Chaperonins GroEL and GroES promote assembly of heterotetramers (α2β2) of mammalian mitochondrial branched-chain α-keto acid decarboxylase in Escherichia coli

R. Max Wynn, James R. Davie, Rody P. Cox, David T. Chuang

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73 Scopus citations

Abstract

We have investigated the possible role of chaperonins groEL and groES in the folding and assembly of xeterotetramers (α2β2) of mammalian mitochondrial branched-chain α-keto acid decarboxylase (E1) in Escherichia coli. The mature E1α subunit fused to maltose-binding protein (MBP) was coexpressed with mature E1β 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-E1α and E1β 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 E1α and E1β assembled into an active 160-kDa species, which was consistent with the α2β2 structure of E1. The present results demonstrate that chaperonins groEL and groES promote folding and assembly of heterotetrameric proteins of mammalian mitochondrial origin.

Original languageEnglish (US)
Pages (from-to)12400-12403
Number of pages4
JournalJournal of Biological Chemistry
Volume267
Issue number18
StatePublished - Jun 25 1992

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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