Positively charged residues, the helical conformation and the structural flexibility of the leader sequence of pALDH are important for recognition by hTom20.

Tom20, a mitochondrial outer membrane receptor necessary for protein translocation, was found to interact specifically with mitochondrial preproteins. The interaction of proteins containing an N-terminal matrix targeting signal was enhanced in an hydrophobic environment and the dependence of this interaction on the alpha helical conformation of the presequence was postulated. In order to test this hypothesis and to gain insights about the features of a matrix targeting signal necessary to be recognized by the receptor machinery including Tom20, the interaction of pALDH and signal sequence mutants to Tom20 in the absence and presence of a hydrophobic environment was investigated. Here we present evidence to show that in a hydrophobic environment the interaction between Tom20 and the leader sequence is strongly dependent on the positive charges within the signal sequence as well as on the flexibility of this signal.

The loss in hydrophobic surface area resulting from a Leu to Val mutation at the N-terminus of the aldehyde dehydrogenase presequence prevents import of the protein into mitochondria.

An apparent conservative mutation, Leu to Val, at the second residue of the rat liver mitochondrial aldehyde dehydrogenase (ALDH) presequence resulted in a precursor protein that was not imported into mitochondria. Additional mutants were made to substitute various amino acids with nonpolar side chains for Leu2. The Ile, Phe, and Trp mutants were imported to an extent similar to that of the native precursor, but the Ala mutant was imported only about one-fourth as well. It was shown that the N-terminal methionine was removed from the L2V mutant in a reaction catalyzed by methionine aminopeptidase. The N-terminal methionine of native pALDH and the other mutant presequences was blocked, presumably by acetylation. Because of the difference in co-translational modification, the L2V mutant sustained a significant loss in the available hydrophobic surface of the presequence. Import competence was restored to the L2V mutant when it was translated using a system that did not remove Met1. The removal of an Arg-Gly-Pro helix linker segment (residues 11-14) from the L2V mutant, which shifted three leucine residues toward the N-terminus, also restored import competence. These results lead to the conclusion that a minimum amount of hydrophobic surface area near the N-termini of mitochondrial presequences is an essential property to determine their ability to be imported. As a result, both electrostatic and hydrophobic components must be considered when trying to understand the interactions between precursor proteins and proteins of the mitochondrial import apparatus.

In vivo mitochondrial import. A comparison of leader sequence charge and structural relationships with the in vitro model resulting in evidence for co-translational import.

The positive charges and structural properties of the mitochondrial leader sequence of aldehyde dehydrogenase have been extensively studied in vitro. The results of these studies showed that increasing the helicity of this leader would compensate for reduced import from positive charge substitutions of arginine with glutamine or the insertion of negative charged residues made in the native leader. In this in vivo study, utilizing the green fluorescent protein (GFP) as a passenger protein, import results showed the opposite effect with respect to helicity, but the results from mutations made within the native leader sequence were consistent between the in vitro and in vivo experiments. Leader mutations that reduced the efficiency of import resulted in a cytosolic accumulation of a truncated GFP chimera that was fluorescent but devoid of a mitochondrial leader. The native leader efficiently imported before GFP could achieve a stable, import-incompetent structure, suggesting that import was coupled with translation. As a test for a co-translational mechanism, a chimera of GFP that contained the native leader of aldehyde dehydrogenase attached at the N terminus and a C-terminal endoplasmic reticulum targeting signal attached to the C terminus of GFP was constructed. This chimera was localized exclusively to mitochondria. The import result with the dual signal chimera provides support for a co-translational mitochondrial import pathway.

A regional net charge and structural compensation model to explain how negatively charged amino acids can be accepted within a mitochondrial leader sequence.

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.

The role of positive charges and structural segments in the presequence of rat liver aldehyde dehydrogenase in import into mitochondria.

Most mitochondrial proteins are nucleus-encoded and translated in the cytosol. They have an N-terminal presequence that allows recognition by the mitochondrial import apparatus and subsequent import into mitochondria. These presequences are rich in positive charges, mainly arginines. The role of these positive charges in the 19-amino acid presequence of rat liver aldehyde dehydrogenase was investigated by systematically replacing them with the polar but uncharged residue, glutamine. The single substitution of any of the four Arg residues in the helical segments did not affect import. Substitution of both Arg residues in the N-terminal segment (R3Q/R10Q) caused a dramatic decrease in import competence. This could be restored by using the mutant lacking the three-amino acid (RGP) linker that separates the two helical domains, determined by two-dimensional NMR (Thornton, K., Wang, Y., Weiner, H., and Gorenstein, D. G. (1993) J. Biol. Chem. 268, 19906-19914). CD and NMR spectra of the peptide corresponding to the linker-deleted presequence showed that it was substantially more prone to helix formation than the native peptide over its entire length. A similar analysis of the peptide corresponding to the R3Q/R10Q presequence revealed that this peptide was only somewhat more helical than the native peptide and that the greater helicity did not include the residues near the N terminus. It is concluded that positively charged residues in the presequence play a vital role in the import of precursor aldehyde dehydrogenase. One of the positive charges in the N-terminal helical segment of the presequence is necessary for import competence. However, if both positive charges are removed, import competence can be retained as long as the presequence is capable of forming a relatively more stable alpha-helix near its N terminus.