Investigation of the roles of the conserved residues in human mitochondrial aldehyde dehydrogenase and the characterization of the mechanism of rat mitochondrial aldehyde dehydrogenase import

Li Ni, Purdue University

Abstract

Sequence alignment of 16 aldehyde dehydrogenases (ALDH) showed that only 23 residues were strictly conserved, of which only five are residues with potentially reactive side chains, except for Cys302 and Glu268, residues previously shown to be in the active site. Mutational analysis was performed on these five residues, and another two partially conserved residues in human mitochondrial ALDH2 in order to study the roles of these residues in the catalytic process. No residue was found to be essential. Mutations of the two NAD+-ribose binding residues, Lys192 and Glu399, led to a change in the rate limiting step such that hydride transfer became rate limiting, not deacylation, when aliphatic, but not when aromatic substrates were used with the E399K/Q and K192Q mutants. Glu399 most likely is involved in stabilizing the transition state by binding to the ribose ring of NAD+. The positively charged residues in the rat pALDH2 leader sequence were replaced by glutamine, and the effect of these mutations on in vivo mitochondrial import in Hela cells was determined. The in vivo import results using rat pALDH2 leader sequence fused to the N-terminus of green fluorescent protein were approximately the same as the in vitro results. Both in vivo and in vitro results suggested that the positive charges in the N-terminal segment of the leader, not in the total leader sequence, were important for the mitochondrial import. It was impossible to determine the role of the secondary structure in the in vivo import from this study. The results also suggested that import might be co-translational in vivo. A chimera (SP-EGFP-ER) that has the mitochondrial signal peptide (SP) at the N-terminus and the endoplasmic reticulum (ER) targeting signal at the C-terminus was constructed. GFP was used as the carrier protein to study whether the mitochondrial import was co- or post-translational in vivo. The results suggested that the chimera expressed in Hela cells was exclusively localized in the mitochondria, with no proteins being found on the ER membrane, consistent with the co-translational mitochondrial import. We propose, at least for the rat pALDH2, in vivo mitochondrial import is a co-translational process.

Degree

Ph.D.

Advisors

Weiner, Purdue University.

Subject Area

Biochemistry|Cellular biology

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