Synthesis in vitro of a Putative Precursor of Mitochondrial Malate Dehydrogenase

Abstract

<p>Although mitochondria have their own genetic system, their DNA codes for less than 10% of their protein mass. The bulk of mitochondrial protein is coded for by the nucleus, synthesized in the cytoplasm and imported. Much attention has recently been focussed on the question of how these proteins are specifically directed to the mitochondrion.</p> <p>It has been found that many mitochondrial proteins are synthesized as higher molecular weight precursors, which might suggest a role in directing their uptake by mitochondria - thus it was decided to determine if rat liver mitochondrial malate dehydrogenase (mMDH) is also first synthesized as a higher molecular weight precursor.</p> <p>Rat liver mMDH was purified by a rapid procedure developed here and antibodies to the denatured rat liver and beef heart (commercial) enzyme were raised in white rabbits. These antisera were shown to cross-react with denatured rat and beef mMDH and were highly specific for these antigens. Total RNA and free polysomes isolated from rat liver were translated in a micrococcal nuclease-treated reticulocyte lysate cell free protein synthesizing system in the presence of [35s]methionine. The translation products were denatured and incubated with antiserum after which antigen-antibody complexes were removed using Protein A Sepharose. The complexes were subsequently stripped from the Protein A then examined by SDS polyacrylamide gel electrophoresis and fluorography.</p> <p>It was found that a protein 1,500-2,000 daltons larger than mature rat liver mMDH (34,500 daltons) was immuno-precipitated using either free polysome or total RNA to prime synthesis. The recovery of this protein could be prevented by competition with excess unlabelled mMDH. This suggests that this 36,000 dalton protein is a higher molecular weight precursor of rat liver mMDH. It was also found that ten times more of this protein could be immunoprecipitated using free polysomes rather than total RNA suggesting that the free polysome fraction is involved in the synthesis of this enzyme.</p>