MDH1-CIT1 interaction under respiration, fermentation, and mixed-respiration conditions.

Yeast cells were grown in the minimum media containing acetate (SD-Acet), glucose (SD-Gluc), and raffinose (SD-Raff) to the exponential growth phase. (A) Luciferase signal indicating MDH1-CIT1 complex interaction. (B) Cellular oxygen consumption rate. (C) MDH1 and CIT1 protein levels detected by Western blotting. In A-B, data are presented as mean ± s.d. and the differences between conditions were tested by Student’s t-test. Asterisks indicate significant differences with p<0.05.

MDH1-CIT1 complex association, mitochondrial microenvironments, and cellular metabolite levels during Crabtree effect induction.

Cells were cultured in fresh SD-Raff media in the control condition (black). The Crabtree effect was induced by the 2% glucose application to the SD-Raff-grown cells at 0 min (red). (A) NanoBIT signal indicating MDH1-CIT1 interaction. Relative luciferase unit (RLU) was calculated by normalizing the luciferase signals by the average signals during three pre-treatment time points. SD-Raff-grown cells were also co-treated with 2% glucose and a fermentation inhibitor, 100 mM phosphate, at 0 min (gray). (B) MDH1 protein levels monitored by the luminescence of MDH1 fused with full-length nanoLUC luciferase. (C) Western blot analysis of MDH1 and CIT1 protein levels after 0, 30, 60, and 90 min of Crabtree induction. Phosphoglycerate kinase (PGK) was detected as a loading control. (D) Mitochondrial matrix pH. (E) Mitochondrial matrix redox states as GSH/GSSG equivalent (mV). (F) Mitochondrial matrix ATP level indicated by the ratio between 560 and 510 nm emission signals. All data in A-F are presented as mean ± s.d. (G) Cellular metabolite levels at 80 min. The boxes, lines, error bars, and points indicate interquartile range, median, minimum, and maximum values, and outliers, respectively. Statistical differences against the control samples were assessed using the Student’s t-test at each time point. Asterisks indicate significant differences with p<0.05.

Growth rate and enzyme activity of nanoBIT reporter strain.

(A) Extractable cellular MDH and CS enzyme activities in the wildtype (black) and nanoBIT reporter strain (Split-luc line, red). (B) Cellular growth of cells in SD-raff media monitored as culture OD600. Data is presented as mean ± s.d. Statistical differences against the wildtype samples were assessed by Student’s t-test at each time point. n.s., not significant (p>0.05).

Effects of sugars on MDH1-CIT1 complex assembly and oxygen consumption rate.

(A-D) NanoBIT signal indicating effect of galactose, sucrose, fructose, and glucose on MDH1-CIT1 interaction. Cells were cultured in fresh SD-Raff media in the control condition (black). The cells are treated with 2% sugar application to the SD-Raff-grown cells at 0 min (red). Relative luciferase unit (RLU) was calculated by normalizing the luciferase signals by the average signals during three pre-treatment time points. (E) Effects of glucose and inorganic phosphate (fermentation inhibitor) on oxygen consumption rate. Basal O2 consumption rate of SD-Raff grown cells was measured. Glucose and inorganic phosphate were added and O2 consumption rate was measured for 5 minutes. All data in A-E are presented as mean ± s.d. Statistical differences against the control samples were assessed by Student’s t-test at each time point. Asterisks indicate significant differences with p<0.05.

Biosensors indicate mitochondria microenvironments.

(A-D) Subcellular localizations of fluorescent biosensors. The yeast strains expressing Mito-roGFP1 (upper panels), pHluorin (middle panels), and Mito-GoAteam2 (lower panels) were observed by a fluorescent microscopy in the SD-Raff media. The cells were stained with Mitotracker orange prior to the analysis. (A) Bright field image. (B) Mito tracker signal. (C) Biosensor signals. (D) Merged images of the A to C. (E) Effect of raffinose addition on MDH1-CIT1 interaction. Cells were cultured in SD-Raff media in the control condition (black). The cells are applied with 2% raffinose to the SD-Raff-grown cells at 0 min (red). Relative luciferase unit (RLU) was calculated by normalizing the luciferase signals by the average signals during three pre-treatment time points. (F) Mitochondria matrix pH in control cells (black) and cells applied with additional raffinose (red). (G) Mitochondrial matrix redox state reported as redox potential of roGFP1 (mV). (H) Mitochondrial matrix ATP level indicated by the ratio between 560 and 510 nm emission signals of mito-GoATeam2 sensor. Data in E-H are presented as mean ± s.d.

MDH1-CIT1 complex association, mitochondrial matrix microenvironments, and cellular metabolite levels following TCA cycle activation and inhibition.

Cells were cultured in SD-Raff media in the control condition (black). The TCA cycle activator (acetate, dark red, A-D) and inhibitors (arsenite, blue, E-H and aminooxyacetate, pink, I-L) were applied at 0 min. (A, E, I) NanoBIT signal indicating MDH1-CIT1 interaction. Relative luciferase unit (RLU) was calculated by normalizing the luciferase signals by the average signals during three pre-treatment time points. (B, F, J) Mitochondrial matrix pH in control cells (black) and cells treated with acetate (dark red), arsenite (blue) and AOA (pink). (C, G, K) Mitochondrial matrix redox states as GSH/GSSG equivalent (mV). (D, H, L) Mitochondrial matrix ATP level indicated by the ratio between 560 and 510 nm emission signals of mito-GoATeam2 sensor. All data in A-L are presented as mean ± s.d. (M) Cellular metabolite levels after 80 min of treatment. The boxes, lines, error bars, and points indicate interquartile range, median, minimum, and maximum values, and outliers, respectively. Statistical differences against the control samples were assessed using the Student’s t-test at each time point. Asterisks indicate significant differences with p<0.05.

MDH1-CIT1 complex association, mitochondria microenvironments, and cellular metabolite levels following mitochondrial electron transport chain (ETC) inhibition.

Cells were cultured in SD-Raff media in the control condition (black). The ETC inhibitors for complex II (malonate, purple, A-D), complex IV (cyanide, green, E-H), and complex III (antimycin, orange, I-L) were applied at 0 min. (A, E, I) NanoBIT signal indicating MDH1-CIT1 interaction. Relative luciferase unit (RLU) was calculated by normalizing the luciferase signals by the average signals during three pre-treatment time points. (B, F, J) Mitochondrial matrix pH. (C, G, K) Mitochondrial matrix redox states as GSH/GSSG equivalent (mV). (D, H, L) Mitochondrial matrix ATP level indicated by the ratio between 560 and 510 nm emission signals of mito-GoATeam2 sensor. All data in A-L are presented as mean ± s.d. (M) Cellular metabolite levels after 30 min for malonate and cyanide and after 80 min for antimycin treatment. The boxes, lines, error bars, and points indicate interquartile range, median, minimum, and maximum values, and outliers, respectively. Statistical differences against the control samples were assessed using the Student’s t-test at each time point. Asterisks indicate significant differences with p<0.05.

Effect of ETC inhibitors on O2 consumption rate.

Basal O2 consumption rate was measured, then inhibitor was added and O2 consumption rate was measured for 5 minutes. Data is presented as mean ± s.d. Statistical differences against the control samples were assessed by Student’s t-test. Asterisks indicate significant differences with p<0.05.

Effects of Complex V inhibition on MDH1-CIT1 complex association, mitochondrial microenvironments, and cellular metabolite levels.

Cells were cultured in SD-Raff media in the control condition (black). Oligomycin was applied at 0 min (red). (A) NanoBIT signal indicating MDH1-CIT1 interaction. Relative luciferase unit (RLU) was calculated by normalizing the luciferase signals by the average signals during three pre-treatment time points. (B) Mitochondrial matrix pH. (C) Mitochondrial matrix redox states as GSH/GSSG equivalent (mV). (D) Mitochondrial matrix ATP level indicated by the ratio between 560 and 510 nm emission signals of mito-GoATeam2 sensor. All data in A-D are presented as mean ± s.d. (E) Cellular metabolite levels after 80 min of oligomycin treatment. The boxes, lines, error bars, and points indicate interquartile range, median, minimum, and maximum values, and outliers, respectively. Statistical differences against the control samples were assessed by Student’s t-test at each time point. Asterisks indicate significant differences with p<0.05.

Effects of pH and metabolites on the yeast MDH1-CIT1 multienzyme complex affinity.

The affinity of the MDH-CS multienzyme complex was analyzed by microscale thermophoresis (MST) using fluorescently labeled MDH1 as the target and CIT1 as the ligand. Curves represent the response (fraction bound) against CIT1 concentration. Data is presented as mean ± s.e.m. (A) Effects of pH. The MDH1-CIT1 interaction was determined in the buffer with pH 7.2 (pink), 6.8 (orange), 6.4 (olive green), 6.0 (green), and 5.8 (blue). (B) Effects of 10 mM malate (red), α-ketoglutarate (green), succinate (brown), citrate (blue), aspartate (purple), glutamate (pink), and fumarate (orange). The Kd values of MDH1-CIT1 interaction were shown next to the legend.

Effects of metabolites and ATP on the yeast MDH1-CIT1 multienzyme complex affinity.

The affinity of the MDH1-CIT1 multienzyme complex was analyzed by microscale thermophoresis (MST) using fluorescently labeled MDH1 as the target and CIT1 as the ligand. Curves represent the response (fraction bound) against CIT1 concentration. Points represent the means of fraction bound, and the error bars represent the standard deviations of three measurements. (A) Effects of metabolites. The MDH1-CIT1 interaction was determined in the buffer (control; black) with 10 mM α-ketoglutarate (green), 10 mM succinate (blue), 10 mM glutamate (pink), and 10 mM aspartate (dark red). (B) Effects of 1.25 mM (brown), 2.5 mM (green), and 5 mM (pink) ATP. The Kd values of MDH1-CIT1 interaction were shown next to the legend.

Relationship between the respiratory metabolism and the MDH1-CIT1 metabolon association.

In conditions with low respiratory flux, the MDH1-CIT1 multienzyme complex dissociates, and the TCA cycle flux reduces. Reduced ETC flux results in higher mitochondrial matrix pH, which reduces MDH1-CIT1 affinity. When the respiratory flux and the TCA cycle flux are high, MDH1-CIT1 metabolon associates and likely channels the intermediate oxaloacetate (OXA). High ETC flux lowers mitochondrial matrix pH and enhances the MDH1-CIT1 interaction. The TCA cycle intermediates affect MDH1-CIT1 metabolon formation; fumarate and malate enhance (red arrows with dotted lines), and citrate inhibits (blue arrows with dotted lines) the interaction. The arrow thickness represents the metabolic fluxes.