Abstract
Mitochondrial leader sequences have been found to be statistically enriched for positively charged residues, with only a few known leader sequences possessing negatively charged residues. Mutational studies that have introduced negatively charged residues into various leader sequences have shown a general, but not absolute, trend toward reduced import. The leader sequence of rat liver aldehyde dehydrogenase has been previously determined by NMR to form a helix-linker-helix structure. A negative charge introduced into this leader did not prevent import, provided that a net positive charge remained in the N-helical segment. When the net charge of the N-terminal helical segment was reduced to zero, import could be recovered by removing the linker, which resulted in a longer, more stable leader. This structural recovery of import was effective enough to compensate for a net charge of zero within the first 10 residues, even when a glutamate is the first charged side chain presented in the sequence.
Highlights
From a statistical basis, negatively charged amino acid residues are infrequently found in leader sequences [1]
Based on the positive charge versus structural compensation model developed from our previous studies, glutamic and aspartic acid residues were systematically introduced into the pALDH leader to ascertain how negative charges may be tolerated
Neutral and Negatively Charged Amino Acid Mutations in the Precursor Aldehyde Dehydrogenase Sequence—The pALDH leader sequence is structurally comprised of two amphiphilic helical segments joined by a flexible, three-amino acid linker segment (Fig. 1)
Summary
Negatively charged amino acid residues are infrequently found in leader sequences [1]. The enhanced stability of the linker-deleted leader has been successfully used to study structural aspects as a compensating factor for the loss of positive charges through arginine to glutamine mutational substitutions. Each helical segment contains an equal number of positive charges, the N-terminal helical segment has been shown to provide the majority of the necessary charge for efficiently targeting the leader to the matrix If both of the N-helical segment arginines were substituted with glutamine residues, the ability to import was essentially eliminated. Based on the positive charge versus structural compensation model developed from our previous studies, glutamic and aspartic acid residues were systematically introduced into the pALDH leader to ascertain how negative charges may be tolerated
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