Co-translational Binding of GroEL to Nascent Polypeptides Is Followed by Post-translational Encapsulation by GroES to Mediate Protein Folding

Bei-Wen Ying,Hideki Taguchi,Takuya Ueda

doi:10.1074/jbc.m603091200

Bei-Wen Ying, Hideki Taguchi + Show 1 more

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https://doi.org/10.1074/jbc.m603091200

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Abstract

The eubacterial chaperonins GroEL and GroES are essential chaperones and primarily assist protein folding in the cell. Although the molecular mechanism of the GroEL system has been examined previously, the mechanism by which GroEL and GroES assist folding of nascent polypeptides during translation is still poorly understood. We previously demonstrated a co-translational involvement of the Escherichia coli GroEL in folding of newly synthesized polypeptides using a reconstituted cell-free translation system (Ying, B. W., Taguchi, H., Kondo, M., and Ueda, T. (2005) J. Biol. Chem. 280, 12035-12040). Employing the same system here, we further characterized the mechanism by which GroEL assists folding of translated proteins via encapsulation into the GroEL-GroES cavity. The stable co-translational association between GroEL and the newly synthesized polypeptide is dependent on the length of the nascent chain. Furthermore, GroES is capable of interacting with the GroEL-nascent peptide-ribosome complex, and experiments using a single-ring variant of GroEL clearly indicate that GroES association occurs only at the trans-ring, not the cis-ring, of GroEL. GroEL holds the nascent chain on the ribosome in a polypeptide length-dependent manner and post-translationally encapsulates the polypeptide using the GroES cap to accomplish the chaperonin-mediated folding process.

Highlights

GroEL executes two consecutive processes: binding of substrate proteins to prevent irreversible aggregation and release of the arrested protein to complete fold
The prevailing view assumes that trigger factor (TF) and the DnaK system act as co-translational chaperones to prevent protein aggregation, whereas GroEL acts as a post-translational chaperone to help polypeptides that have been released from the ribosome to reach their native state [4, 19]
Stable Co-translational Association of GroEL with the Nascent MetK Peptide Is Dependent on the Polypeptide Length—We have used the essential E. coli protein MetK [35, 36] as a model substrate for further investigation of the translation-coupled folding process since our previous study using the PURE system clearly demonstrated that GroEL was able to associate with the nascent MetK polypeptide on the ribosome [29]

Summary

Introduction

GroEL executes two consecutive processes: binding of substrate proteins to prevent irreversible aggregation (the holder function) and release of the arrested protein to complete fold-. The prevailing view assumes that TF and the DnaK system act as co-translational chaperones to prevent protein aggregation, whereas GroEL acts as a post-translational chaperone to help polypeptides that have been released from the ribosome to reach their native state [4, 19] This model of the roles of the major chaperones is essentially based on the well designed in vitro order-of-addition experiments [20], on genetic experiments showing that simultaneous deletion of TF and DnaK is lethal [9, 21], and on the fact that TF is a ribosome-tethered chaperone [22,23,24,25,26]. How GroEL associates with the translation complex and how GroES participates in the folding process remain to be clarified

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