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 calibration 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 (Fig. 1 and Fig. S1 ). When photobleaching step distribution of YFP–mTOR in mTORC1 to cell lysates from this line were applied to single-molecule im- – the calibration data suggested that nearly all mTORC1 complexes aging chambers coated with an antibody against raptor, YFP (>95%) contain two copies of YFP–mTOR, assuming the mat- mTOR fluorescence spots were observed, well above the back- uration level of YFP for all experiments is the same. ground level of fluorescence seen in the control channel without We also transiently coexpressed YFP–mTOR and HA–raptor, C D the antibody (Fig. 1 and ), illustrating specific pulldown of which assembled into functional mTORC1 complexes (Fig. S1B). mTOR in complex with raptor, or mTORC1. Single-molecule fluorescence photobleaching analysis for YFP– To assess the sensitivity of SiMPull, we compared detection of mTOR pulled down with HA–raptor revealed that the complexes YFP–mTOR by Western blotting and by SiMPull with cell each contained two copies of YFP–mTOR (Fig. S3 A and B). lysates at the same concentrations. Remarkably, even at 1,000- Similarly, when YFP–raptor and Flag–mTOR were coexpressed fold dilution of the lysates SiMPull detected YFP–mTOR spe- (Fig. S1 C and D), two copies of YFP–raptor were found in each cifically (Fig. S2A), whereas at 100-fold dilution of the same mTOR–raptor complex (Fig. 1 F and G). Additionally, the lysates, there was no longer any signal on Western blots (Fig. S2B). mTORC1 inhibitors PRAS40 and DEPTOR were also present in Furthermore, in SiMPull, we were able to detect YFP–mTOR two copies per mTORC1 (Figs. S1 E–G and S3 C–F). pulled down via endogenous raptor using a 100-fold dilution of the To confirm that the complexes captured via SiMPull represent lysate (Fig. S2 C and D), whereas no signal was detected in con- physiological state of mTORC1 and are not assembled/dis- ventional coimmunoprecipitation using the same dilution of lysate assembled upon cell lysis, we followed the strategy of Riley et al.

A YFP-mTOR 293 BC Insulin -- - + + YFP-mTOR shRNA: Scr mTOR - FBS - + - + - AA + + + - - YFP-mTOR Raptor mTOR pS6K1 T389 Lysate mTOR IP pAKT S473 Anti-Raptor 293 YM 293 YM p4EBP1 T37/46 YFP-mTOR S6K1 Anti-Rabbit mTOR Biotin Fig. 1. mTORC1 is dimeric. (A) Expression of mTOR Rictor AKT NeutrAvidin PEG – Raptor 4EBP1 Quartz slide in the YFP mTOR stable cells (YM) (Upper) and in- 80 teraction with raptor and rictor assessed by co-IP 300 60 D E N (Lower) are shown. Scr, scramble. (B) YM cells were YFP-mTOR = 2939 40 stimulated with insulin, serum, or amino acids (AA). Raptor 40 N f f YFP

N f (C) Schematic depiction of mTORC1 SiMPull. (D) Anti-Raptor N 20 Anti-Rabbit % Endogenous raptor was pulled down from YM cells, 0 0 0 followed by SiMPull analysis. Shown are schematic Steps 1234 0 200 400 600 F IP Raptor - IP Raptor - Intensity (a.u.) diagram (Left), representative YFP fluorescence 60 100 images (Center), and average number of molecules HA-YFP-Raptor 200 G N = 2840 per imaging area (Nf). (E) Distribution of fluorescence 40 f

N 50 photobleaching steps (Left) and corresponding in- Flag-mTOR N f N f YFP % 20 tensity (a.u., arbitrary units) distribution (Right)for Anti-Flag samples described in D. N, total number of molecules 0 0 0 Steps 1234 0 400 800 analyzed. (F) Flag–mTOR and HA–YFP–raptor were IP Flag T7 IP Flag T7 Intensity (a.u.) H coexpressed and Flag–mTOR was pulled down, fol- 100 100 100 100 100 mYFP only 79% mYFP 55% mYFP 29% mYFP tdYFP only lowed by analyses similar to those in D.(G) Same 21% tdYFP 45% tdYFP 71% tdYFP I 100 y = 2.15x - 32 f analyses as in E for samples described in F. (Scale bar, N 50 50 50 50 50 R2=0.997 μ

% 5 m.) (H) Monomeric YFP (mYFP) and tandem di- 50 meric YFP (tdYFP) were mixed in the ratios as in-

0 0 0 0 0 %tdYFP Steps 1234 1234 1234 1234 1234 dicated and single-molecule fluorescence time trajectories were analyzed. (Top) Number of fluo- 300 300 200 250 250 0 15 30 45 60 rescence photobleaching steps. (Bottom)Intensity %2-step (Int., arbitrary units) of molecules exhibiting one or 150 150 100 125 125

N f 1-step 2-step two photobleaching steps. (I) Observed fraction of Discarded two-step bleaching events changes linearly with the 0 0 0 0 0 Int. 048048048048048 fraction of tdYFP.

17834 | www.pnas.org/cgi/doi/10.1073/pnas.1419425111 Jain et al. Downloaded by guest on September 24, 2021 (17) and mixed lysates from cells expressing YFP–raptor and (Fig. 2E). Furthermore, YFP–DEPTOR in mTORC2 was found Flag–mTOR separately, and compared them to lysates of cells to display photobleaching distribution that corresponded to a mix- coexpressing the same proteins at similar expression levels (Fig. ture of monomers and dimers (∼60% dimer) (Fig. S6 C and D), S3G). At least 10-fold more mTORC1 complexes were detected indicating that each mTORC2 can harbor up to two copies in the lysates from cotransfected cells than the mixed lysates of DEPTOR. (Fig. S3H). Similar results were obtained when intact cells Once again, cell mixing (Fig. S6 E and F) and DSP cross- were mixed before lysis (Fig. S3I). Thus, postlysis association linking (Fig. S6 G and H) experiments were performed, the of mTORC1 components does not significantly contribute to results of which confirmed that the dimeric stoichiometry most the complexes detected here via SiMPull. likely reflects physiological assembly of mTORC2 in live cells. In SiMPull requires dilution of cell lysates to achieve low pull- conclusion, our results unequivocally reveal mTORC2 as a di- down density suitable for single-molecule analysis, which could mer, in which each subunit is present in two copies. lead to loss of weakly associated physiological complexes. To address this issue, we treated YFP–mTOR-expressing cells with mTORC1 and mTORC2 Are Mostly Distinct but Cocomplexes Exist. the cross-linker dithiobis(succinimidylpropionate) (DSP) before Biochemical characterizations so far suggest that mTORC1 cell lysis. Lysis of cells using a Triton X-100–containing buffer and mTORC2 are mutually exclusive, but functional cross-talk disrupted mTORC1 complexes as expected (18), whereas DSP between these two complexes at multiple levels is increasingly cross-linking before cell lysis preserved the complex under the same apparent (20–22). Even a small fraction of mTORC1/mTORC2 lysis condition (Fig. S4 A and B), validating our cross-linking con- cocomplex could be functionally significant, but may have es- ditions. Importantly, cross-linked mTORC1 complexes exhibited caped detection by conventional biochemical methods. Multi- a photobleaching step distribution corresponding to that of a di- color SiMPull should allow direct visualization of such hybrid mer (Fig. S4C), suggesting that the physiological complexes are complexes, if any. As a positive control, we coexpressed Flag– intact during SiMPull analysis without cross-linking. In summary, mTOR, mCherry–raptor, and YFP–PRAS40. Upon capturing we find that mTORC1 is dimeric, containing two copies of each Flag–mTOR, we observed both mCherry–raptor and YFP– component. These findings are consistent with the previous cryo- PRAS40 fluorescence spots (Fig. 3A); ∼49% of YFP–PRAS40 EM data (11), thus validating our experimental system for anal- spots colocalized with mCherry–raptor, indicating their coexistence ysis of mTOR complexes by SiMPull. in the same complexes. Incomplete colocalization between the two likely arises from incomplete chromophore maturation (∼40% for mTORC2 Is Dimeric. mTORC2 assembly requires mSin and mLST8 mCherry and ∼75% for YFP) (23, 24) and the participation of (14, 19) in addition to mTOR and rictor, but the oligomeric state endogenous untagged proteins. of mTORC2 is under debate (7, 10, 13, 14). When all four core Next, we coexpressed mCherry–raptor and YFP–rictor to- components of mTORC2 (mTOR, rictor, mSin, and mLST8) were gether with Flag–mTOR, mLST8, and mSin. An anti-Flag anti- coexpressed, each component self-associated as indicated by body captured both mTORC1 and mTORC2 as visualized by coimmunoprecipitation of the recombinant protein with two dif- mCherry and YFP fluorescent spots, respectively, and about 7% ferent tags (Fig. 2A and Fig. S5A), suggesting that assembled of mCherry–raptor spots reproducibly colocalized with YFP– mTORC2 is oligomeric. To determine the oligomeric state of rictor (Fig. 3B). Taken into consideration the incomplete chro- mTOR in mTORC2, we captured mTORC2 complexes from the mophore maturation mentioned above, it is likely that the true YFP–mTOR stable cell line on SiMPull surfaces using an antibody fraction of the mTORC1/mTORC2 cocomplex is higher than against endogenous rictor (Fig. 2B). Intriguingly, once again we 7%. Under these experimental conditions, the colocalization by observed a photobleaching pattern characteristic for dimeric chance was ∼1% (Fig. S7A). Additionally, when YFP instead of YFP–mTOR (Fig. 2C). Similar results were obtained when YFP– YFP–rictor was expressed, only a background level of YFP fluo- mTOR was coexpressed with recombinant mTORC2 compo- rescent spots was observed upon Flag–mTOR pulldown, and these nents (Fig. S6 A and B), which also assembled into functional spots did not colocalize with mCherry–raptor (Fig. S7B). Impor- mTORC2 (Fig. S5 B and C). tantly, raptor coexpression did not alter mTORC2 stoichiometry, Next, we probed the stoichiometry of rictor in mTORC2. YFP– as rictor was still present in two copies (Fig. S7C). rictor was coexpressed with Flag–mTOR, mSin–HA and HA– To verify that the hybrid complex was not an artifact of cell mLST8, and the assembly of mTORC2 was verified by ensemble lysis, we performed cell mixing experiments. mTORC1 compo- pulldown (Fig. S5 D and E). Upon single-molecule pulldown of nents (Flag–mTOR and mCherry–raptor) and mTORC2 com- mTORC2 via Flag–mTOR (Fig. 2D), we observed YFP–rictor ponents (Flag–mTOR, YFP–rictor, mSin–HA, and HA–mLST8) photobleaching consistent with two copies of rictor per mTORC2 were expressed in two separate pools of cells, which were mixed

AC Lysate HA IP B YFP-mTOR 60 80 HA-mTOR + + ++ 400 BIOCHEMISTRY N = 2290 Flag-mTOR + + + + Rictor 40

Flag-Rictor - + - + f 40 Flag-mLST8 - + - + Anti-Rictor 200 N f N f YFP - + - + %N mSin-Flag Anti-Rabbit 20 Flag-mTOR 0 0 HA-mTOR 0 IP Rictor - IP Rictor - Steps 1234 0 250 500 Intensity (a.u.) 500 D YFP-Rictor E 60 100 mSin N = 2588 40 250 f N f

Flag-mTOR YFP 50 N f

mLST8 %N Anti-Flag 20

0 0 0 IP Flag - Flag IP Flag- Flag Steps 1234 0 200 400 600 800 mTOR + + - mTOR ++- Intensity (a.u.)

Fig. 2. mTORC2 is dimeric. (A) mTOR oligomerizes in mTORC2 shown by co-IP. (B and C) SiMPull for endogenous rictor from YM cells and analysis similar to Fig. 1 D and E.(D and E) YFP–rictor was coexpressed with Flag–mTOR, mSin, and mLST8, and Flag–mTOR was pulled down. SiMPull data are presented as in B and C. (Scale bar, 5 μm.)

Jain et al. PNAS | December 16, 2014 | vol. 111 | no. 50 | 17835 Downloaded by guest on September 24, 2021 YFP mCherry Overlay Effect of Rapamycin on mTOR Complexes. Several models have been YFP-PRAS40 A 49 ± 2% proposed for the mechanism by which rapamycin inhibits mTOR. mCherry-Raptor For example, rapamycin may limit substrate access to the kinase Flag-mTOR domain (25, 26), may induce raptor dissociation from mTOR Anti-Flag (11, 18, 27), or displace a key regulator (28). We investigated the effect of rapamycin on the stoichiometry of mTOR complexes. As 7 ± 3% B mCherry-Raptor YFP-Rictor reported (29), short-term treatment of cells with rapamycin dis- mSin sociated raptor from mTOR and inhibited mTORC1 signaling, Flag-mTOR whereas on prolonged treatment, mTORC2 assembly was also Anti-Flag mLST8 affected (Fig. S9 A and B). When YFP–mTOR stable cells were treated with increasing concentrations of rapamycin (2–100 nM) for 30 min and mTORC1 complexes were captured from cell Fig. 3. mTORC1 and mTORC2 colocalization. (A) Flag–mTOR cocaptures lysates through endogenous raptor, the number of YFP–mTOR YFP–PRAS40 (Left) and mCherry–raptor (Center). Overlay of the two images A ± – – pulled down decreased with increasing rapamycin dose (Fig. 6 ): (Right) showed 49 2% colocalization. (B) Flag mTOR, mCherry raptor, treatment with 2 nM of rapamycin led to reduction by 56%, and YFP–rictor, HA–mLST8, and mSin–HA were coexpressed. Flag–mTOR was a maximal reduction of ∼90% was reached by 10 nM and 100 nM pulled down, and YFP (rictor, Left) and mCherry (raptor, Center) were im- C aged. Overlay of the two images (Right) showed 7 ± 3% colocalization across rapamycin (Fig. 6 ). The residual mTORC1 appeared to be six independent experiments. (Scale bar, 10 μm.) resistant to rapamycin, consistent with previously reported observations (18). Thus, acute rapamycin treatment at low dose disrupted the interaction between mTOR and raptor. during lysis (Fig. S7 D–F). After accounting for false colocalization Additionally, we found that the fraction of dimers decreased by chance, only 2% colocalization between mCherry–raptor and as the rapamycin dose was increased. At 2 nM of rapamycin, YFP–rictor was observed from mixed lysates. Therefore, although about 47% of the molecules bleached in two steps, whereas at the two mTOR complexes are predominantly distinct, hybrid mTOR 100 nM, only 29% of the molecules exhibited two-step bleaching (Fig. 6B). The photobleaching analysis was performed at similar complexes containing both raptor and rictor or higher order as- − semblies of mTORC1/mTORC2 exist, albeit at a low level. surface densities of the complexes (∼0.1 molecules·μm 2), pre- cluding any artifacts due to differences in the immobilization mTORC1 and mTORC2 Components Are Monomeric. Because both density. This observation of transient monomeric mTOR–raptor mTORC1 and mTORC2 are dimeric, we asked if mTOR or complexes indicates that rapamycin disrupts mTORC1 in at least other core components could self-dimerize. To that end, each two steps, displacing one mTOR (or mTOR–raptor monomeric component tagged with YFP was individually expressed. When subcomplex) at a time. Consistent with these results, mTOR YFP–mTOR was captured using an anti-mTOR antibody, nearly bound to FKBP12–rapamycin is monomeric, as indicated by the 75% of the molecules bleached in a single step, whereas 20% single-step photobleaching exhibited by YFP–mTOR pulled bleached in two steps, indicating that a majority of overexpressed down via surface immobilized FKBP12–rapamycin (Fig. S8 E mTOR was monomeric (Fig. 4A). Furthermore, when YFP–mTOR and F). The reduction in mTORC1 signaling activity (pS6K1) and HA–mLST8 were coexpressed and the mTOR–mLST8 sub- corresponded with the loss of intact dimeric mTORC1 com- complexes were captured through the HA tag, a photobleaching plexes (Fig. 6C), implying that disruption of mTORC1 dimeric step distribution characteristic of monomers was observed for YFP– architecture contributes significantly to rapamycin-induced ab- mTOR (Fig. 4B). Similar analysis for HA–YFP–raptor and HA– rogation of mTORC1 signaling. YFP–rictor, pulled down through the HA tag, also revealed mo- Next, we investigated the effect of prolonged rapamycin treat- nomeric distributions (Fig. 4 C and D). In addition, YFP–PRAS40 ment on mTORC2 assembly by capturing endogenous rictor from bound to raptor was monomeric (Fig. S8 A and B). The observed YFP–mTOR cell lysates. The number of mTORC2 complexes small fractions of dimer may arise due to incorporation of YFP- decreased by 40% upon 6-h treatment with 100 nM of rapamycin tagged proteins in endogenous mTOR complexes. Taken to- and was reduced by 79% after 24 h (Fig. 6D). Interestingly, in gether, our results show that although both mTORC1 and contrast to the transient mTORC1 monomers, mTORC2 mTORC2 are exclusively dimeric, individual mTORC compo- remained a dimer at all time points (Fig. 6E). Rapamycin treat- nents and subcomplexes are predominantly monomeric. Thus, ment did not affect mTOR expression levels (Fig. S9 C and D), no single mTORC subunit serves as a dimerizing component. whereas the amount of mTORC1 decreased (Fig. S9E). Thus, our

mTORC Stoichiometry Is Unchanged Under Various Physiological Conditions. Next we asked if the oligomerization of mTOR N N complexes was affected by upstream signals or physiological A YFP-mTOR 300 100 = 2723 B YFP-mTOR 300 100 = 1073 conditions known to regulate mTOR signaling, including growth f factors, nutrient availability, and cellular energy levels. To ex- HA-mLST8 f

Anti-mTOR N N N f – N f

amine the effect of energy sufficiency, we starved the YFP % Anti-HA % mTOR stable cells of glucose and glutamine, which leads to Anti-IgG energy depletion and inhibition of mTORC1 signaling (12), and 0 0 0 0 1234 1234 briefly (1 h) restimulated them with growth medium containing IP mTOR V5 IP HA IgG glucose and glutamine. As shown in Fig. 5A, a nearly equal number C 500 100 N = 2232 D 200 100 N = 1921 of mTORC1 complexes pulled down through endogenous raptor HA-YFP-Raptor HA-YFP-Rictor were detected by SiMPull, with similar photobleaching step dis- f f N N N f tribution, under starvation and stimulation conditions. Similarly, N f % neither amino acids (Fig. 5B) nor leucine (Fig. S8 C and D)stim- Anti-HAHA Anti-HA % ulation had any effect on the number or stoichiometry of 0 0 0 0 mTORC1. In addition, insulin stimulation did not affect mTORC1 IP HA Flag 1234 IP HA Flag 1234 (Fig. 5C) or mTORC2 assembly (Fig. 5D). The starvation and Steps Steps stimulation conditions impacted mTOR signaling as expected (Fig. Fig. 4. mTORC components are monomeric. (A) YFP–mTOR, (B) YFP–mTOR 1B and Fig. S1A). These observations directly establish that in- and HA–mLST8, (C)HA–YFP-raptor, or (D)HA–YFP–rictor were expressed in hibition of mTOR activity by energy stress or nutrient- or growth- HEK293 cells and captured on SiMPull surfaces as depicted (diagrams on factor depletion can be achieved without disassembly of the Left). The number of molecules observed per imaging area (Center) and the mTOR complexes. distribution of fluorescence photobleaching steps (Right) are shown.

17836 | www.pnas.org/cgi/doi/10.1073/pnas.1419425111 Jain et al. Downloaded by guest on September 24, 2021 subunit. Interestingly, all individual subunits of mTORCs in- A 60 N N B 60 250 = 357 = 414 250 N = 437 N = 411 cluding mTOR, when expressed alone, are monomeric. This - Gluc + Gluc - AA + AA /gln /gln excludes a commonly presumed role of the HEAT repeats in 125 N f 125 N f mediating mTOR self-association. Interaction between mTOR % N f % N f and raptor alone is sufficient to form mTORC1 dimers, but 0 0 0 0 mTORC2 assembly and dimerization require coexpression of α-Raptor + + - 1234 1234 α-Raptor + + - 1234 1234 Gluc/gln - + - Steps AA - + - Steps mSin and mLST8 in addition to mTOR and rictor. Under physiological conditions, there is no evidence of monomeric in- 60 C 300 D 450 70 N = 480 N = 604 N = 1238 N = 561 teraction between mTOR and raptor or mTOR and rictor. - Insulin + Insulin 300 - Insulin + Insulin Hence, we propose a model for mTORC assembly where the

150 N f N f

% N f interaction between monomeric subunits is unstable; assembly % N f 150 requires multiple subunits to accumulate at high local concen- 0 0 0 0 trations (Fig. 6F), which may be facilitated by membrane local- α-Raptor + + - 1234 1234 α-Rictor + + - 1234 1234 Insulin - + - Steps Insulin - + - Steps ization, subcellular compartmentalization, or scaffolding proteins. The small but significant fraction of mTORC1– Fig. 5. Effect of physiological stimulations on mTORCs. YFP–mTOR stable mTORC2 cocomplex revealed by SiMPull suggests a potential cells were starved (−)of(A) glucose and glutamine (Gluc/gln) for 12 h, (B) physical cross-talk between the two complexes that may have amino acids (AA) for 2 h, or (C and D) serum for 24 h, followed by restim- evaded detection by conventional biochemical methods. Future ulation (+) with glucose/glutamine for 1 h, amino acids for 30 min, or 100 nM – investigation examining the biological function of this cocomplex insulin for 30 min, respectively. mTORC1 (A C) or mTORC2 (D) was pulled down is warranted. via endogenous raptor or rictor, respectively, followed by SiMPull analysis. Of significance, our SiMPull assays have captured the exis- tence of monomeric mTORC1 (mTOR–raptor complex) upon results suggest that rapamycin does not directly affect mTORC2 acute rapamycin treatment, before complete disruption of the mTOR–raptor interaction (Fig. 6F). The capture of this in- stoichiometry. Instead, rapamycin may sequester free mTOR and termediate state of mTORC1, which was not detected by cryo- subsequently impair mTORC2 assembly over time. EM analysis (11), further attests to the exquisite sensitivity of the Discussion SiMPull method. We also provide direct evidence that cellular stress conditions that abrogate mTORC1- or mTORC2-medi- Using SiMPull we have determined the stoichiometry and as- ated signaling do not alter the number or oligomerization state sembly of mTOR complexes captured from whole cell lysates. of mTOR complexes, indicating that effective inhibition of In addition to confirming the dimeric structure of mTORC1 mTOR signaling can be achieved without disassembling mTOR as previously revealed by cryo-EM studies, we find that complexes. Of note, Kim et al. recently reported that glucose and mTORC2 assembles into a dimer, with two copies of each glutamine deprivation results in monomeric mTORC1 (12), which

Rap (nM) 0 2 10 100 A 700

N f 350 IP: Raptor IP:

0 B 80 80 80 80 Rap (nM) 0 2 10 100 Con N = 2971 N = 2805 N = 2274 N = 1367 C mTOR-raptor (total) 40 mTOR % N f mTOR-raptor (dimer) 150 pS6K1 T389 Fig. 6. Effect of rapamycin on mTORCs. YFP– 0 0 0 0 Steps 1234 1234 1234 1234 mTOR stable cells were treated with (A–C)in- 100 175 1-step 150 150 75 creasing doses of rapamycin (Rap) for 30 min or (D 2-step 50 Discarded and E) 100 nM rapamycin over the indicated time

N f 0 course. mTORC1 (A and B) and mTORC2 (D and E) % relative to 0 nM 0 5 10 100 0 5 10 100 were captured via endogenous raptor and rictor, Rap (nM) 0 0 0 0 04 8 respectively. Representative SiMPull images and Int. 048048048 BIOCHEMISTRY Rap TPT (h) 0 6 12 24 600 number of molecules observed per imaging area D are shown in A and D. (Scale bar, 5 μm.) Dis- tributions of fluorescence photobleaching steps

N f 300 are shown in B and E. N, total number of molecules analyzed. (C, Left) Relative levels of mTOR, IP: Rictor mTOR–raptor (total), and mTOR–raptor (dimer) 0 Rap (h) 0 6 12 24 Con were obtained from SiMPull. (Right) Phosphory- 75 75 75 75 lation of S6K1 (pT389) was measured by Western E N = 1348 N = 1850 N = 1303 N = 1553 mTORC1 F Rapamycin- blotting under similar conditions and quantified 50 Raptor FKBP12 by densitometry. TPT, time posttreatment. (F)A

% N f 25 model for the assembly of mTOR complexes. In- dividual mTORC components are monomeric mTOR 0 0 0 0 Steps1234 1234 1234 1234 and assemble into homodimeric holocomplexes, 50 80 70 mLST8 mTORC1 or mTORC2. Rapamycin directly disrupts 60 the mTOR–raptor interaction leading to monomeric mTORC1 and single proteins. Rapamycin–

N f Rictor FKBP12-associated mTOR cannot be incorporated 0 0 0 mTORC2 into mTORC1 or mTORC2, resulting in indirect 0 mSin Int. 048048048048 depletion of mTORC2 over time.

Jain et al. PNAS | December 16, 2014 | vol. 111 | no. 50 | 17837 Downloaded by guest on September 24, 2021 is not observed in our SiMPull assays. Again, direct experimenta- Materials and Methods tion with fresh cell lysates containing near-endogenous proteins, Antibodies, Plasmids, and Other Reagents. These are all described in SI Materials without any additional manipulation such as those required for and Methods. conventional coimmunoprecipitation, may be advantageous in capturing physiologically relevant protein complexes. Dimerization Cell Culture. The maintenance and transfection of HEK293 cells, and the gen- of other PIKKs such as ATM and DNA-PK play important roles in eration of YFP–mTOR stable cells are described in SI Materials and Methods. the regulation of their activities (4, 6). Our data reveals differences in assembly mode and regulation of mTOR compared with these Single-Molecule Analyses. Single-molecule imaging and spot counting, pho- members of the PIKK family. tobleaching analysis, and single-molecule colocalization were performed as Over 75% of the proteins in a cell can oligomerize (30). previously reported (15, 31). Detailed procedures are described in SI Mate- SiMPull is a powerful method to investigate oligomeric protein rials and Methods. assemblies. This method provides direct and quantitative read- out of the assembly state of the proteins, expressed at endoge- Western Blotting, Immunoprecipitation, and in Vitro mTOR Kinase Assays. nous levels directly in their native context. Proteins are captured These assays followed previously published procedures (32) and are described in detail in SI Materials and Methods. from freshly lysed cells and probed at single-molecule resolution without requiring removal of other proteins, minimizing loss of Statistical Analysis. All data are presented as mean ± SD, or representative interactions due to stringent wash steps associated with con- images, of at least three sets of independent experiments. Whenever nec- ventional immunoprecipitation. SiMPull worked wherever en- essary, statistical significance of the data was analyzed by performing one- semble immunoprecipitation worked across the variety of sample or paired t tests. constructs and samples tested in this study, highlighting the versatility of the assay. By performing SiMPull and biochemical ACKNOWLEDGMENTS. We thank Kaushik Ragunathan, Benjamin Leslie, Kyu analysis on the same samples, we were able to correlate the Young Han, Kyung Suk Lee, Reza Vafabakhsh, and the members of the J.C. complex architecture with its functional activity. The advent of laboratory for helpful discussions. This work was supported by National Science Foundation Grant PHY 1430124 (to T.H.) and National Institutes of genetic engineering at endogenous loci and developments in Health Grants AI083025 (to T.H.), GM089771 (to J.C.), AR048914 (to J.C.), and short genetically encoded fluorescent tags should enable pow- AG042332 (to J.C. and T.H.). T.H. is an Investigator of the Howard Hughes erful applications of this technology to near-endogenous systems. Medical Institute.

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