Identification of hydrophobic residues in the signal sequence of mitochondrial preornithine carbamyltransferase that enhance the rate of precursor import.
Previous studies employing circular dichroism and resonance energy transfer techniques have demonstrated that the signal peptide of mitochondrial preornithine carbamyltransferase (pOCT) has the potential to interact with the surface of an anionic phospholipid membrane via a short amphiphilic helical domain. Here we have used predictive secondary structure computations as a guide to localize the putative membrane binding region in the pOCT signal sequence and demonstrate that replacement of leucine residues at positions 5, 8, and 9 with the less hydrophobic residue, alanine, significantly reduces the rate of precursor import (4-5-fold compared to wild type); the amino acid substitutions had little effect, however, on the ability of a mitochondrial matrix extract to process the mutant precursor polypeptide. The mutant precursor bound to anionic liposomes with a lower affinity compared to wild-type pOCT and was inhibited to a lesser extent than pOCT during import into mitochondria in the presence of varying concentrations of liposomes. Taken together, the results suggest that this small region of the pOCT signal sequence, containing a limited number of critical hydrophobic residues, contributes to the optimal rate of precursor import, perhaps by functioning as a membrane surface-seeking entity.
Skerjanc
I S
IS
Department of Biochemistry, McGill University, Montreal, Canada.
Sheffield
W P
WP
Silvius
J R
JR
Shore
G C
GC
D000595:Amino Acid Sequence; D000818:Animals; D004247:DNA / Q000235:genetics; D004792:Enzyme Precursors / Q000235:genetics*; D005796:Genes; D008081:Liposomes; D008929:Mitochondria, Heart / Q000201:enzymology*; D008969:Molecular Sequence Data; D009954:Ornithine Carbamoyltransferase / Q000235:genetics*; D010957:Plasmids; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D051381:Rats; D015183:Restriction Mapping
D004792:Enzyme Precursors; D008081:Liposomes; D004247:DNA; C052289:preornithine carbamoyltransferase; D009954:Ornithine Carbamoyltransferase