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.