INTRAMITOCHONDRIAL FOLDING AND ASSEMBLY OF MEDIUM-CHAIN ACYL-COA DEHYDROGENASE (MCAD) - DEMONSTRATION OF IMPAIRED TRANSFER OF K304E-VARIANTMCAD FROM ITS COMPLEX WITH HSP60 TO THE NATIVE TETRAMER

We incubated in vitro translated precursor of medium-chain acyl-CoA de hydrogenase (MCAD) with isolated rat liver mitochondria and fractionat ed the solubilized mitochondria on gel filtration. After a 5-min impor t into mitochondria, MCAD was recovered exclusively as a high molecula r weight (hM(r)) complex (700,000), while after a 10 min import, it wa s recovered mainly in the hM(r) complex and mature tetramer, with a sm all amount in monomer. Either a further 15-min chase or exposure to AT P caused a marked decrease of MCAD in the hM(r) complex and an increas e in the mature tetramer in comparable amounts, suggesting that the hM (r) complex was the precursor of tetramer. No monomer was detected in either case. Using specific antibodies, we have shown that the hM(r) c omplex 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 (hsp70(mi t)) and then transferred to hsp60 to complete its folding into an asse mbly-competent conformation. We also examined the assembly of K304E MC AD, which is a prevalent variant enzyme among patients with MCAD defic iency. The assembly of the K304E into its tetrameric form was severely impaired. The binding of K304E with hsp70(mit) and its transfer from hsp70(mit) to hsp60 were normal. However, the hsp60 complex of K304E w as 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 o f K304E subunit to tetramer from, the complex with hsp60 was impaired.