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

Nat Commun. 2019 Jun 28;10(1):2865. 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Chaperonin Containing TCP-1 / physiology*
  • Cryoelectron Microscopy
  • Gene Expression Regulation / physiology
  • Gene Knockdown Techniques
  • HEK293 Cells
  • Hep G2 Cells
  • Humans
  • Mass Spectrometry
  • Models, Molecular
  • Protein Binding
  • Protein Conformation
  • Protein Folding
  • Regulatory-Associated Protein of mTOR / genetics
  • Regulatory-Associated Protein of mTOR / metabolism*
  • TOR Serine-Threonine Kinases / genetics
  • TOR Serine-Threonine Kinases / metabolism*
  • mTOR Associated Protein, LST8 Homolog / genetics
  • mTOR Associated Protein, LST8 Homolog / metabolism*

Substances

  • MLST8 protein, human
  • RPTOR protein, human
  • Regulatory-Associated Protein of mTOR
  • mTOR Associated Protein, LST8 Homolog
  • MTOR protein, human
  • TOR Serine-Threonine Kinases
  • Chaperonin Containing TCP-1