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Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA
Reprint requests to: Dr. Henry Weiner, Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA; e-mail: hweiner{at}purdue.edu; fax: (765) 494-7897.
Human liver cytosolic (ALDH1) and mitochondrial (ALDH2) aldehyde dehydrogenases are both encoded in the nucleus and synthesized in the cytosol. ALDH1 must fold in the cytosol, but ALDH2 is first synthesized as a precursor and must remain unfolded during import into mitochondria. The two mature forms share high identity (68%) at the protein sequence level except for the first 21 residues (14%); their tertiary structures were found to be essentially identical. ALDH1 folded faster in vitro than ALDH2 and could assemble to tetramers while ALDH2 remained as monomers. Import assay was used as a tool to study the folding status of ALDH1 and ALDH2. pALDH1 was made by fusing the presequence of precursor ALDH2 to the N-terminal end of ALDH1. Its import was reduced about 10-fold compared to the precursor ALDH2. The exchange of the N-terminal 21 residues from the mature portion altered import, folding, and assembly of precursor ALDH1 and precursor ALDH2. More of chimeric ALDH1 precursor was imported into mitochondria compared to its parent precursor ALDH1. The import of chimeric ALDH2 precursor, the counterpart of chimeric ALDH1 precursor, was reduced compared to its parent precursor ALDH2. Mature ALDH1 proved to be more stable against urea denaturation than ALDH2. Urea unfolding improved the import of precursor ALDH1 and the chimeric precursors but not precursor ALDH2, consistent with ALDH1 and the chimeric ALDHs being more stable than ALDH2. The N-terminal segment of the mature protein, and not the presequence, makes a major contribution to the folding, assembly, and stability of the precursor and may play a role in folding and hence the translocation of the precursor into mitochondria.
Keywords: Aldehyde dehydrogenase; N-terminal portion; chimeric proteins; folding; import into mitochondria
Abbreviations: ALDH, aldehyde dehydrogenase • ALDH2, mitochondrial mature aldehyde dehydrogenase • pALDH2, mitochondrial aldehyde dehydrogenase precursor • ALDH1, cytosolic aldehyde dehydrogenase • pALDH1, the presequence of pALDH2 fused to the ALDH1 • M-ALDH1, chimeric protein of ALDH1 in which the first 21 amino acid residues of the mature protein were replaced by the corresponding residues from ALDH2 • pM-ALDH1, the presequence of pALDH2 fused to M-ALDH1 • C-ALDH2, chimeric protein of ALDH2 in which the first 21 amino acid residues of the mature protein were replaced by the corresponding residues from ALDH1 • pC-ALDH2, the presequence of pALDH2 fused to C-ALDH2 • HEPES, N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid) • HEPPS, N-2-hydroxyethylpiperazine-N'-3-propanesulfonic acid • DTT, dithiotreitol • PAGE, polyacrylamide gel electrophoresis • PCR, polymerase chain reaction
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