 and Glu-132, amino acids flanking the transmembrane domain, to lysines resulted in a protein with normal membrane topology and function.
Vergères
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Department of Anesthesia, University of California, Veterans Administration Medical Center, San Francisco 94121.
Waskell
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L
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D015786:Cytochromes b5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004247:DNA; D006418:Heme / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012995:Solubility
D008565:Membrane Proteins; D010455:Peptides; C027819:polyleucine; D006418:Heme; D004247:DNA; D015786:Cytochromes b5