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Stoichiometry and Assembly of Mtor Complexes Revealed by Single-Molecule Pulldown

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Stoichiometry and Assembly of Mtor Complexes Revealed by Single-Molecule Pulldown Stoichiometry and assembly of mTOR complexes revealed by single-molecule pulldown Ankur Jaina,b,1, Edwin Arauzc,1, Vasudha Aggarwala,b, Nikita Ikonc, Jie Chenc,2, and Taekjip Haa,b,d,e,2 aCenter for Biophysics and Computational Biology, bInstitute for Genomic Biology, cDepartment of Cell and Developmental Biology, dDepartment of Physics, and eHoward Hughes Medical Institute, University of Illinois at Urbana–Champaign, Urbana, IL 61801 Edited by Melanie H. Cobb, University of Texas Southwestern Medical Center, Dallas, TX, and approved November 11, 2014 (received for review October 11, 2014) The mammalian target of rapamycin (mTOR) kinase is a master mTORC2, which has been proposed to be monomeric, dimeric, regulator of cellular, developmental, and metabolic processes. De- or multimeric (7, 10, 13, 14). High-resolution structural analysis regulation of mTOR signaling is implicated in numerous human of mTORC2 has not been possible thus far, likely owing to its diseases including cancer and diabetes. mTOR functions as part of large size and multiplicity of interaction partners. either of the two multisubunit complexes, mTORC1 and mTORC2, Ensemble biochemical methods have inherent limitations in but molecular details about the assembly and oligomerization of analyzing multicomponent heterogeneous protein assemblies. mTORCs are currently lacking. We use the single-molecule pull- These methods do not directly reveal the stoichiometry of in- down (SiMPull) assay that combines principles of conventional teraction and offer low-resolution estimates of the sizes of pro- pulldown assays with single-molecule fluorescence microscopy tein complexes. Additionally, the lengthy procedures often to investigate the stoichiometry and assembly of mTORCs. After associated with biochemical characterization may lead to loss or validating our approach with mTORC1, confirming a dimeric as- alteration of physiological protein complexes. We recently reported sembly as previously reported, we show that all major compo- a single-molecule pulldown (SiMPull) technology that combines nents of mTORC2 exist in two copies per complex, indicating that the principles of conventional pulldown assays with single-molecule mTORC2 assembles as a homodimer. Interestingly, each mTORC fluorescence microscopy (15). In SiMPull, protein complexes are pulled down from freshly lysed cells directly onto chambers for component, when free from the complexes, is present as a mono- single-molecule fluorescence microscopy. When proteins are stoi- mer and no single subunit serves as the dimerizing component. chiometrically labeled for example using fluorescent protein tags, Instead, our data suggest that dimerization of mTORCs is the result SiMPull can reveal the stoichiometry of the protein complexes via of multiple subunits forming a composite surface. SiMPull also single-molecule fluorescence photobleaching step analysis (15). allowed us to distinguish complex disassembly from stoichiometry We have used SiMPull to investigate the oligomeric assembly changes. Physiological conditions that abrogate mTOR signaling of mTORCs. Upon validating our approach by demonstrating such as nutrient deprivation or energy stress did not alter the dimeric assembly of mTORC1, we find that mTORC2 is also stoichiometry of mTORCs. On the other hand, rapamycin treat- dimeric and contains two molecules of mTOR and rictor per ment leads to transient appearance of monomeric mTORC1 before complex. Individual mTORC components are predominantly complete disruption of the mTOR–raptor interaction, whereas monomeric, but under physiological conditions there is no evi- mTORC2 stoichiometry is unaffected. These insights into assembly dence of monomeric interaction between mTOR and raptor or of mTORCs may guide future mechanistic studies and exploration rictor. Multicolor imaging of individual complexes revealed that of therapeutic potential. although the two complexes are predominantly distinct, small fractions of mTORC1 and mTORC2 components coexist in the mTOR | mTORC | single molecule | stoichiometry | rapamycin same complex. Physiological perturbations that abrogate mTOR signaling had no effect on the stoichiometry of mTOR com- he mammalian target of rapamycin (mTOR) is a master plexes, indicating that inhibition of mTOR signaling can be Tregulator of crucial cellular and developmental processes. As a serine/threonine protein kinase belonging to the phosphatidy- Significance linositol-3-kinase (PI3K)-related kinase family, mTOR integra- tes the sensing of nutrients, growth factors, oxygen, energy, and The mammalian target of rapamycin (mTOR) kinase is a central different types of stress to regulate a myriad of biological pro- regulator of cell growth, differentiation, and metabolism. cesses such as cell growth, proliferation, differentiation, and me- mTOR is assembled into two distinct complexes, mTORC1 and tabolism (1). mTOR functions as part of at least two biochemically mTORC2. Using single-molecule pulldown (SiMPull), we have and functionally distinct complexes—mTORC1 and mTORC2 determined the stoichiometric composition of mTORCs under BIOCHEMISTRY (2). mTORC1, better characterized of the two complexes, is the growth and stress conditions. We find that both mTORC1 and rapamycin-sensitive complex, composed of the proteins raptor and mTORC2 form obligate dimers, in which major components mLST8, and it is regulated by the inhibitory proteins PRAS40 exist in two copies per complex. Importantly, SiMPull allowed and DEPTOR (2, 3). mTORC1 is activated by nutrients (such as us to distinguish complex disassembly from stoichiometry amino acids), growth factors, and cellular energy among other changes, providing insights into the effects of physiological stimuli (1, 2). mTORC2 contains rictor, mLST8, and mSin, as well conditions and the drug rapamycin on mTOR complexes. as the negative regulator DEPTOR (2, 3). PI3K-related kinases (PIKKs) such as ataxia telangiectasia Author contributions: A.J., E.A., J.C., and T.H. designed research; A.J., E.A., V.A., and N.I. mutated (ATM), ATM and Rad3-related protein (ATR), and performed research; A.J. and E.A. contributed new reagents/analytic tools; A.J., E.A., and DNA-dependent protein kinase (DNA-PK) are known to oli- V.A. analyzed data; and A.J., E.A., J.C., and T.H. wrote the paper. gomerize (4–6). Biochemical and genetic analyses have identi- The authors declare no conflict of interest. fied self-association of mTOR and its orthologs in yeast and This article is a PNAS Direct Submission. Drosophila (7–10). A cryoelectron microscopy (cryo-EM) study 1A.J. and E.A. contributed equally to this work. revealed that mTORC1 self-associates into a dimeric structure 2To whom correspondence may be addressed. Email: [email protected] or tjha@illinois. (11). Oligomerization of mTORC1 has been reported to be sensi- edu. tive to nutrient status based on biochemical analyses of recombinant This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. proteins (10, 12). Consensus is lacking on the oligomeric state of 1073/pnas.1419425111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1419425111 PNAS | December 16, 2014 | vol. 111 | no. 50 | 17833–17838 Downloaded by guest on September 24, 2021 achieved without requiring disassembly of mTOR complexes or and raptor antibody (Fig. S2E); SiMPull required only 50 μLof changing their oligomeric state. On the other hand, treatment with the extract as opposed to 500 μL used for the corresponding rapamycin led to transient mTOR–raptor complexes containing one coimmunoprecipitation. Hence, the SiMPull method is highly mTOR before complete disassembly of the interaction, whereas sensitive compared with conventional biochemical methods. mTORC2 stoichiometry was unaffected. We then analyzed the fluorescence time trajectories of YFP– mTOR pulled down with raptor. Most molecules (96%) bleached Results in either one or two steps, indicating that mTORC1 contains one – E Assay Validation and mTORC1 Stoichiometry. To study mTOR or two molecules of fluorescently active YFP mTOR (Fig. 1 ). complexes by SiMPull, we deemed it important to establish Nearly 60% of the molecules exhibited two-step bleaching, a system where a fluorescently tagged mTOR can incorporate whereas 36% bleached in a single step. The molecules bleaching into endogenous complexes. To that end, we established a cell in two steps were nearly twice as bright as one-step bleachers – on average, indicating a reliable classification based on photo- line stably expressing YFP mTOR in which the endogenous E mTOR was silenced by short hairpin RNA (Fig. 1A, henceforth bleaching steps (Fig. 1 ). Previous studies have determined that “ – ” – fluorescent proteins may not all mature to completion and the called YFP mTOR stable cell line ). The YFP mTOR protein ∼ associated with endogenous raptor and rictor (Fig. 1A). More fraction of fluorescently active YFP is 75% (15, 16). In a cali- bration experiment, photobleaching step distribution of mono- importantly, the cell line faithfully recapitulated known regu- meric and dimeric YFP or a mixture of the two proteins was lations of mTOR signaling, such as insulin- and serum-stimu- measured, which revealed that the fraction of two-step photo- lated phosphorylation of mTORC1 targets S6K1 and 4E-BP1, bleaching spots is linearly proportional to the fraction of dimeric and mTORC2 target Akt, as well as amino acid dependence of H I B A YFP included (Fig. 1 and ). A comparison of the observed S6K1 and 4E-BP1 phosphorylation
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