The role of arginine residues in the rat mitochondrial malate dehydrogenase transit peptide.
Arginine residues in the transit peptides of mitochondrial precursors are proposed to be important for uptake into mitochondria. To study this further, we have used cassette mutagenesis to create site-specific amino acid replacements within the transit peptide of rat mitochondrial malate dehydrogenase. Plasmids containing mutant sequences were expressed in vitro and tested in a mitochondrial uptake system utilizing isolated rat liver mitochondria. Substitution for arginine at position 14 with asparagine, glutamine, or alanine decreased the relative import level by 20-30% compared to the wild-type sequence when assayed in 1-h uptake experiments. Although lysine substitution did not alter import, substitution with glutamic acid decreased import by 40%. Alanine substitution for arginines at both positions 14 and 15 also dramatically decreased import. Uptake was partially restored in this mutant when positive charge was inserted at a new location within the transit peptide. Time course experiments showed that the initial rates of import were decreased in these mutants, as were the relative amounts of incorporated protein. These results were best explained by the loss of positive charge following amino acid substitutions for the arginine residues and suggest that the role of the charge is to enhance the efficiency of membrane translocation.
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000502:physiology*; D001692:Biological Transport; D004247:DNA / Q000235:genetics; D004792:Enzyme Precursors / Q000378:metabolism*; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000378:metabolism*; D009154:Mutation; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D004792:Enzyme Precursors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D004247:DNA; D001120:Arginine; D008291:Malate Dehydrogenase