Proteolysis prevents in vivo chimeric fusion protein import into yeast mitochondria. Cytosolic cleavage and subcellular distribution.
The in vivo import of liver mitochondrial aldehyde dehydrogenase was investigated in yeast by constructing fusion proteins between its leader sequence and beta-galactosidase. Only 7% of the protein was imported. If 21 or 71 amino acids from the mature portion of aldehyde dehydrogenase were included in the construct, 40% was imported. The protein remaining in cytosol was sequenced. When the leader was fused directly to beta-galactosidase, the first 7 residues of the leader were missing. When 21 residues of mature aldehyde dehydrogenase were included, the entire leader plus 6 residues of the mature portion were missing; if 71 residues of mature aldehyde dehydrogenase were included, the first residue found corresponds to the 66th residue of the mature portion. When the leader was fused directly to beta-galactosidase, no processing of the imported protein occurred, and the N-terminal amino acid was blocked, presumably by acetylation. If the 21-amino acid insert was included, processing occurred. A modified leader sequence lacking the three-amino acid linker (RGP) was imported but not processed, just as we found in vitro (Thornton, K., Wang, Y., Weiner, H., and Gorenstein, D.G. (1993) J. Biol. Chem. 268, 19906-19914). The less than 100% import of pre-aldehyde dehydrogenase was due to the action of a post-translational protease attack which prevented import by destroying the leader peptide segment.
Zhou
J
J
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA.
Bai
Y
Y
Weiner
H
H
D000444:Aldehyde Dehydrogenase / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003600:Cytosol / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D000444:Aldehyde Dehydrogenase; D001616:beta-Galactosidase