Structural and functional analysis of the role of the chaperonin CCT in mTOR complex assembly

Jorge Cuéllar,Nicole C Tensmeyer,Sarah Franklin,Aman Makaju,M Teresa Bueno-Carrasco,César Santiago,Michael J Mann,Rebecca L Plimpton,Barry M Willardson,José M Valpuesta,W Grant Ludlam,Madhura Dhavale,Takuma Aoba

doi:10.1038/s41467-019-10781-1

Abstract

The mechanistic target of rapamycin (mTOR) kinase forms two multi-protein signaling complexes, mTORC1 and mTORC2, which are master regulators of cell growth, metabolism, survival and autophagy. Two of the subunits of these complexes are mLST8 and Raptor, β-propeller proteins that stabilize the mTOR kinase and recruit substrates, respectively. Here we report that the eukaryotic chaperonin CCT plays a key role in mTORC assembly and signaling by folding both mLST8 and Raptor. A high resolution (4.0 Å) cryo-EM structure of the human mLST8-CCT intermediate isolated directly from cells shows mLST8 in a near-native state bound to CCT deep within the folding chamber between the two CCT rings, and interacting mainly with the disordered N- and C-termini of specific CCT subunits of both rings. These findings describe a unique function of CCT in mTORC assembly and a distinct binding site in CCT for mLST8, far from those found for similar β-propeller proteins.

Highlights

The mechanistic target of rapamycin kinase forms two multi-protein signaling complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), which are master regulators of cell growth, metabolism, survival and autophagy
To begin to examine the possible role of CCT in mTORC assembly and function, we sought to confirm the interaction of mammalian lethal with SEC13 protein 8 (mLST8) and Raptor with CCT reported in human interactome studies[10]
We tested the binding of mTOR and the other core components of mTORC2, rapamycininsensitive companion of mTOR (Rictor), and MAP kinase-interacting protein 1 (mSIN1) to CCT by coimmunoprecipitation and found no interaction (Supplementary Fig. 1)

Summary

Introduction

The mechanistic target of rapamycin (mTOR) kinase forms two multi-protein signaling complexes, mTORC1 and mTORC2, which are master regulators of cell growth, metabolism, survival and autophagy. A high resolution (4.0 Å) cryo-EM structure of the human mLST8-CCT intermediate isolated directly from cells shows mLST8 in a nearnative state bound to CCT deep within the folding chamber between the two CCT rings, and interacting mainly with the disordered N- and C-termini of specific CCT subunits of both rings. These findings describe a unique function of CCT in mTORC assembly and a distinct binding site in CCT for mLST8, far from those found for similar β-propeller proteins. Overexpression of CCT subunits suppressed phenotypes associated with temperature sensitive mutations of yeast

Methods

Results

Conclusion