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.
Hammen
P K
PK
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA.
Waltner
M
M
Hahnemann
B
B
Heard
T S
TS
Weiner
H
H
D000444:Aldehyde Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D004792:Enzyme Precursors / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D008081:Liposomes; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D017208:Rats, Wistar; D014357:Trypsin / Q000378:metabolism
D004792:Enzyme Precursors; D008081:Liposomes; D010455:Peptides; D000444:Aldehyde Dehydrogenase; D014357:Trypsin