Branched-chain amino acid synthesis is coupled to TOR activation early in the cell cycle in yeast

Abstract

How cells coordinate their metabolism with division determines the rate of cell proliferation. Dynamic patterns of metabolite synthesis during the cell cycle are unexplored. We report the first isotope tracing analysis in synchronous, growing budding yeast cells. Synthesis of leucine, a branched-chain amino acid (BCAA), increases through the G1 phase of the cell cycle, peaking later during DNA replication. Cells lacking Bat1, a mitochondrial aminotransferase that synthesizes BCAAs, grow slower, are smaller, and are delayed in the G1 phase, phenocopying cells in which the growth-promoting kinase complex TORC1 is moderately inhibited. Loss of Bat1 lowers the levels of BCAAs and reduces TORC1 activity. Exogenous provision of valine and, to a lesser extent, leucine to cells lacking Bat1 promotes cell division. Valine addition also increases TORC1 activity. In wild-type cells, TORC1 activity is dynamic in the cell cycle, starting low in early G1 but increasing later in the cell cycle. These results suggest a link between BCAA synthesis from glucose to TORC1 activation in the G1 phase of the cell cycle.

Keywords: BCAA; BCAT; TORC1; cell size; isotope tracing.

Figure 1. Overview of the approach to obtain samples for metabolic flux analysis in highly synchronous, dividing, budding yeast cells

1. A

   For each experiment, early G1 daughter cells of a prototrophic strain (CEN.PK; see Materials and Methods) were obtained by centrifugal elutriation. The elutriated culture was split into eight aliquots and cultured for a varying amount of time, from 20 to 160 min, in minimal, [U‐¹²C]‐glucose medium. They were then transferred to a medium with [U‐¹³C]‐glucose for 20 min (pulse) and then incubated for another 20 min in [U‐¹²C]‐glucose medium (chase). Metabolite extracts from these cells were analyzed by mass spectrometry. Five such independent experiments were performed. The figure was generated with Biorender.com.

2. B

   Boxplots showing the cell size (y‐axis) over time (x‐axis) of all the samples as they progressed in the cell cycle.

3. C

   Boxplots showing the percentage of budded cells (y‐axis) from the same samples shown in (B). The boxplot graphs were generated with R language functions. Each box is drawn from the first to the third quartile, with a horizontal line drawn in the middle to denote the median. The whiskers show the interquartile range (IQR), and they were drawn at 1.5xIQR. The replicates were all biological ones.

Source data are available online for this figure.

Figure 2. Relative abundance of intracellular metabolite isotopomers in the cell cycle

Each plot shows the relative mass isotopomer distribution (MID) values for metabolite species detected from cell extracts in each of the five independent experiments shown in Fig 1. Each value was divided by the average value of the entire time series (x‐axis) for that species from the same experiment, and Log2‐transformed (y‐axis). Loess curves and the std errors at a 0.95 level are shown. The Log2(relative abundance) values of pyruvate(M1) and leucine(M6) showed the most significant changes (P < 0.1, see Dataset EV1/Sheet2) in cells across the cell cycle. Source data are available online for this figure.

Figure EV1. Steady‐state amino acid levels in the cell cycle

Intracellular amino acid levels were measured as described in Materials and Methods from synchronous, elutriated cultures in SMM media. The relative abundance of each amino acid is on the y‐axis. Source data are available online for this figure.

Figure 3. BCAA supplementation suppresses the growth defect of bat1Δ mutants

1. A

   Diagram of the reactions leading to BCAAs from the corresponding alpha‐keto (α‐k) acids, catalyzed by Bat1,2p. A more detailed diagram leading to the of valine (M5) and leucine (M6) isotopomers is in Fig EV1.

2. B

   The indicated strains (all in the prototrophic CEN.PK background; see Materials and Methods) were spotted at 10‐fold serial dilutions on solid Synthetic Minimal Medium (SMM) agar plates. Exogenous amino acids were added at 1 mM final concentration, as indicated in each case. The plates were incubated at 30°C and photographed after 3‐days.

3. C

   DNA content profiles of BAT1 and bat1Δ cells from asynchronous cultures, in SMM medium. Where indicated, exogenous amino acids were added at 1 mM final concentration. On the x‐axis of the histograms is fluorescence per cell, while on the y‐axis is the cell number. Peaks corresponding to cells in G1 with unreplicated (1 N) and cells in G2 and M phases with fully replicated (2 N) DNA are indicated. The percentage of cells with G1 DNA content (%G1) from 3 independent measurements is shown in each case (mean and sd).

Source data are available online for this figure.

Figure EV2. Alpha‐keto acid supplementation does not suppress the growth defect of bat1Δ mutants

The experiment was done as in Fig 3. Exogenous alpha‐keto acids were added at 1 mM final concentration, as indicated in each case. Source data are available online for this figure.

Figure EV3. Amino acid levels in BAT1 ⁺ and bat1Δ cells

Intracellular amino acid levels were measured as described in Materials and Methods and Fig EV1, from exponentially growing cultures in SMM media. The fraction of each amino acid is on the y‐axis. The four amino acids (Gly, Ile, Val, Ala) with significantly altered fractional abundance (> 2‐fold, P < 0.05; from six independent samples in each case) are indicated. The boxplot graphs were generated with R language functions. Each box is drawn from the first to the third quartile, with a horizontal line drawn in the middle to denote the median. The whiskers show the interquartile range (IQR), and they were drawn at 1.5xIQR. The replicates were all biological ones. Source data are available online for this figure.

Figure EV4. Changes in primary and biogenic amine metabolites in cells lacking Bat1

Metabolites whose levels changed were identified from the magnitude of the difference (x‐axis; Log2‐fold change in BAT1 ⁺: bat1Δ cells) and statistical significance (y‐axis), indicated by the red lines. The analytical and statistical approaches are described in Materials and Methods. The labels indicate selected metabolites. Source data are available online for this figure.

Figure EV5. Cells lacking Bat1 are smaller, increase in size slower, and are delayed in the G1 phase of the cell cycle

1. A

   Boxplots showing the size of the cells (in fL). The measurements were taken from asynchronous cultures in synthetic minimal media (SMM) without amino acid supplementation. The boxplot graphs were generated with R language functions. Each box is drawn from the first to the third quartile, with a horizontal line drawn in the middle to denote the median. The whiskers show the interquartile range (IQR), and they were drawn at 1.5xIQR. The replicates were all biological ones.

2. B

   Plots of the percentage of budded cells (y‐axis) as a function of size (x‐axis). The measurements were from daughter cells of the indicated strains, obtained by centrifugal elutriation, progressing in the cell cycle in SMM medium. Loess curves and the std errors at a 0.95 level are shown.

3. C

   From the same experiments as in (B), the specific rate of the increase in size (k) was calculated, from the slope of the regression lines plotting the Ln‐transformed cell size values (y‐axis) against time (x‐axis).

Source data are available online for this figure.

Figure 4. Bat1 is functionally linked to TORC1 activation in early G1

1. A

   The indicated strains were spotted at 10‐fold serial dilutions on solid Synthetic Minimal Medium (SMM) agar plates. Rapamycin was added at 30 ng/ml, as shown in each case. The plates were incubated at 30°C and photographed after 5 and 7 days, as indicated.

2. B

   Immunoblots of total cell extracts from asynchronous BAT1 and bat1Δ cells, from four independent experiments. The signal from Pgk1 (α‐Pgk1) is shown on the blot at the bottom, and from phosphorylated Rps6 (α‐Rps6‐P) is on the blot above, indicated for two exposures (2 min, top; 20 s, middle).

3. C

   On the left are immunoblots from wild‐type (CEN.PK) cells treated (+), or not (−), with rapamycin (Rapa) at 200 ng/ml for 1 h before cell extract preparation. On the right are immunoblots of wild‐type (CEN.PK) cell extracts. The extracts were treated (+), or not (−), with λ‐phosphatase for 1 h (see Materials and Methods). The levels of phosphorylated Rps6 (α‐Rps6‐P) and Pgk1 (α‐Pgk1) are shown in each case.

4. D

   Exogenous addition of valine leads to sustained activation of TORC1 and phosphorylation of Rps6 in cells lacking Bat1. Wild type (BAT1 ⁺) and bat1∆ (two independent isolates, #4, and #5) strains were grown overnight in minimal (SMM) medium, diluted to 1E+06 cells/ml in fresh SMM media containing the indicated amino acid (at 1 mM), and harvested when they reached 5E+06 cells/ml. The levels of phosphorylated Rps6 (α‐Rps6‐P) and Pgk1 (α‐Pgk1) are shown in immunoblots from total cell extracts in each case. The relative levels of Rps6‐P/Pgk1 are shown in each case at the bottom. The boxplot graphs were generated with R language functions. Each box is drawn from the first to the third quartile, with a horizontal line drawn in the middle to denote the median. The whiskers show the interquartile range (IQR), and they were drawn at 1.5xIQR. The replicates were all biological ones.

Source data are available online for this figure.

Figure 5. TORC1 activity increases as cells progress in the cell cycle

1. Immunoblots of total cell extracts from synchronous, elutriated wild‐type (CEN.PK) cells in minimal (SMM) medium with exogenous Leu or Val added at 1 mM immediately after elutriation. At the top, the percent of budded cells (%B), cell size (in fL), and time (in min) are indicated. The levels of phosphorylated Rps6 (α‐Rps6‐P) and Pgk1 (α‐Pgk1) are shown in each case.

2. Quantification of the levels of phosphorylated Rps6 and Pgk1 from independent experiments done as in A. The relative levels of each protein across the cell cycle is shown on the y‐axis, as a function of cell size (x‐axis). Loess curves and the std errors at a 0.95 level are shown.

3. Schematic of a possible model to explain our findings, linking BCAA synthesis to TORC1 activation early in the cell cycle.

Source data are available online for this figure.