1. mTOR Inhibition Restores Amino Acid Balance in Cells Dependent on Catabolism of Extracellular Protein 
Michel Nofal, Kevin Zhang, Seunghun Han, Joshua D. Rabinowitz 
Molecular Cell 
Volume 67, Issue 6, Pages e5 (September 2017)
DOI: /j.molcel
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2. Molecular Cell 2017 67, 936-946.e5DOI: (10.1016/j.molcel.2017.08.011)
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3. Figure 1
Isotope-Tracer Method for Measuring Rate of Amino Acid Release Due to Protein Scavenging
(A) Outline of isotope labeling scheme for measuring protein scavenging flux. Cells are grown for five doublings in medium with uniformly labeled amino acids without added BSA (13C-AA medium), before being switched to 13C-AA medium supplemented with 5% BSA. At this point, the appearance of unlabeled amino acids can be attributed to degradation of supplemented BSA, and this degradative flux can be computed.
(B) Flux model for amino acid metabolism. This model only applies to amino acids that cannot be synthesized de novo.
(C) Kinetic labeling data generated by application of the isotope labeling scheme described in (A) to K-RasG12D MEFs.
(D) Quantitation of rates of amino acid release from extracellular protein catabolism. Differences across amino acids reflect their relative abundances in BSA.
(E) Associated rate of albumin catabolism. Data for each individual amino acid from (D) are corrected for the number of appearances of that amino acid in BSA.
(F) iBMK-Ras cells show higher rates of protein scavenging than iBMK-parental and iBMK-Akt cells. Multiple estimates of each absolute protein scavenging flux (left) were supported by independent data from different amino acids, as in (E). Relative scavenging fluxes (right) were computed by averaging these independent estimates and normalizing to parental cells.
Error bars indicate 95% confidence intervals (n = 3).
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4. Figure 2
Cells Adapted to Growth on Extracellular Protein Maintain Active mTORC1 Signaling
(A) KRPC cells continuously cultured in leucine-free medium supplemented with 5% BSA for 10 or 100 generations show enhanced proliferation in this condition.
(B) Comparison of protein scavenging fluxes in parental and adapted KRPC cells cultured in amino acid-replete medium. Multiple estimates of each absolute protein scavenging flux (left) were supported by independent data from different amino acids, as in Figure 1E. Relative scavenging fluxes (right) were computed by averaging these independent estimates, then normalizing to parental cells.
(C) Comparison of mTORC1 activity in parental and adapted KRPC cells cultured in amino acid-replete medium, as measured by phosphorylation of S6K1 (T389), S6 (S240/244), and 4E-BP1 (S37/46). Protein was extracted from cells grown in medium with or without Torin1 (1,000 nM) for 24 hr.
Error bars indicate 95% confidence intervals (n = 3).
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5. Figure 3
Moderate Inhibition of mTORC1 Facilitates Growth on Extracellular Protein, but Excessive Inhibition Slows It
Parental KRPC cells (A) and adapted KRPC (KRPCA) cells (B) were cultured in amino acid-replete and amino acid-deficient media and in varying Torin1 concentrations. Cell growth was measured using a viability assay based on resazurin reduction after 24 hr (replete medium) or 72 hr (amino acid-deficient media). Error bars indicate 95% confidence intervals (n = 3).
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6. Figure 4
Amino Acid Deprivation Induces Extracellular Protein Catabolism Despite Persistent mTORC1 Activity
(A) Torin1 treatment increases protein scavenging in KRPCA cells and K-RasG12D MEFs growing in amino acid-replete medium. Multiple estimates of each absolute scavenging flux (left) were supported by independent data from different amino acids, as in Figure 1E. Relative scavenging fluxes (right) were computed by averaging these independent estimates, then normalizing to the flux of untreated cells.
(B) Amino acid deprivation induces extracellular protein catabolism in KRPCA cells or K-RasG12D MEFs in the presence or absence of Torin1. Relative scavenging fluxes were computed as in (A).
(C) mTORC1 activity persists in KRPCA cells and K-RasG12D MEFs cultured in amino acid-deficient media, as shown by phosphorylation of S6K1 (T389), S6 (S240/244), and 4E-BP1 (S37/46). Cellular protein was extracted after 24 hr in the specified condition. Torin1 was added at 1,000 nM.
Error bars indicate 95% confidence intervals (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p <
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7. Figure 5
mTOR Inhibition Moderately Enhances Overall DQ-BSA Fluorescence While Substantially Increasing Bright Punctate Regions
(A) Catabolism of extracellular protein in K-RasG12D MEFs in the absence or presence of 250 nM Torin1, as measured by DQ-BSA fluorescence. Images were taken after 6 hr.
(B) Histograms of pixel intensities of DQ-BSA fluorescence from untreated or Torin1-treated cells.
(C) Total fluorescence per cell in untreated and Torin1-treated cells.
(D) The sum of pixel intensities within the ranges specified.
(E) Total thresholded fluorescence per cell in Torin1-treated relative to untreated cells. For each threshold, pixel intensities below the threshold were set to zero, and the remaining pixel intensities were summed. The effect size is equal to the ratio of the sums of these pixels in Torin1-treated versus untreated cells.
(F) DQ-BSA images in (A) discretized by applying the same color to all pixels within each range in (D).
Error bars indicate 95% confidence intervals (n = 5). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p <
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8. Figure 6
Translation Inhibition of Amino Acid-Deprived Cells Relieves Amino Acid Depletion and Rescues Cell Viability
(A) K-RasG12D MEFs cultured in leucine-free medium with or without 5% BSA for 6 hr accumulate ATF4 and CHOP, whose expression is induced in response to depletion of amino acid pools (i.e., the integrated stress response). mTOR inhibition prevents ATF4 and CHOP accumulation. Note that the Torin1 concentration required to inhibit mTORC1 is higher in the presence of 5% BSA, presumably due to protein binding of the drug.
(B and C) Cell viability of K-RasG12D MEFs cultured in leucine-free medium is rescued by either Torin1 (B) or harringtonin (C). Apoptosis and cell death after 48 hr in leucine-free medium ± 5% BSA were measured using flow cytometry after incubation with CellEvent Caspase-3/7 Green Detection Reagent and SYTOX AADvanced Dead Cell Stain. Error bars indicate 95% confidence intervals (n = 3). p values were calculated using the sums of the apoptotic and dead cell fractions. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p <
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9. Figure 7
Optimal Cell Growth Dependent on Extracellular Proteins as Nutrients Requires Intermediate mTORC1 Activity
Excessive mTORC1 activity results in amino acid shortage. Insufficient mTORC1 activity results in amino acid excess. At intermediate mTORC1 activity, amino acid supply matches demand, amino balance is achieved, and growth is maximized.
Molecular Cell  , e5DOI: ( /j.molcel )
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