Premium
Structural analysis of CCT‐bound folding Intermediates for mTORC1 subunits Raptor and mLST8
Author(s) -
Ludlam William Grant,
Aoba Takuma,
Cuéllar Jorge,
Makaju Aman,
Franklin Sarah,
Valpuesta José M,
Willardson Barry M
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.598.5
Subject(s) - biophysics , chaperone (clinical) , folding (dsp implementation) , chemistry , microbiology and biotechnology , crystallography , biology , medicine , electrical engineering , pathology , engineering
The mTOR signalling complex 1 (mTORC 1) is a master regulator of cell growth and a high‐value drug target. While mTORC1 function has been studied extensively, little is known about how its subunits are folded and assembled together. Using a combination of cryo‐electron microscopy (cryo‐EM) and chemical crosslinking coupled with tandem mass‐spectrometry (XL‐MS), we investigated the mechanism by which the chaperonin CCT and its co‐chaperone, PhLP1, participate in the folding of mTORC1 subunits Raptor and mLST8. 2D cryo‐EM reconstructions of the raptor‐CCT complex show a mass completely filling the CCT folding cavity and extending out from it. This evidence supports the claim that CCT folds large proteins, such as Raptor, that do not fit inside its cavity by accommodating individual domains inside the cavity while the rest of the protein remains outside the cavity. 2D cryo‐EM reconstructions of the PhLP1‐mLST8‐CCT complex show that mLST8 sits inside the cavity while PhLP1 spans the cavity. XL‐MS data for the mLST8‐CCT complex indicate that mLST8 is oriented on one side of the cavity in contact the CCTα and CCTγ subunits. These results suggest different mechanisms for mLST8 and Raptor folding by CCT, one involving PhLP1 and the other involving a single domain Raptor. Support or Funding Information Grants from the National Institute of Health: EY012287, GM07855