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

• Published in: Nature communications Vol. 10; no. 1; pp. 2865 - 14
• Main Authors: Cuéllar, Jorge, Ludlam, W. Grant, Tensmeyer, Nicole C., Aoba, Takuma, Dhavale, Madhura, Santiago, César, Bueno-Carrasco, M. Teresa, Mann, Michael J., Plimpton, Rebecca L., Makaju, Aman, Franklin, Sarah, Willardson, Barry M., Valpuesta, José M.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 28.06.2019; Nature Publishing Group; Nature Portfolio
• Subjects: 101/28; 101/58; 13; 13/89; 38; 631/45/275; 631/45/470/1981; 631/535/1258/1259; 96; 96/1; 96/106; 96/109; Amino Acid Sequence; Assembly; Autophagy; Binding Sites; Cell survival; Chaperonin Containing TCP-1 - physiology; Cryoelectron Microscopy; Folding; Functional analysis; Gene Expression Regulation - physiology; Gene Knockdown Techniques; HEK293 Cells; Hep G2 Cells; Humanities and Social Sciences; Humans; Kinases; Mass Spectrometry; Metabolism; Models, Molecular; mTOR Associated Protein, LST8 Homolog - genetics; mTOR Associated Protein, LST8 Homolog - metabolism; multidisciplinary; Phagocytosis; Protein Binding; Protein Conformation; Protein Folding; Proteins; Rapamycin; Regulators; Regulatory-Associated Protein of mTOR - genetics; Regulatory-Associated Protein of mTOR - metabolism; Science; Science (multidisciplinary); Signaling; Structure-function relationships; Substrates; TOR protein; TOR Serine-Threonine Kinases - genetics; TOR Serine-Threonine Kinases - metabolism
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-019-10781-1

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. β-propeller domains are an important class of folding substrates for the eukaryotic cytosolic chaperonin CTT. Here the authors find that CTT contributes to the folding and assembly of two β-propeller proteins from mTOR complexes, mLST8 and Raptor, and determine the 4.0 Å cryoEM structure of a human mLST8-CCT intermediate that shows mLST8 in a near-native state.