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1. chinaXiv:201605.01474 [pdf]

Protomer Roles in Chloroplast Chaperonin Assembly and Function

Bai, Cuicui; Guo, Peng; Zhao, Qian; Lv, Zongyang; Zhang, Shijia; Gao, Fei; Tian, Zhixi; Liu, Cuimin; Gao, Liyan; Wang, Yingchun; Bai, Cuicui; Guo, Peng; Zhao, Qian; Zhang, Shijia; Wang, Jifeng; Yang, Fuquan; Wang, Jifeng; Yang, Fuquan
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

The individual roles of three chloroplast CPN60 protomers (CPN60 alpha, CPN60 beta 1, and CPN60 beta 2) and whether and how they are assembled into functional chaperonin complexes are investigated in Chlamydomonas reinhardtii. Protein complexes containing all three potential subunits were identified in Chlamydomonas, and their co-expression in Escherichia coli yielded a homogeneous population of oligomers containing all three subunits (CPN60 alpha beta 1 beta 2), with a molecular weight consistent with a tetradecameric structure. While homo-oligomers of CPN60 beta could form, they were dramatically reduced when CPN60 alpha was present and homo-oligomers of CPN60 beta 2 were readily changed into hetero-oligomers in the presence of ATP and other protomers. ATP hydrolysis caused CPN60 oligomers to disassemble and drove the purified protomers to reconstitute oligomers in vitro, suggesting that the dynamic nature of CPN60 oligomers is dependent on ATP. Only hetero-oligomeric CPN60 alpha beta 1 beta 2, containing CPN60 alpha, CPN60 beta 1, and CPN60 beta 2 subunits in a 5: 6: 3 ratio, cooperated functionally with GroES. The combination of CPN60 alpha and CPN60 beta subunits, but not the individual subunits alone, complemented GroEL function in E. coli with subunit recognition specificity. Down-regulation of the CPN60 alpha subunit in Chlamydomonas resulted in a slow growth defect and an inability to grow autotrophically, indicating the essential role of CPN60 alpha in vivo.

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