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.
Hammen
P K
PK
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA.
Heard
T S
TS
Waltner
M
M
Weiner
H
H
D000444:Aldehyde Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D003198:Computer Simulation; D003510:Cyclohexanes; D004591:Electrophoresis, Polyacrylamide Gel; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D008099:Liver / Q000201:enzymology; D008928:Mitochondria / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D051381:Rats; D012717:Sesquiterpenes
D000900:Anti-Bacterial Agents; D003510:Cyclohexanes; D005231:Fatty Acids, Unsaturated; D012717:Sesquiterpenes; C026211:fumagillin; D000444:Aldehyde Dehydrogenase