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. 2022 Apr 19;11:e67659. doi: 10.7554/eLife.67659

Figure 1. Interconversion and active synthesis of adenine nucleotides are important for adenosine triphosphate (ATP) homeostasis.

(A) Adk1 and Snf1 synergistically control cellular ATP levels. QUEEN ratio images of ATP homeostasis mutant cells grown in medium containing 2% glucose. The asterisk indicates an example of cells with significantly reduced ATP levels. (B) The mean QUEEN ratios of cells were translated to ATP levels and shown in a dot plot. The horizontal bar indicates the mean of each population. Asterisks indicate p values less than 0.0001 versus WT (Dunnett’s multiple comparison). CV: coefficient of variance. N = 134–276 cells were scored. (C) Biochemical measurements of cellular ATP levels. ATP levels in cells of the indicated genotypes were measured as described in Materials and methods. Data are the mean ± 1SD (error bars) (N = 4). An asterisk indicates a p value of 0.022 versus WT (Dunnett’s multiple comparison). (D) QUEEN ratio images of bas1∆ cells grown in 2% glucose medium. Growth in media supplemented with 0.11 mg/ml adenine partially restored the low ATP phenotype of bas1∆. (E) ATP levels in cells shown in D were plotted. Asterisks indicate p values: *p < 0.0001 (versus WT in glucose, Dunnett’s multiple comparison); **p = 3.6 × 10−20 (versus bas1∆ in glucose). N = 186–296 cells were scored. (F) ATP levels in WT and bas1∆ cells were measured as described in C. Data are the mean ± 1SD (error bars) (N = 4). An asterisk indicates a p value of 8.5 × 10−5 versus WT.

Figure 1—source data 1. Raw data for Figure 1.

Figure 1.

Figure 1—figure supplement 1. AMP-activated protein kinase (AMPK) is involved in the maintenance of cellular adenosine triphosphate (ATP) levels in nonstarving cells.

Figure 1—figure supplement 1.

(A) Visualization of intracellular ATP levels in wild-type (WT) and snf1∆ cells using the ATP sensor QUEEN. Cells were grown to the mid-log phase in SC-H medium containing the indicated levels of glucose and then imaged. White scale bar = 5 µm. (B) snf1∆ cells had lower ATP levels than wild-type cells. The mean QUEEN ratio inside a single cell (mean QUEEN ratio/cell), which represents the intracellular ATP level of the cell, was quantified for each cell from the ratio image. Data are the mean of the cell population ± 1SD (error bar) normalized to wild-type cells. N = 105–193 cells were scored. Asterisks indicate a significant difference from WT (p < 0.05). (C) ATP levels in single-deletion mutants of AMPK β subunits. Data are the mean of the cell population ± 1SD (error bar) normalized to wild-type cells. N = 177–223 cells were scored. Asterisks indicate a significant difference (p < 0.0001 versus WT, Dunnett’s multiple comparison). (D) mig1∆ cells had slightly lower ATP levels than wild-type cells. Data are the mean of the cell population ± 1SD (error bar) normalized to wild-type cells. N = 190–231 cells were scored. Asterisks indicate p values versus mig1∆: *p = 0.0003; **p < 0.0001, Dunnett’s multiple comparison.
Figure 1—figure supplement 1—source data 1. Raw data for Figure 1—figure supplement 1.
Figure 1—figure supplement 2. Adenylate kinase Adk1 is involved in the maintenance of cellular adenosine triphosphate (ATP) levels.

Figure 1—figure supplement 2.

(A) QUEEN ratio images of wild-type and adk1∆ cells grown in 2% glucose or 2% galactose. White scale bar = 5 µm. (B) Quantification of data shown in (A). The mean QUEEN ratio/cell was quantified for each cell from ratio images. Values are the mean of the cell population ± 1SD (error bar). N = 182–236 cells were scored. Asterisks indicate a significant difference from WT (p < 0.05).
Figure 1—figure supplement 2—source data 1. Raw data for Figure 1—figure supplement 2.
Figure 1—figure supplement 3. Biochemical measurements of adenosine triphosphate (ATP) and ADP.

Figure 1—figure supplement 3.

(A) Biochemical measurements of cellular ADP levels. ADP levels in cells of the indicated genotypes were measured as described in Materials and methods. Data are the mean ± 1SD (error bars) (N = 4). Asterisks indicate p values less than 0.01 versus WT (Dunnett’s multiple comparison). (B) 2-Deoxyglucose (2DG) depletes cellular ATP. ATP levels in WT cells incubated with medium containing 2% glucose or 40 mM 2DG for 10 min were biochemically measured. Relative ATP levels were expressed as the ratios of ATP levels in glucose-treated cells. Data are the mean ± 1SD (error bars) (N = 4). (C) ATP/ADP ratios in WT and ATP-mutant cells. ATP/ADP ratios were calculated from ATP and ADP levels measured using biochemical assays. Data are the mean ± 1SD (error bars) (N = 4). An asterisk indicates a p value of 0.036 versus WT (Dunnett’s multiple comparison). (D) The sums of ATP and ADP levels in WT and ATP-mutant cells were calculated from ATP and ADP levels measured using biochemical assays. Data are the mean ± 1SD (error bars) (N = 4). Asterisks indicate p values versus WT: *p < 0.01, **p < 0.001, Dunnett’s multiple comparison.
Figure 1—figure supplement 3—source data 1. Raw data for Figure 1—figure supplement 3.
Figure 1—figure supplement 4. Adenosine triphosphate (ATP) levels in bas1∆ snf1∆ cells.

Figure 1—figure supplement 4.

(A) QUEEN ratio images of wild-type, bas1∆, snf1∆, and bas1∆ snf1∆ cells. (B) Quantification of data shown in (A). The mean QUEEN ratio/cell was quantified for each cell from ratio images. Data are shown as a dot plot. The horizontal bar in the plot indicates the mean of each population. Significance between two sets of data was tested using the Dunnett’s multiple comparison test and indicated by p values. N.S., no significance (p value >0.3).
Figure 1—figure supplement 4—source data 1. Raw data for Figure 1—figure supplement 4.