A study on the transport of mitochondrial aldehyde dehydrogenase into rat liver mitochondria
Abstract
Mitochondrial aldehyde dehydrogenase (ALDH) is a major enzyme involved in oxidation of biogenic aldehydes and acetaldehyde. Like other mitochondrial proteins, ALDH is synthesized as a higher molecular weight precursor in cytosol, and transported into mitochondria with energy- and receptor-dependent processes. Rat and beef pALDHs showed similar binding, import, and processing efficiencies even though their signal peptides are different in sizes and sequences. In vivo ethanol inhibition on pALDH import was not observed with mitochondria isolated from ethanol administered rats, while in vitro inhibition by alcohol on import was reported. Using two dimensional NMR, a helix-turn-helix structure of the synthetic pALDH signal peptide in dodecylphosphocholine micelle was elucidated. The helix at C-terminus (helix C) was more stable than was the helix at N-terminus (helix N) and was associated with the micelle. A turn structure existed between the two helices. Proline in the turn region and hypothetical hydrogen bonds between helices were found to be not essential for import function. The helix C is proposed to first bind to mitochondrial membrane then migrates to a receptor/translocator at contact site along the membrane. The receptor/translocator recognizes the helix N and allows the precursor proteins to be translocated into mitochondria. Unlike precursor proteins, the synthetic pALDH and pOTC signal peptides were imported rapidly in the absence of a membrane potential and a receptor protein. It was concluded that the synthetic signal peptide could be imported differently than the precursor proteins. The imported signal peptides were associated with both outer and inner membranes. The inhibition of precursor import by the signal peptides might result from association of signal peptides with inner membrane near or at translocator sites. Chicken and rabbit anti-idiotypic antibodies against the anti-pALDH signal peptide inhibited pALDH import. The rabbit anti-idiotypic antibody could recognize 66, 60, 42, 34 and 29 kDa mitochondrial proteins. The 66, 42, and 60 kDa (minor) proteins were cross-linked with the chicken antibody. The 66, 34, and 42 kDa (minor) proteins were cross-linked to the rabbit antibody. The pALDH was cross-linked to the same sizes of proteins as was the chicken antibody. These proteins which appear to be contact site proteins were isolated by either cross-linking to a immobilized anti-idiotypic antibody or a hydroxylapatite chromatography.
Degree
Ph.D.
Advisors
Weiner, Purdue University.
Subject Area
Biochemistry
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