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 figures S1A, S1B.

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 S1D.

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 x 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. Serial dilution growth assay in high glucose in the presence/absence of sodium azide (1 mM) are shown. Also see figure S1F.

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 S2A.

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, figures S2B, S2C.

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 S3A.

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 S3B.

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) Mitochondrial Pi amounts in WT vs ubp3Δ. Mitochondria were isolated by immunoprecipitation from WT and ubp3Δ and mitochondrial Pi estimated. Mitochondrial Pi levels (normalised to Idh1) are shown. Data represent mean ± SD from three biological replicates (n=3). Also see figure S4A, 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 S4C.

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 S4D, S4F.

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 two independent experiments (n=2). 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 S5A.

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 S5B.

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.

G) Mitochondrial Pi transport requirement 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.

H) 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.