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.
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
R M
RM
Wynn
J R
JR
Davie
R P
RP
Cox
D T
DT
Chuang
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)