Direct role of chaperonins groel and groes in mediating heteroougomeric protein assembly

D. T. Chuany, J. L. Chtung, R. M. Wynn

Research output: Contribution to journalArticle

Abstract

The El decarboxylase component of the human mitochondrial branchedchain a-ketoacid dehydrogenase complex is a thiamine pyrophosphatedependent enzyme, comprising two a (M.W. 45,500) and two β(M.W. 37,500) subunits. In the present study, (His)6-tagged El tetramers denatured in 8 M urea were reconstituted with high yields in vitro at 25°C, with an absolute requirement for chaperonins GroEL/GroES and Mg-ATP. However, the kinetics of El reconstitution was very slow with a 11/2 of 5 h. Reconstitution of El activity with a similarly slow kinetics was also achieved using stable GroEL-α and GroEL-β complexes as combined substrates. The assembly state between the a and the βsubunits during renaturation was analyzed by Ni- NTA affinity chromatognphy and sucrose density gradient centrifugation. The results showed that die a and the βsubunits released from GroEL assembled into an inactive αβ dimeric Intermediate, which subsequently dimerized to produce the active ojfe tetramer. The αβ dimer, but not the α2β2 tetramer, isolated from E. coli lysates was found to bind to GroEL, resulting in a stable GroEL-α-β ternary complex. Incubation of this novel ternary complex with GroES and Mg-ATP resulted in the assembly of the active tetramer. Enzyme assays again showed a slow kinetics of reconstituted El activity from the GroEL-α-β complex. The study provides the first evidence that chaperonins directly mediate the "maturation" of an assembly intermediate, rendering it dimerization-competent during the tetrameric assembly. This step appears rate-limiting in the chaperonin-dependent reconstitution of El α2β2 tetramers. Supported by grant DK-26758 from the NIH and grant 1-1286 from the Welch Foundation.

Original languageEnglish (US)
Pages (from-to)A1419
JournalFASEB Journal
Volume12
Issue number8
StatePublished - Dec 1 1998

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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