Article

Human CCT4 and CCT5 chaperonin subunits expressed in E. coli form biologically active homo-oligomers.

Massachusetts Institute of Technology, United States
Journal of Biological Chemistry (Impact Factor: 4.6). 04/2013; DOI: 10.1074/jbc.M112.443929
Source: PubMed

ABSTRACT Chaperonins are a family of chaperones that encapsulate their substrates and assist their folding in an ATP-dependent manner. The ubiquitous eukaryotic chaperonin, TCP-1 Ring Complex (TRiC), is a hetero-oligomeric complex composed of two rings each formed from eight different CCT (Chaperonin Containing TCP-1) subunits. Each CCT subunit may have distinct substrate recognition and ATP-hydrolysis properties. We have expressed each human CCT subunit individually in E. coli to investigate whether they form chaperonin-like double ring complexes. CCT4 and CCT5, but not the other six CCT subunits, formed high molecular weight complexes within the E.coli cells that sedimented about 20S in sucrose gradients. When CCT4 and CCT5 were purified, they were both organized as two back-to-back rings of eight subunits each, as seen by negative stain and cryo-electron microscopy. This morphology is consistent with that of the hetero-oligomeric double-ring TRiC purified from bovine testes and HeLa cells. Both CCT4 and CCT5 homo-oligomers hydrolyzed ATP at a rate similar to human TRiC, and were active as assayed by luciferase refolding and human γD-crystallin aggregation suppression and refolding. Thus both CCT4 and CCT5 homo-oligomers have the property of forming eight-fold double rings absent the other subunits, and these complexes carry out chaperonin reactions without other partner subunits.

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