Abstract
We have investigated the possible role of chaperonins groEL and groES in the folding and assembly of heterotetramers (alpha 2 beta 2) of mammalian mitochondrial branched-chain alpha-keto acid decarboxylase (E1) in Escherichia coli. The mature E1 alpha subunit fused to maltose-binding protein (MBP) was coexpressed with mature E1 beta on the same vector in ES- and EL- mutant strains. Only small or trace amounts of active E1 component were obtained. Cotransformation of the ES- mutant host with a second vector overexpressing groEL and groES resulted in a greater than 500-fold increase in E1-specific activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the content of both MBP-E1 alpha and E1 beta polypeptides was markedly increased in the presence of overexpressed chaperonin proteins. The time course studies showed that the increase in E1-specific activity and subunit levels correlated with the increase in groEL and groES until the concentration of the chaperonins reached a saturating level in the cell. The functional MBP-E1 fusion protein from ES- double transformants were purified by amylose resin affinity chromatography. The MBP moiety was removed by subsequent digestion with Factor Xa endoprotease, followed by Sephacryl S-300HR chromatography. It was found that E1 alpha and E1 beta assembled into an active 160-kDa species, which was consistent with the alpha 2 beta 2 structure of E1. The present results demonstrate that chaperonins groEL and groES promote folding and assembly of heterotetrameric proteins of mammalian mitochondrial origin.
Highlights
Molecular chaperones are aclass of oligomericproteins that facilitate the proper folding of certain other proteins, usually by inhibiting aggregation or suppressing kinetic “trapping” along the folding pathway [1].The role of bacterial chaper
Mammalian Mitochondrial Branched-chain a-Keto Acid Decarboxylase in ones such asgroEL and groES in catalyzing the correct folding of a variety of denatured or newly synthesized polypeptides has become firmly established within thepast few years
Our approach was to thatEla and E l 8 assembled into an active 160-kDa cotransform the E. coli mutant host with species, which was consistent with the a& structure a vector expressing maltose-binding protein (MBP)-Ela and E1P and a second vector of E l
Summary
Molecular chaperones are aclass of oligomericproteins that facilitate the proper folding of certain other proteins, usually by inhibiting aggregation or suppressing kinetic “trapping” along the folding pathway [1].The role of bacterial chaper-. Mammalian Mitochondrial Branched-chain a-Keto Acid Decarboxylase in ones such asgroEL and groES in catalyzing the correct folding of a variety of denatured or newly synthesized polypeptides has become firmly established within thepast few years. Examples include the single subunit folding of rhodanese [2], Escherichia coli*. Folding and dimeric assembly of citrate synthase [3, 4], and the folding and assembly of the large subunit of ribulose 1,5-. (Received for publication, April 3, 1992) bisphosphate carboxylase/oxygenase [5]. Chaperonin proteins in the folding and assembly of mamma-.
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