Stoichiometry of Mammalian Target of Rapamycin Complex 1 (mTORC1) And Complex 2 (mTORC2) deciphered by Single-Molecule Pulldown (SiMPull)

The mammalian target of rapamycin (mTOR) is a master regulator of essential cellular and developmental processes, including cell growth, proliferation, differentiation, and metabolism. mTOR is found in at least two biochemically and functionally distinct complexes named mTORC1 and mTORC2. Previous biochemical studies have indicated that mTOR complexes may oligomerize, and structural studies of purified mTORC1 indicate it is a dimer. However, the molecular organization and subunit composition of mTORC2 remains unclear. Here we utilize the recently developed single-molecule pulldown (SiMPull) assay, which combines the principles of the conventional pull-down assay with single molecule fluorescent microscopy, to probe the stoichiometry of both mTOR complexes. We have determined the copy number of the subunits of each complex in crude cell lysates. We find that the subunits Raptor, PRAS40, mLST8 and mTOR of mTORC1 are each present in a copy number of two, consistent with the dimeric organization of mTORC1 revealed by a reported cryo-EM study. Our data suggest that mTORC2 is also dimeric, with the subunits Rictor, mSin1, mLST8, and mTOR each present in a copy number of two. Interestingly, the mTOR-mLST8, Raptor-PRAS40, and Rictor-mSin1 sub-complexes are all monomeric, suggesting that no single subunit serves as the dimerizing component but rather subcomplexes interact to form the dimeric holocomplex. Our results provide insights into mTOR complexes assembly, which may guide future mechanistic studies and exploration of therapeutic potentials.