title: Human CCT4 and CCT5 chaperonin subunits expressed in Escherichia coli form biologically active homo-oligomers.
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 Escherichia 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 8-fold double rings absent the other subunits, and these complexes carry out chaperonin reactions without other partner subunits.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Oksana A
authors.initials: OA
authors.lastname: Sergeeva
authors.forename: Bo
authors.initials: B
authors.lastname: Chen
authors.forename: Cameron
authors.initials: C
authors.lastname: Haase-Pettingell
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Ludtke
authors.forename: Wah
authors.initials: W
authors.lastname: Chiu
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: King
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D002499:Centrifugation, Density Gradient; D018834:Chaperonin 60 / Q000648:ultrastructure; D056404:Chaperonin Containing TCP-1 / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure; D002850:Chromatography, Gel; D020285:Cryoelectron Microscopy; D004926:Escherichia coli; D006801:Humans; D006868:Hydrolysis; D008156:Luciferases / Q000737:chemistry; D055503:Protein Multimerization; D058849:Protein Refolding; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000096:biosynthesis / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry; D044366:Transition Temperature; D038222:gamma-Crystallins / Q000737:chemistry
chemical_list: C533300:CCT4 protein, human; C533301:CCT5 protein, human; C504903:CRYGD protein, human; D018834:Chaperonin 60; D021122:Protein Subunits; D038222:gamma-Crystallins; D012710:Serum Albumin, Bovine; D000255:Adenosine Triphosphate; D008156:Luciferases; D056404:Chaperonin Containing TCP-1
keywords: CCT; Protein Assembly; Protein Expression; Protein Folding; Protein Self-assembly; Protein-Protein Interactions; TRiC