Expression and activity of a gene encoding rat cytochrome c in the yeast Saccharomyces cerevisiae.
A rat-processed pseudogene, which encodes normal rat cytochrome c, has been expressed in the yeast, Saccharomyces cerevisiae. The translated region of the chromosomal CYC1+ locus, which encodes yeast iso-1-cytochrome c, was replaced by the translated region of the gene encoding rat cytochrome c (CYC1-RAT), thus preserving the proper CYC1 transcription initiation and termination signals. Although the levels of transcription of the normal CYC1+ gene and the CYC1-RAT gene in yeast were equivalent, rat cytochrome c was produced at approx. 40% of the level of iso-1-cytochrome c. In addition, the specific activity in vivo was estimated to be approx. 60% that of the yeast iso-1-cytochrome c. N-terminal processing of indigenous rat cytochrome c, in which the N-terminal methionine residue is cleaved and the penultimate glycine residue is acetylated, also occurred in yeast. Methionine cleavage was complete, while acetylation proceeded to only 70% completion. Lys-72 was trimethylated to 66% completion in the rat cytochrome c produced in yeast. The near normal expression (40%) and specific activity (60%) in vivo indicates that the 40% difference in amino acid sequence is not critical for mitochondrial import, heme attachment and interactions with redox partners.
Clements
J M
JM
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, NY 14642.
O'Connell
L I
LI
Tsunasawa
S
S
Sherman
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002851:Chromatography, High Pressure Liquid; D003574:Cytochrome c Group / Q000096:biosynthesis / Q000235:genetics*; D004274:DNA, Recombinant; D015870:Gene Expression; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011544:Pseudogenes; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D003574:Cytochrome c Group; D004274:DNA, Recombinant; D012333:RNA, Messenger; D011994:Recombinant Proteins