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 b y the mitochondrial import apparatus and subsequent import into mitoch ondria. These presequences are rich in positive charges, mainly argini nes. The role of these positive charges in the 19-amino acid presequen ce of rat liver aldehyde dehydrogenase was investigated by systematica lly replacing them with the polar but uncharged residue, glutamine. Th e single substitution of any of the four Arg residues in the helical s egments did not affect import. Substitution of both Arg residues in th e N-terminal segment (R3Q/R10Q) caused a dramatic decrease in import c ompetence. This could be restored by using the mutant lacking the thre e-amino acid (RGP) linker that separates the two helical domains, dete rmined by two-dimensional NMR (Thornton, K., Wang, Y., Weiner, H., and Gorenstein, D. G. (1993) J. Biol. Chem. 268, 19906-19984), CD and NMR spectra of the peptide corresponding to the linker deleted presequenc e showed that it was substantially more prone to helix formation than the native peptide over its entire length. A similar analysis of the p eptide corresponding to the R3Q/R10Q presequence revealed that this pe ptide 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 p lay a vital role in the import of precursor aldehyde dehydrogenase. On e of the positive charges in the N terminal helical segment of the pre sequence 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-heli x near its N terminus.