Chaperonins groEL and groES promote assembly of heterotetramers (alpha 2 beta 2) of mammalian mitochondrial branched-chain alpha-keto acid decarboxylase in Escherichia coli.
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.
Wynn
R M
RM
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
Davie
J R
JR
Cox
R P
RP
Chuang
D T
DT
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D018528:ATP-Binding Cassette Transporters; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D018835:Chaperonin 10; D018834:Chaperonin 60; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism*; D007658:Ketone Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008928:Mitochondria / Q000201:enzymology*; D009004:Monosaccharide Transport Proteins; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014169:Transformation, Bacterial
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009097:Multienzyme Complexes; C461599:maltose transport system, E coli; D008320:Maltose; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)