Expression of a continuous open reading frame encoding subunits 1 and 2 of cytochrome c oxidase in the mitochondrial DNA of Acanthamoeba castellanii

We have investigated the expression of a continuous open reading frame (ORF) present in the mitochondrial genome of Acanthamoeba castellanii and specifying the two largest subunits (COX1 and COX2) of the cytochrome c oxidase complex. Northern hybridization and primer extension analysis demonstrated that this ORF (cox1/2, 873 codons) is transcribed as part of a 4.7 kb RNA that also includes the upstream small subunit rRNA sequence. Between the cox1 and cox2 portions of the transcript, RNA sequence exactly matches gene sequence, excluding the possibility that a standard cowl termination codon is created by post-transcriptional RNA processing or editing. Western analysis revealed an A. castellanii COX2 protein with a mobility matching that of mature COX2 from yeast (Saccharomyces cerevisiae) mitochondria. These observations indicate that although A. castellanii COX1 and COX2 are apparently translated from the same ORF, they do not exist in mature form as a COX1-COX2 ''fusion'' protein. Whereas translation of COX2 could potentially be initiated from an internal AUG codon in the cox1/2 ORF, COX1 must be generated either through an unusual translation termination mechanism acting between the cox1 and cox2 coding regions of the cox1/2 mRNA, or by co-translational or post-translational proteolytic processing of a translation product whose synthesis continues into the cox2 coding region. Because the cox2 nucleotide sequence predicts a COX2 protein considerably larger than that observed by Western analysis, A. castellanii COX2 may undergo additional post-translational processing to its final form. (C) 1996 Academic Press Limited