TORC1 organized in inhibited domains (TOROIDs) regulate TORC1 activity - PubMed (original) (raw)
. 2017 Oct 12;550(7675):265-269.
doi: 10.1038/nature24021. Epub 2017 Oct 4.
Affiliations
- PMID: 28976958
- PMCID: PMC5640987
- DOI: 10.1038/nature24021
TORC1 organized in inhibited domains (TOROIDs) regulate TORC1 activity
Manoël Prouteau et al. Nature. 2017.
Abstract
The target of rapamycin (TOR) is a eukaryotic serine/threonine protein kinase that functions in two distinct complexes, TORC1 and TORC2, to regulate growth and metabolism. GTPases, responding to signals generated by abiotic stressors, nutrients, and, in metazoans, growth factors, play an important but poorly understood role in TORC1 regulation. Here we report that, in budding yeast, glucose withdrawal (which leads to an acute loss of TORC1 kinase activity) triggers a similarly rapid Rag GTPase-dependent redistribution of TORC1 from being semi-uniform around the vacuolar membrane to a single, vacuole-associated cylindrical structure visible by super-resolution optical microscopy. Three-dimensional reconstructions of cryo-electron micrograph images of these purified cylinders demonstrate that TORC1 oligomerizes into a higher-level hollow helical assembly, which we name a TOROID (TORC1 organized in inhibited domain). Fitting of the recently described mammalian TORC1 structure into our helical map reveals that oligomerization leads to steric occlusion of the active site. Guided by the implications from our reconstruction, we present a TOR1 allele that prevents both TOROID formation and TORC1 inactivation in response to glucose withdrawal, demonstrating that oligomerization is necessary for TORC1 inactivation. Our results reveal a novel mechanism by which Rag GTPases regulate TORC1 activity and suggest that the reversible assembly and/or disassembly of higher-level structures may be an underappreciated mechanism for the regulation of protein kinases.
Conflict of interest statement
The authors have no competing financial interests.
Figures
Extended data Figure 1. Stoichiometric colocalization of TORC1 subunits to a vacuole-associated focus.
a, and b, Differential interference contrast (DIC) microscopy and confocal images of yeast cells exiting exponential growth. Cells express GFP- and/or mCherry-tagged TORC1 subunits as indicated. Vph1-mCherry marks the membrane of the vacuole. We note, however, that the Lst8-GFP and Tco89-mCherry strains presented major growth phenotypes, which for Lst8 were so severe that we were unable to generate the strains necessary to assess its presumptive colocalization with other TORC1 components. c, Purification of GFP-Ypk1 used for GFP calibrations, quantifications and localisations. Left panel, Gel filtration plot showing void volume and GFP-Ypk1 monomer peaks. Right panel, Coommassie stained gel of purified fractions obtained by gel filtration. d, Distribution of single GFP brightness values calculated from GFP calibration using epi-fluorescence images of GFP-Ypk1. e, Anti-GFP western blot analysis of the GFP tagged TORC1 subunits expressed in the presence (+Glc) or absence (-Glc) of glucose. Hog1 is used as loading control. f, g, Boxplots of the number of GFP molecules per cell (f) and per focus (g) for the indicated GFP-tagged TORC1 subunits. Error bars represent s.d. for values obtained on at least 100 cells. h, Boxplot quantifying the number of GFP molecules per WT cell expressing GFP-Kog1 in the presence (+Glc) or absence (-Glc) of glucose. i, Boxplot quantifying the number of GFP molecules per focus in WT cells expressing GFP-Kog1 starved for glucose (-Glc) or grown into stationary phase (Stat.). f-i, Error bars represent s.d. for values obtained with ≥100 cells.
Extended data Figure 2. TORC1 focus formation occurs upon glucose-starvation but not nitrogen or leucine starvation independently of external pH.
a, The majority of cells grown into stationary phase display a prominent TORC1 focus as determined by confocal microscopy imaging of GFP-Kog1. Addition of glucose, but not amino acids (AA) or ammonium sulphate (AS) triggered rapid disassembly of these foci. b, TORC1 focus formation monitored by confocal microscopy using a microfluidic device demonstrates that foci can be observed within 2-3 minutes after glucose depletion. c-e, TORC1 focus formation monitored by confocal microscopy after Glucose (c), Ammonium sulphate (d), or Leucine (e) starvation and subsequent re-addition. f, Confocal images of WT cells expressing GFP-Kog1 and Gln1-mCherry grown in pH-controlled medium before (+Glucose) and 1 hour after (-Glucose) glucose starvation. White arrows show Gln1 foci.
Extended data Figure 3. Snf1 does not contribute significantly to TORC1 focus dynamics.
a, Growth phenotypes on YP-Dextrose or YP-Glycerol plates of WT and Δ_snf1_ cells expressing GFP-Tor1. Δ_snf1_ cells show a characteristic defect in using glycerol as a carbon source. b, Representative confocal images of TORC1 focus formation in WT and Δ_snf1_ cells following glucose depletion (-Glucose, 0 min) and subsequent re-addition (+Glucose, 30 min). c, Percentage of cells displaying a TORC1 focus as measured from (b). Data are mean ± s.d. and represent three independent experiments. d, Western blot assessing the extent of Sch9 Ser-758 phosphorylation as a proxy of TORC1 activity, at the time points monitored in (b). Hog1 is used as loading control.
Extended data Figure 4. TORC1 focus dynamics, but not size, are different in WT vs Δgtr1, Δgtr2 cells.
a, 3D reconstruction of WT cells expressing GFP-Kog1 in the presence of glucose or after 5 and 30 minutes of glucose starvation. Red arrows show granular TORC1 localisation while Blue arrows show TORC1 foci. b, Representative confocal images of exponentially growing WT and Δgtr1, Δgtr2 cells expressing the indicated GFP-tagged TORC1 subunits. c, Boxplot quantifying the number of GFP-Kog1 molecules per focus in stationary phase WT cells (Stat.) and in exponentially growing Δgtr1, Δgtr2 cells. Error bars represent s.d. for values obtained with ≥100 cells. d, Quantitative analysis of TORC1 focus disassembly after dilution of stationary phase WT (light grey) and Δgtr1, Δgtr2 (dark grey) cells expressing GFP-Kog1 into fresh complete synthetic medium. Half-life of TORC1 foci in both backgrounds is indicated. Data are mean ± s.d. and represent at least three independent experiments (150-800 cells each).
Extended data Figure 5. Gtr1GDP/Gtr2GTP conformation favours focus formation while the Gtr1GTP/Gtr2GDP conformation antagonizes focus formation.
a, TORC1 focus formation (GFP-Kog1; dark grey bars on left graph) was assessed in Δgtr1 Δgtr2 cells expressing all combinations of plasmid-borne wild-type or nucleotide-locked (GDP/GTP) variants of Gtr1 and Gtr2, both in exponentially growing or saturated cultures. Using growth state [State: exponential = -1 and saturated = 1] and nucleotide loading of the Gtrs [Gtr1 and Gtr2: GDP bound = -1, WT = 0 and GTP bound =1] as variables, the data was analysed by multiple linear regression to obtain a model explaining the percentage of cells displaying a TORC1 focus (equation). The obtained model explains 83.7% of the total observed variance (R2, residual error = 16.3%) and has a high statistical significance (Fisher test, p-value < 10-5; summarized in table on right). Simulation with the model is shown (light grey bars on left graph). The model shows that contribution of both growth state and Gtrs variables are statistically significant (β1, β2 and β3: Student test, p-values < 0.05). Gtr1 and Gtr2 participate together to account for 47.3% of the variation of TORC1 focus formation and have opposing contributions, Gtr1-GTP disfavours while Gtr2-GTP favours TORC1 focus formation (β2 = -0.076 and β3 = 0.056). The rest of variation of focus formation is Gtr-independent. b-f, The amount of active TORC1 is lower, but Sch9 phosphorylation is faster, in Δgtr1, V_gtr2_ cells compared to WT cells. b, Plot showing the proportion of cells without TOROIDs and the relative Sch9-phosphorylation in WT and Δgtr1, Δgtr2 cells. Sch9 phosphorylation is partially compromised in Δgtr1, Δgtr2 cells (~75% of WT and not 40% as would be suggested by the fraction of cells lacking focus) suggesting that the remaining active TORC1 entities in Δgtr1, Δgtr2 cells must be ~2-fold more active compared to the active TORC1 entities in WT cells. c, Growth curves of Δgtr1, Δgtr2 and WT cells treated with increasing concentrations of BGW867 in liquid culture, (estimated EC50: 0.12 μM and 0.27 µM respectively). d, Spot assays of WT and Δgtr1, Δgtr2 cells on increasing concentrations of BGW867. On plate, the Δgtr1, Δgtr2 cells appear to be about twice more sensitive compared to WT. e, Measures of Sch9 dephosphorylation (phosphatase activity) and rephosphorylation (TORC1 activity) rates in WT and Δgtr1, Δgtr2 cells. To first monitor dephosphorylation rates, we treated cells with 1.0 μM BGW867 and collected samples, at the indicated time points, over a period of 10 minutes for western blot analyses (0-10 min.). The remaining cells were washed and resuspended into fresh medium containing varying concentrations of BGW867. Samples, at the indicated time points, were subsequently collected over the next 20 minutes for western blot analyses (10-30 min.). f, Plot of relative Sch9 rephosphorylation rates versus increasing concentrations of BGW867 measured in WT and Δgtr1, Δgtr2 cells in panel (e). In the absence of BGW867, the rate of Sch9 rephosphorylation is 1.75 higher in Δgtr1, Δgtr2 cells than in WT. However, when released into BGW867, the rate of Sch9 rephosphorylation is more strongly affected in Δgtr1, Δgtr2 cells compared to WT (estimated EC50: 0.1 μM and 0.2 µM respectively).
Extended data Figure 6. STORM supplementary data.
a, Correlation plot between A647 and GFP integrated intensities, measured for A647-conjugated anti-GFP nanobody-labelled TORC1 foci segmented from cytosolic signal. b, Graphical representation of A647 localisation precision along x and y axes, derived from A647-conjugated anti-GFP nanobody calibration. c, Gallery of reconstructed clusters classified according to their eccentricity (Ecc < 2 and Ecc > 2). Data corresponds to TORC1 foci imaged in wild-type cells expressing GFP-Kog1 grown into stationary phase. Scale bar 100 nm.
Extended data Figure 7. Negative-stain Electron Microscopy analyses of TORC1 purification.
a-b, assembled TEM micrographs showing 50 µm2 of an negative-stained TORC1 purification from exponentially growing (a) or starved (b) Kog1-TAP expressing cells. Red stars show extended and end-on views of TORC1 helices. Scale bar 500 nm. c, 2D class averaging analyses reveal similar TORC1 single particle features and organised pattern of subunits in the tubular structures. Green circles correspond to the picked images used for 2D classification and averaging. Red arrows highlight a repeated pattern in the 2D class. Scale bar 25 nm. d, Fourier filtration of the tubular structure suggests an helical organisation. Scale bar 50 nm.
Extended data Figure 8. 3D TOROID reconstruction and comparison between the cryo-EM map and the mTORC1 atomic model.
a, Symmetry refinement of TORC1 filaments. The pitch of the helix (left), and the number of units per turn (right) were refined by 30 iterations of IHRSR using 29 different starting choices for the number of subunits per turn (5.1 to 7.9 in steps of 0.1). Numbers on the right indicate the refined solutions for the number of units per turn at the end of the refinement. For each of these solutions, the number of refinement cycles culminating in the converged solution are indicated in parentheses. b, Side-by-side display of experimental versus simulated power spectra from the 3D reconstruction. Experimental power spectrum is the sum of the power spectrum of aligned segments after projection matching. c, Resolution assessment for the TORC1 helical reconstruction determined using SPRING - the Fourier Shell correlation was computed from two reconstructions containing half of the dataset each. The resolution corresponding to the 0.5 and 0.143 cut-offs is indicated in the plot. d, Image processing statistics of the reconstruction. e, Side (upper row) and cross-section views (lower row) of the cryo-EM maps for the 3 possible refined solutions for the number of units per helix turn with helix pitch of 211Å as deduced from the analysis of the convergence points of the IHRSR refinements. The maps shown at 30Å resolution were rendered at a threshold 1.0 σ. The maps in the left and middle panel are inconsistent with the expected density profile of the TORC1 subunit from panel (f). In rightmost panel, a dashed ellipse encloses one TORC1 dimer. f, Comparison of the density models of mTORC1 dimer generated from the atomic model (pdb ID 5FLC) filtered to 27Å resolution and that extracted from the final cryo-EM map. g, Estimation of the docking precision. Shown is the correlation of the fitted model with the map upon rotation of the mTORC1 dimer around its principal axes of inertia x,y and z as indicated in the inset. h, Comparison of the cryo-EM map and electron density maps computed from the models suggests that the features of the map are in agreement with the two variants of the docked model at ~30Å resolution and confirms the resolution assessment. The FSC between the helically arranged models and the experimental cryo-EM map is shown together with the FSC used for assessing the cryo-EM map resolution.
Extended data Figure 9. Simulation of STORM data using EM particle reconstruction data.
a, Left panel, End-on view of reconstructed TORC1 helix fitted with 9 TORC1 particles in dark green, dark red and purple (c.f. Figure 3). Middle panel, Crystal structure of GFP (light green) was added to the reconstructed helix at sites identified as Raptor/Kog1 N-termini. Right panel, Crystal structure of anti-GFP nanobody (red) was added to a subset of the GFP structures, according to the labelling efficiency estimated for our experimental conditions (~20%). The localisation precision is indicated as a red cloud. b, 3D representation of the simulated starting model according to a. Red and blue spheres denote GFP positions. c, Simulated STORM images. Helices of different lengths were generated based on the distribution of focus sizes observed in vivo (Figure 2 and bar graph). Orientations of these helices were randomized in 3D (XYZ) space and then projected in 2D (XY). Coloured spheres denote GFP positions, a random fraction of which were considered labeled according to our experimentally determined labelling efficiency (see SMLM simulation in Methods for more details). d, Reconstructions of individual simulations from c, classified according to their eccentricity and presence of a cavity. Scale bar 100 nm. e, Particle averages obtained from the images in d. f, Plot of the eccentricity distribution of the simulated and experimental STORM dataset.
Extended data Figure 10. Disruption of TORC1-TORC1 contact interfaces abolish TOROID assembly.
a, Quantitative analysis of TORC1 focus disassembly after dilution of stationary phase WT cells expressing GFP-Kog1 into either fresh complete synthetic medium containing rapamycin, BGW867 or drug vehicle (Mock). b, Quantitative analysis of TORC1 focus formation after treatment of late log-phase WT cells expressing of GFP-Kog1 with either rapamycin, BGW867 or drug vehicle (Mock). B and c, black diamonds represent mock treatment, grey squares rapamycin treatment (200 nM) and grey triangles BGW867 treatment (800 nM). Data are mean ± s.d. and represent three independent experiments (150-800 cells each). c, Representative confocal images of WT or Δ_gtr1_, Δ_gtr2_ cells expressing GFP-Tor1 or Tor1D330::3xGFP in exponential or stationary growth phases.
Figure 1. Glucose signals mediate TORC1 focus formation and activity in WT but not Δgtr1 Δgtr2 cells.
a/g, Confocal images of TORC1 (GFP-Kog1) in WT (a) and Δgtr1 Δgtr2 cells (g) following dilution of saturated cultures into fresh complete medium. b/h, Quantifications from a/g and a linear regression plot between the percentage of cells containing a focus and the optical density of the culture. c/i, Confocal images of TORC1 in WT (c) and Δgtr1 Δgtr2 (i) cells following glucose depletion and subsequent re-addition. d/j, Distribution of TORC1 focus sizes (number of GFP-Kog1 molecules) during glucose starvation / repletion from c/i. e/k, Western blot analysis of TORC1 activity (Sch9 Ser-758 phosphorylation) at the time points monitored in (c/i). Hog1 is a loading control. f/l, TORC1 activity is anti-correlated with focus formation in WT (f) but not Δgtr1 Δgtr2 (l) cells. Activity values are normalised to initial activity before glucose depletion. Data are mean ± s.d. and represent at least three independent biological experiments with at least 100 cells measured per experiment.
Figure 2. TORC1 foci present a regular cylindrical shape.
a-e, STORM imaging of GFP-Kog1 in Δgtr1 Δgtr2 cells. a upper row, Epi-fluorescence images from Alexa647-conjugated anti-GFP nanobody and GFP. lower left, Plot of an A647 localisation cluster obtained by STORM. lower right, Histogram of 771 STORM image clusterss containing more than 300 localisations plotted according to their eccentricity (ecc). b, Gallery of STORM cluster reconstructions classified by eccentricity and the presence of a central cavity. c, Averaged images of aligned clusters. d, Line profiles (arbitrary units) from images in c, across the circular cavity (red) and along the short axis of the rod (black). e, Correlation plot between the number of localisations and cluster eccentricity. f, Silver-stained SDS-PAGE of Kog1-TAP purification showing TORC1 subunits (and tobacco etch virus protease: TEV). g, Negative-stain electron micrograph of TORC1 oligomerised into a cylindrical structure. h, Cryo-micrographs of TORC1 tubules. Boxed areas in left micrograph contain aggregated tubules. The starred box is shown at higher magnification in the right micrograph. Inset: power spectrum of the selected tubule fragment showing layer lines corresponding to bessel order of n=1 (pitch distance) and n=2 at about 1/215 Å-1 and 1/107 Å-1, respectively. The red arrow denotes the highest layer line detected at about 1/60 Å-1.
Figure 3. TORC1 Oligomerised in Inhibited Domains (TOROIDs).
a, 3D TOROID reconstruction at 27 Å resolution. b, Outer and Inner views of the mTORC1 fitted into the TOROID map. c, Nine consecutive mTORC1 protomers fitted into the TOROID map. d, Overview of mTORC1 protomer contact interfaces. e-g, Zoom in of contact interfaces depicted in d (red, green and blue squares). e, Inter-coil interface between the Horn of TOR and the WD40 repeat of Raptor. Approximate locations, according to Baretic et al., of the N-terminus (N-term) and residue 330 of Tor1 (ǂ) are indicated; c.f. GFP-insertions in Figure 4. f, Intra-coil interfaces between adjacent CASPase domains of Raptor, and the armadillo repeat (ARM) of Raptor and the Bridge of TOR. g, Intra-coil interface between the FRB domain of TOR and ARM of Raptor. h, In a TOROID the ARM domain of Raptor occludes the TOR kinase cleft similarly to FKBP12-Rapamycin.
Figure 4. TORC1-TORC1 contact interfaces are necessary for TOROID formation and proper TORC1 regulation.
a, Confocal images of TORC1 in GFP-TOR1 Δ_tor2_ p_YPK2D239A_ or TOR1D330::3xGFP Δ_tor2_ p_YPK2D239A_ cells following glucose depletion and subsequent re-addition. b, Western blot analyses of TORC1 activity at the time points monitored in (a). c,TORC1 activity is anti-correlated with focus formation in GFP-TOR1 but not TOR1D330::3xGFP expressing cells. Data are mean ± s.d. and represent at least three independent biological experiments with at least 100 cells measured per experiment. d, Cartoon illustrating TORC1 regulation downstream of glucose signals via reversible helical assembly.
References
- Loewith R, et al. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Molecular cell. 2002;10:457–468. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases