Expression and activity of a gene encoding rat cytochrome c in the yeast Saccharomyces cerevisiae

Abstract

A rat-processed pseudogene, which encodes normal rat cytochrome c, has been expressed m 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.