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Intramitochondrial folding and assembly of medium-chain acyl-CoA dehydrogenase (MCAD). Demonstration of impaired transfer of K304E-variant MCAD from its complex with hsp60 to the native tetramer

We incubated in vitro translated precursor of medium-chain acyl-CoA dehydrogenase (MCAD) with isolated rat liver mitochondria and fractionated the solubilized mitochondria on gel filtration. After a 5-min import into mitochondria, MCAD was recovered exclusively as a high molecular weight (hMr) compl...

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Bibliographic Details
Published in:The Journal of biological chemistry 1994-02, Vol.269 (6), p.4401-4408
Main Authors: SAIJO, T, WELCH, W. J, TANAKA, K
Format: Article
Language:English
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Summary:We incubated in vitro translated precursor of medium-chain acyl-CoA dehydrogenase (MCAD) with isolated rat liver mitochondria and fractionated the solubilized mitochondria on gel filtration. After a 5-min import into mitochondria, MCAD was recovered exclusively as a high molecular weight (hMr) complex (700,000), while after a 10-min import, it was recovered mainly in the hMr complex and mature tetramer, with a small amount in monomer. Either a further 15-min chase or exposure to ATP caused a marked decrease of MCAD in the hMr complex and an increase in the mature tetramer in comparable amounts, suggesting that the hMr complex was the precursor of tetramer. No monomer was detected in either case. Using specific antibodies, we have shown that the hMr complex represented a complex of MCAD and heat-shock protein 60 (hsp60), and, that upon import into mitochondria, unfolded MCAD first formed a transient complex with mitochondrial heat-shock protein 70 (hsp70mit) and then transferred to hsp60 to complete its folding into an assembly-competent conformation. We also examined the assembly of K304E MCAD, which is a prevalent variant enzyme among patients with MCAD deficiency. The assembly of the K304E into its tetrameric form was severely impaired. The binding of K304E with hsp70mit and its transfer from hsp70mit to hsp60 were normal. However, the hsp60 complex of K304E was much more stable than the wild-type counterpart upon a 15-min chase or exposure to ATP, suggesting that the folding in, or the transfer of K304E subunit to tetramer from, the complex with hsp60 was impaired.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(17)41794-7