MOLECULAR EVOLUTION OF ALANINE/GLYOXYLATE AMINOTRANSFERASE-1 INTRACELLULAR TARGETING - ANALYSIS OF THE FELINE GENE

The subcellular distribution of hepatic alanine:glyoxylate aminotransferase 1 (AGT) has changed, under the influence of dietary selection pressure, on several occasions during the evolution of mammals. In some species (e.g. human and rabbit) ACT is entirely peroxisomal; in other species (e.g. marmoset and rat) this enzyme is found in similar amounts in peroxisomes and mitochondria; in yet other species (e.g. cat) it is mainly mitochondrial. The molecular basis of the species-specific dual intracellular targeting of AGT has been partially elucidated in the human and rabbit (as examples of the first group), and in the rat and marmoset (as examples of the second group). As part of a wider study on the molecular evolution of ACT intracellular targeting, we report in the present paper the results of an investigation into the molecular basis of the subcellular distribution of AGT in the cat (as an example of the third group). Cat liver ACT cDNA has been cloned and sequenced, and shown to have a high degree of similarity to AGT from human, rabbit, marmoset and rat. Southern-blotting analysis showed that AGT in the cat is probably encoded by a single gene, as it is in other species. Transcript analysis by RNase protection indicated that almost all of the ACT mRNA would possess an open reading frame encoding a polypeptide of 414 amino acids and a molecular mass of 45508 Da. The N-terminal 22 amino acids comprised the putative mitochondrial-targeting sequence (by analogy with the equivalent sequence in marmoset and rat pre-mitochondrial ACT). The very low level of peroxisomal ACT in cat liver is compatible with the absence of any RNase-protected transcripts initiating downstream of the first putative translation initiation codon (i.e. absence of any transcripts in which the mitochondrial-targeting sequence is excluded from the open reading frame). In. vitro studies showed that the 45 kDa polypeptide was imported into rat liver mitochondria and processed to a mature protein of approximate to 43 kDa, compatible with the cleavage of the N-terminal 22 amino acids, as is also the case in rat and marmoset. A polypeptide in which the N-terminal 22 amino acids was absent could not be imported into mitochondria in vitro.