A deubiquitinase deletion screen identifies Ubp3 as a regulator of glucose-mediated mitochondrial repression

A) Schematic depicting glucose-mediated mitochondrial repression (Crabtree effect).

B) Schematic describing the screen with a yeast DUB KO library to identify regulators of Crabtree effect.

C) Identifying DUB knockouts with altered mitochondrial potential. Heat map shows relative mitochondrial membrane potential of 19 deubiquitinase deletions in high glucose, from 2 biological replicates. Also see Figure 1-figure supplements 1A, B.

D) The deubiquitinase activity of Ubp3 and repression of mitochondrial membrane potential. WT, ubp3Δ, and Ubp3C469A were grown inhigh glucose and relative mitochondrial membrane potential was measured. Data represent mean ± SD from three biological replicates (n=3). Also see Figure 1-figure supplement 1 D.

E) Effect of loss of Ubp3 on ETC complex IV subunit Cox2. WT and ubp3Δ were grown in high glucose, and Cox2 was measured (western blot using an anti-Cox2 antibody). A representative blot (out of 3 biological replicates, n=3) and their quantifications are shown. Data represent mean ± SD.

F) Basal oxygen consumption rate (OCR) in high glucose in ubp3Δ. WT and ubp3Δ were grown in high glucose, and OCR was measured. Basal OCR corresponding to ∼3 × 10^5 cells, from two independent experiments (n=2), normalized to the OD600 is shown. Bar graph representations are shown in the inset. Data represent mean ± SD.

G) Total ATP levels in ubp3Δ and WT. WT and ubp3Δ were grown in high glucose, and total ATP were measured. Data represent mean ± SD from three biological replicates (n=3).

H) Dependence of ubp3Δ on mitochondrial ATP. WT and ubp3Δ cells were grown in high glucose, and treated with 1 mM sodium azide for 45 minutes. Total ATP levels in sodium azide treated and untreated cells were measured. Data represent mean ± SD (n=3).

I) Requirement for mitochondrial respiration in high glucose in ubp3Δ. A growth curve of WT and ubp3Δ in high glucose in the presence of OXPHOS uncoupler FCCP (10 µM), and serial dilution growth assay in high glucose in the presence/absence of sodium azide (1 mM) are shown. Data represent mean ± SD (n=2). Also see Figure 1-figure supplement 1 H, I.

Data information: **p<0.01, ***p<0.001.

Key glycolytic enzymes decrease and glucose flux is rerouted in ubp3Δ cells

A) A schematic illustrating directions of glucose-6-phosphate (G6) flux in cells. Glucose is converted to G6P, a precursor for trehalose, the pentose phosphate pathway (PPP), and glycolysis.

B) Effect of loss of Ubp3 on key glycolytic enzymes. WT and ubp3Δ were grown in high glucose and the Pfk1, Tdh2, and Tdh3 levels were measured by western blot using an anti-FLAG antibody. A representative blot (out of three biological replicates, n=3) and their quantification are shown. Data represent mean ± SD. Also see Figure 2-figure supplement 1A.

C) Steady-state metabolite amounts in WT and ubp3Δ in high glucose. Relative steady-state levels of trehalose, major glycolytic, and PPP intermediates were estimated in WT and ubp3Δ. Data represent mean ± SD from three biological replicates (n=3). Also see Appendix Table S3.

D) Relative glycolytic and trehalose synthesis flux in WT and ubp3Δ. Relative 13C-label incorporation into trehalose and glycolytic intermediates, after a pulse of 1% 13C6 glucose is shown. Data represent mean ± SD from three biological replicates (n=3). Also see Appendix Table S3, Figure 2-figure supplement 1 D, E.

E) Ethanol production in ubp3Δ. WT and ubp3Δ were grown in high glucose and ethanol in the media was measured. Data represent mean ± SD from three biological replicates (n=3).

F) Relative rate of ethanol production in WT vs ubp3Δ. WT and ubp3Δ were grown in high glucose (to OD600 ∼ 0.6), equal numbers of cells were shifted to fresh medium (high glucose) and ethanol concentration in the medium was measured temporally. Data represent mean ± SD from three biological replicates (n=3)

Data information: *p<0.05, **p<0.01, ***p<0.001.

Rerouted glucose flux results in phosphate (Pi) accumulation

A) Changes in protein levels in ubp3Δ (dataset from Isasa et al., 2015). ubp3Δ cells have an increase in proteins involved in mitochondrial respiration and decrease in proteins involved in glucose and phosphate metabolism.

B) Schematic showing maintenance of Pi balance during glycolysis. Trehalose synthesis from G6P releases Pi, and the conversion of G3P to 1,3BPG by GAPDH consumes Pi. In ubp3Δ, trehalose biosynthesis (which releases Pi) increases. ubp3Δ have decreased GAPDH, which will decrease Pi consumption. This increase in Pi release along with decreased Pi consumption could increase cytosolic Pi.

C) Intracellular Pi levels in WT and ubp3Δ. WT and ubp3Δ were grown in high glucose and the total free phosphate (Pi) levels were estimated. WT in high Pi (2% glucose, 10mM Pi) was a positive control. Data represent mean ± SD from three biological replicates (n=3). Also see Figure 3-figure supplement 1A.

D) Pho regulon responses in WT and ubp3Δ. Protein levels of Pho84-FLAG and Pho12-FLAG were compared between WT grown in high glucose and in high Pi, ubp3Δ in high glucose with or without a shift to a no-Pi medium for one hour, by western blot. A representative blot (out of three biological replicates, n=3) and their quantifications are shown. Data represent mean ± SD.

E) Contribution of trehalose synthesis as a Pi source. WT, tps2Δ, ubp3Δ, and ubp3Δtps2Δ were grown in high glucose and the total Pi levels were estimated. Data represent mean ± SD from three biological replicates (n=3). Also see Figure 3-figure supplement 1B.

F) Loss of GAPDH isozymes Tdh2 and Tdh3 and effect on Pi. WT, ubp3Δ, and tdh2Δtdh3Δ were grown in high glucose and total Pi was estimated. Data represent mean ± SD from three biological replicates (n=3).

G) Pi levels in ubp3Δ and tdh2Δtdh3Δ cells in ethanol medium. WT, ubp3Δ, and tdh2Δtdh3Δ cells were grown in ethanol medium and the total Pi levels were estimated. Data represent mean ± SD from three biological replicates (n=3).

Data information: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Mitochondrial Pi availability correlates with mitochondrial activity in ubp3Δ

A) A hypothetical mechanism of cytosolic free Pi controling mitochondrial activity by regulating mitochondrial Pi availability.

B) Cytosolic and mitochondrial Pi amounts in WT vs ubp3Δ. The cytosolic fraction was isolated by centrifugation (see SI appendix), and in separate experiments, mitochondria were isolated by immunoprecipitation from WT and ubp3Δ and mitochondrial Pi estimated. (i) Cytosolic Pi levels (relative as well as absolute) and (ii) mitochondrial Pi levels (normalised to Idh1) are shown. Data represent mean ± SD from three biological replicates (n=3) respectively for the cytosolic and mitochondrial measurements. Also see Figure 4-figure supplement 1A, B.

C) Cox2 protein in tdh2Δtdh3Δ. WT, ubp3Δ, and tdh2Δtdh3Δ were grown in high glucose and Cox2 protein was estimated. A representative blot (out of three biological replicates, n=3) and their quantifications are shown. Data represent mean ± SD.

D) Basal OCR levels in tdh2Δtdh3Δ. WT, ubp3Δ, and tdh2Δtdh3Δ were grown in high glucose and basal OCR was measured from two independent experiments (n=2). Data represent mean ± SD. Also see Figure 4-figure supplement 1C.

E) Comparative Pi amounts and Cox2 levels in ubp3Δ, tdh2Δtdh3Δ, WT cells. WT cells were grown in high glucose, ubp3Δ and tdh2Δtdh3Δ were grown in high glucose and low Pi, and Cox2 protein was estimated. A representative blot (out of three biological replicates, n=3) and their quantifications are shown. Data represent mean ± SD. Also see figure Figure 4-figure supplement 1D, F.

F) Pi amounts and basal OCR in ubp3Δ and tdh2Δtdh3Δ vs WT cells. WT cells were grown in high glucose, ubp3Δ and tdh2Δtdh3Δ were grown in high glucose and low Pi, and basal OCR was measured from three independent experiments (n=3). Data represent mean ± SD.

G) Effect of loss of mitochondrial Pi transporter Mir1 on Cox2protein. WT, ubp3Δ, mir1Δ, and mir1Δubp3Δ were grown in high glucose and Cox2 amounts compared. A representative blot (out of three biological replicates, n=3) and their quantifications are shown. Data represent mean ± SD.

H) Relationship of mitochondrial Pi transport and basal OCR in WT vs ubp3Δ. WT, ubp3Δ, mir1Δ, and mir1Δubp3Δ cells were grown in high glucose and basal OCR was measured from two independent experiments (n=2). Data represent mean ± SD.

Data information: *p<0.05, **p<0.01, ****p<0.0001.

Mitochondrial Pi availability constrains mitochondrial activity under high glucose

A) Relationship of mitochondrial Pi transport and respiration after glucose removal. WT and mir1Δ cells were cultured in high (2%) glucose and shifted to low (0.1%) glucose for 1 hour. The normalized basal OCR, from two independent experiments (n=2) are shown. Data represent mean ± SD.

B) Requirement of mitochondrial Pi transport for switch to respiration upon glycolytic inhibition by 2DG. WT and mir1Δ cells were cultured in high glucose and treated with or without 0.25% 2DG for 1 hour. Basal OCR was measured from two independent experiments (n=2). Data represent mean ± SD. Also see Figure 5-figure supplement 1A.

C) Glucose-dependent regulation of Mir1. Cells (with Mir1-HA) were grown in high glucose and shifted to low glucose (0.1% glucose) for 1 hour, and Mir1 levels compared. A representative blot (out of three biological replicates, n=3) and their quantifications are shown. Data represent mean ± SD. Also see Figure 5-figure supplement 1B.

D) Increasing Pi concentrations and mitochondrial activity in isolated mitochondria. Mitochondria were isolated from WT cells grown in high glucose, incubated with 1 mM pyruvate, 1 mM malate, 0.5 mM ADP and 0-50 mM KH2PO4. The mitochondrial activity was estimated by mitotracker fluorescence intensity, and intensities relative to the sample with 0 mM KH2PO4 is shown. Data represent mean ± SD from three biological replicates (n=3).

E) Effect of overexpressing Mir1 on Cox2 protein. WT (containing empty vector) and Mir1 overexpressing (Mir1OE) cells were grown in high glucose and Cox2 levels were estimated. A representative blot (out of three biological replicates, n=3) and their quantifications are shown. Data represent mean ± SD. Also see figure S5F.

F) Effect of overexpressing Mir1 on basal OCR. The basal OCR in WT (containing empty vector) and Mir1OE in high glucose was measured from three independent experiments (n=3). Data represent mean ± SD.

Data information: *p<0.05, **p<0.01, ***p<0.001.

Repression of mitochondrial respiration via Pi budgeting is conserved in Ubp3 mutants across diverse yeast genetic backgrounds

A) Effect of loss of Ubp3 on mitochondrial membrane potential in different yeast strains. WT and ubp3Δ cells (in CEN.PK, BY4742, W303 and Σ1278 strains of S. cerevisiae) were grown in high glucose and relative mitochondrial membrane potential was measured. Data represent mean ± SD from three biological replicates (n=3).

B) Effect of loss of Ubp3 on ETC complex IV subunit Cox2. WT and ubp3Δ (in BY4742, W303 and Σ1278 strains of S. cerevisiae) were grown in high glucose, and Cox2 was measured. A representative blot (out of 3 biological replicates, n=3) and their quantifications are shown. Data represent mean ± SD.

C) Intracellular Pi levels in WT and ubp3Δ in W303 strain background. WT and ubp3Δ (in W303 strain background) were grown in high glucose and the total free phosphate (Pi) levels were estimated. Data represent mean ± SD from three biological replicates (n=3).

D) Effect of low Pi on the basal OCR in WT and ubp3Δ cells in W303 strain background. WT cells were grown in high glucose and ubp3Δ were grown in high glucose and low Pi, and basal OCR was measured. Data represent mean ± SD (n=3).

E) Requirement of mitochondrial Pi transport for growth after 2DG treatment. Shown are serial dilution growth assays in high glucose in the presence and absence of 0.1% 2DG, using WT and mir1Δ cells. The results after 40hrs incubation/30°C are shown.

F) A model illustrating how mitochondrial Pi availability controls mitochondrial activity. In high glucose, the decreased Pi due to high Pi consumption in glycolysis, along with the glucose-mediated repression of mitochondrial Pi transporters, decreases mitochondrial Pi availability. This reduces mitochondrial activity. In low glucose, increased mitochondrial Pi transporters and lower glycolytic flux increases mitochondrial Pi, leading to enhanced mitochondrial activity. In ubp3Δ cells in high glucose, high trehalose synthesis and lower glycolytic flux results in an increase in Pi. This increases mitochondrial Pi availability and thereby the mitochondrial activity.

Data information: *p<0.05, **p<0.01, ***p<0.001.

DUB screen details and further characterization of Ubp3 functions

A) A list of the known/identified S. cerevisiae deubiquitinase enzymes is shown. The DUBs which were used in this study, and the mitotracker intensity of two biological replicates (n=2) relative to WT are shown.

B) WT cells grown in a respiratory, ethanol medium and mitotracker fluorescence intensity compared to glucose medium. WT cells were grown inhigh glucose medium (2% glucose) and ethanol medium (2% ethanol), and the mitochondrial membrane potential was measured. Data from two independent experiments (n=2) is shown. Data represent mean ± SD.

C) Representative images of WT and ubp3Δ cells treated with mitotracker red CMXROS.

D) Schematic representation of the conserved domains of Ubp3 and its mammalian ortholog Usp10. The catalytic cysteine of Usp10 and Ubp3 is highlighted.

E) The catalytically inactive mutant of Ubp3, Ubp3C469A does not have altered Ubp3 protein levels. WT and Ubp3C469A cells containing endogenous Ubp3 tagged with 3xFLAG (C terminus) were grown in high glucose and the protein levels of Ubp3 were measured. A representative blot (out of three biological replicates, n=3) and their quantification are shown. Data represent mean ± SD.

F) The total mitochondrial volume does not change in ubp3Δ cells. WT and ubp3Δ cells with mitochondria targeted mNeon green were imaged, and the total mitochondrial volume per cell (n=104 for WT, n=107 for ubp3Δ) was calculated. A representative image and quantification are shown. Data represent mean ± SD.

G) Tom70 protein amounts do not change in ubp3Δ cells. WT and ubp3Δ cells containing endogenous Tom70 tagged at the C terminus with a 3xFLAG epitope were grown in high glucose and the protein levels of Tom70 were measured by western blot. A representative blot (out of three biological replicates, n=3) and their quantification are shown. Data represent mean ± SD.

H) Dependence of ubp3Δ on mitochondrial respiration. WT and ubp3Δ cells were grown in high glucose with or without mitochondrial inhibitor oligomycin (25 µM), and the OD600 was measured at different time points. Data represent mean ± SD (n=2).

I) Dependence of ubp3Δ on functional mitochondrial respiration. Ubp3 deletions were done in respiration defective strains -Rho0, cox2-62, atp1Δ and atp10Δ, and serial dilution growth assay was done in high glucose. The results after 24 hours of incubation at 30°C are shown.

J) Changes in global ubiquitination state in WT and ubp3Δ. WT and ubp3Δ cells were grown in high glucose with or without heat stress (42°C/1 hour), and the changes in global total ubiquitination was estimated by western blotting using an anti-ubiquitin antibody.

Further estimates of glycolytic enzymes and flux

A) Loss of Ubp3 does not change the protein levels of enolase isozymes Eno1 and Eno2. WT and ubp3Δ cells containing Eno1 and Eno2 C terminus endogenously tagged with a 3x FLAG epitope, were grown in high glucose and protein amounts were estimated. A representative blot (out of three biological replicates, n=3) is shown in the lower panel, quantifications shown in the upper panel. Data represent mean ± SD.

B) The decreased levels of glycolytic enzymes in ubp3Δ cells is not because of proteasomal degradation. WT and ubp3Δ were grown in high glucose and ubp3Δ cells were treated with or without MG132 (100 μM) for 30 minutes. Pfk1, Tdh2, and Tdh3 levels were measured by western blot using an anti-FLAG antibody. A representative blot (out of three biological replicates, n=3) and their quantification are shown. Data represent mean ± SD.

C) Loss of Ubp3 results in decreased transcription of PFK1, TDH2 and TDH3 genes. WT, ubp3Δ and Ubp3C469A cells were grown in high glucose and the mRNA levels of PFK1, TDH2 and TDH3 were analysed by RT-qPCR. The fold changes in mRNA levels are shown. Data represent mean ± SD.

D) Steady-state pyruvate levels in WT and ubp3Δ in high glucose. Data represent mean ± SD from three biological replicates (n=3). Also see Appendix Table S3.

E) Schematic showing the experimental design for measuring 13C label incorporation into glycolytic intermediates and trehalose using a 13C6 glucose-pulse.

F) Changes in 13C label incorporation into glycolytic intermediates and trehalose with time, and linearity of label incorporation after a pulse of 1% 13C6 glucose is shown.

Estimation of steady state levels and flux of TCA cycle intermediates

A) Steady-state TCA metabolite amounts in WT and ubp3Δ in high glucose. Relative steady-state levels of TCA cycle intermediates were estimated in WT and ubp3Δ. Data represent mean ± SD from three biological replicates (n=3). Also see Appendix Table S3.

B) Schematic showing the experimental design for measuring 13C label incorporation into TCA cycle intermediates using a 13C6 glucose-pulse.

C) Changes in 13C label incorporation into TCA cycle intermediates with time, and linearity of label incorporation after a pulse of 1% 13C6 glucose is shown.

D) Relative TCA cycle flux in WT and ubp3Δ. Relative 13C-label incorporation into TCA cycle intermediates, after a pulse of 1% 13C6 glucose is shown. Data represent mean ± SD from three biological replicates (n=3). Also see Appendix Table S3.

Data information: *p<0.05

Comparisons of phosphate, ethanol and other metabolites in WT, ubp3Δ and GAPDH mutants

A) Loss of Ubp3 deubiquitinase activity and Pi levels. WT, ubp3Δ, and Ubp3C469A cells were grown in high glucose and the total free phosphate (Pi) levels were estimated. Data from two independent experiments (n=2) is shown. Data represent mean ± SD. B) Trehalose levels in WT and ubp3Δ cells in the absence of Tps2. tps2Δ and ubp3Δtps2Δ cells were grown in high glucose, and the trehalose levels were estimated using targeted LC-MS/MS. Data represent mean ± SD from three biological replicates (n=3).

C) Loss of GAPDH isozymes Tdh2 and Tdh3 and ethanol production. WT and tdh2Δtdh3Δ cells were grown in high glucose and the ethanol concentration in the medium was measured. Data are represented as mean ± SD from three biological replicates (n=3). Note: The WT shown here is same as the WT in Figure 2E, the ethanol assays in Figure 2E and Figure S3C were done together and a common WT control was used.

D) Steady state levels of glycolytic intermediates: tdh2Δtdh3Δ cells have unaltered steady state levels of G6P/F6P, increased levels of F1,6 BP and G3P and decreased levels of 3PG and PEP. WT and tdh2Δtdh3Δ cells were grown in high glucose and the steady state amounts of glycolytic intermediates were estimated using targeted LC-MS/MS. Data represent mean ± SD from three biological replicates (n=3). Also see Appendix Table S3.

E) Trehalose levels in tdh2Δtdh3Δ cells. WT and tdh2Δtdh3Δ cells were grown in high glucose and trehalose amounts were estimated using targeted LC-MS/MS. Data represent mean ± SD from three biological replicates (n=3).

Data information: *p<0.05, **p<0.01.

Mitochondrial Pi estimation characterizations and correlations of mitochondrial activity with Pi availability

A) Isolation of cytosolic fraction by centrifugation and lack of vacuolar contamination in mitochondria isolated by immunoprecipitation. Cytosolic fraction was isolated from WT cells grown in high glucose with or without excess Pi and ubp3Δ cells. Eno1 was used as a cytosolic marker and Cox2 as mitochondrial marker to ensure purity of the cytosolic fractions. Mitochondria were isolated from WT cells grown in high glucose and the protein levels of Tom20, Cox2, Idh1 and Vph1 and were measured in cell lysate and immunoprecipitated mitochondria by western blot.

B) Idh1 levels in ubp3Δ cells and WT cells grown in high Pi. WT cells were grown in high glucose, or high glucose and high Pi medium (10mM Pi) and ubp3Δ cells were grown in high glucose, and the Idh1 protein levels were estimated by western blot. A representative blot (out of three biological replicates, n=3) is shown in the lower panel. Quantification is shown in the upper panel. Data represent mean ± SD.

C) Mitochondrial membrane potential in tdh2Δtdh3Δ cells. WT, ubp3Δ, and tdh2Δtdh3Δ cells were grown inhigh glucose and the mitochondrial membrane potential was measured. Data represent mean ± SD from three biological replicates (n=3).

D) Pi levels in ubp3Δ cells grown in low Pi (1mM Pi) vs WT cells. WT cells were grown in high glucose and ubp3Δ cells were grown in high glucose or high glucose-low Pi, and the total Pi levels were estimated. Data represent mean ± SD from three biological replicates (n=3).

E) Ethanol production in ubp3Δ cells grown in a low Pi medium. WT cells were grown in high glucose and ubp3Δ cells were grown in high glucose or high glucose-low Pi, and the ethanol concentration in the medium was measured. Data represent mean ± SD from three biological replicates (n=3).

F) Pi amounts and mitochondrial membrane potential in WT, ubp3Δ, and tdh2Δtdh3Δ cells. The cells were grown in high glucose or high glucose-low Pi, and the mitochondrial membrane potential was measured. Data from two independent experiments (n=2) is shown. Data represent mean ± SD.

G) The Pi mediated change in basal OCR in ubp3Δ is dependent on mitochondrial respiration. WT and Rho0 cells were grown in high glucose, Rho0/ubp3Δ were grown in high glucose and low Pi, and basal OCR was measured from three independent experiments (n=3). Data represent mean ± SD.

H) The protein levels of mitochondrial Pi transporters Mir1 and Pic2 in ubp3Δ cells. WT and ubp3Δ cells containing endogenously tagged Mir1 and Pic2 at their C terminus with a 6x HA epitope tag were grown in high glucose and Mir1 and Pic2 proteins were measured by western blot. A representative blot (out of three biological replicates, n=3) is shown in the lower panel. Quantification is shown in the upper panel. Data represent mean ± SD.

Data information: *p<0.05, **p<0.01, ***p<0.001.

Mitochondrial phosphate and pyruvate transport relationships with mitochondrial activity

A) Requirement of Mir1 for switching to increased mitochondrial activity upon 2-deoxyglucose (2-DG) treatment. WT and mir1Δ cells were grown inhigh glucose and treated with 0.1% and 0.25% 2-DG for one hour. The mitochondrial membrane potential was measured from two independent experiments (n=2). Data represent mean ± SD.

B) Mir1 protein levels and glucose repression. Cells containing Mir1 endogenously tagged at the C terminus with a 6x HA epitope tag were grown in high glucose or a respiratory medium (2% ethanol). Mir1 protein was estimated by western blot. A representative blot (out of three biological replicates, n=3) is shown in the lower panel. Quantification is shown in the upper panel. Data represent mean ± SD.

C) Effect of supplementing Pi in the medium on cytosolic and mitochondrial Pi. WT cells were grown in high glucose, and high glucose-high Pi medium (10 mM Pi). The cytosolic fraction was isolated by centrifugation (see SI appendix), and in separate experiments, mitochondria were isolated by immunoprecipitation from WT and ubp3Δ and mitochondrial Pi estimated. (i) Cytosolic Pi levels (relative and absolute) and (ii) mitochondrial Pi levels normalised to Idh1 protein levels is shown. Data represent mean ± SD from three biological replicates (n=3). Also see Figure 4-figure supplement 1A, B.

D) Effect of supplementing Pi on the total mitochondrial volume. WT cells with mitochondria targeted mNeon green were grown in high glucose, and high glucose, high Pi medium (10 mM Pi), cells were imaged and the total mitochondrial volume per cell was calculated.

E) Increasing Pi concentration in a high glucose medium results in a decrease in basal OCR. WT cells were grown in high glucose medium (YPD-Pi medium), supplemented with 5 mM Pi and at OD600∼0.6, the medium was supplemented with Pi (10 mM and 25 mM final concentrations). The basal OCR was measured one hour after Pi supplementation. Data represent mean ± SD from three biological replicates (n=3).

E) Effect of increasing the Pi concentration in a high glucose medium, in the presence of 2DG, on basal OCR. WT cells were grown in high glucose medium (YPD-Pi medium), supplemented with 5 mM Pi and at OD600∼0.6, the medium was supplemented with Pi (25 mM final concentration) and 2DG (0.25%) for one hour and the basal OCR was measured. Data represent mean ± SD from three biological replicates (n=3).

F) Overexpression of Mir1. C terminal 6x HA epitope-tagged Mir1 was expressed under G6PD promoter in cells with Mir1 endogenously tagged at the C terminus with a 6x-HA epitope tag. WT cells (carrying an empty vector, and expressing Mir1-HA under the endogenous Mir1 promoter) and Mir1-OE cells were grown in high glucose. The protein levels of Mir1 were estimated by western blot, and clones with an ∼2-fold increase in Mir1 were selected. A representative blot (out of three biological replicates, n=3) is shown in the lower panel. Quantification is shown in the upper panel. Data represent mean ± SD.

G) Mpc3 protein levels in ubp3Δ cells. WT and ubp3Δ cells containing endogenously tagged Mpc3 at the C terminus with a 3x FLAG epitope tag, were cultured in high glucose (2% glucose) and high glucose-low Pi (2% glucose, 1 mM Pi). The Mpc3 protein levels were measured by western blot. A representative blot (out of three biological replicates, n=3) is shown in the lower panel, quantifications shown in the upper panel. Data represent mean ± SD.

H) Mpc3 protein amounts upon shifting WT and mir1Δ cells to a low-glucose medium. WT and mir1Δ cells containing Mpc3-FLAG, were cultured in high glucose (2% glucose) and shifted to low (0.1%) glucose for 1 hour. Mpc3 protein was measured by western blot. A representative blot (out of three biological replicates, n=3) is shown in the lower panel, quantifications shown in the upper panel. Data represent mean ± SD.

I) Requirement of Mpc3 for the increase in basal OCR upon shifting to low glucose. WT and mpc3Δ cells were cultured in high glucose (2% glucose) and shifted to low (0.1%) glucose for 1 hour. The basal OCR from two independent experiments (n=2), normalized to the OD600 is shown. Data represent mean ± SD.

J) Mpc3 levels and Mir1 overexpression. WT cells and Mir1-OE cells containing endogenously tagged Mpc3 ate the C terminus with a 3x FLAG tag were grown in high glucose. The protein levels of Mpc3 were estimated by western blot. A representative blot (out of three biological replicates, n=3) is shown in the panel. Quantification is shown in the upper panel. Data represent mean ± SD. Data information: *p<0.05, **p<0.01, ***p<0.001.