Synthetic transit peptides inhibit import and processing of mitochondrial precursor proteins.
We have demonstrated that a synthetic peptide corresponding to the rat mitochondrial malate dehydrogenase (mMDH) transit peptide (TP-28) inhibits the binding of pre-mMDH to isolated mitochondria. Synthetic peptides derived from chloroplast transit peptide sequences, which have a similar net charge, did not inhibit import. In addition, this peptide (TP-28) inhibits import of ornithine transcarbamylase, another mitochondrial matrix protein, thus suggesting that common import pathways exist for both mMDH and ornithine transcarbamylase. A smaller synthetic peptide corresponding to residues 1-20 of the mMDH transit peptide (TP-20) also inhibits binding. However, several substitutions for leucine-13 in the smaller peptide relieve import inhibition, thus providing evidence that this neutral residue plays a crucial role in transit peptide binding to the mitochondrial surface. Proteolytic processing of pre-mMDH by a mitochondrial matrix fraction to both the mature and intermediate forms of mMDH was also inhibited by TP-28. The ability of synthetic peptides to inhibit distinct steps in the import of mitochondrial precursor proteins corresponds precisely to their ability to interact with the same components used by transit peptides on intact precursors. Furthermore, inhibition at multiple points along the import pathway reflects the functions of several independent structures contained within transit peptides.
Chu
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TW
Department of Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110.
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive; D001692:Biological Transport / Q000187:drug effects; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D006868:Hydrolysis; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology* / Q000378:metabolism; D008969:Molecular Sequence Data; D009954:Ornithine Carbamoyltransferase / Q000037:antagonists & inhibitors; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology*; D011498:Protein Precursors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D051381:Rats
D010455:Peptides; D011498:Protein Precursors; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D008291:Malate Dehydrogenase; D009954:Ornithine Carbamoyltransferase