Yeast AMP-activated protein kinase monitors glucose concentration changes and absolute glucose levels

Abstract

Analysis of the time-dependent behavior of a signaling system can provide insight into its dynamic properties. We employed the nucleocytoplasmic shuttling of the transcriptional repressor Mig1 as readout to characterize Snf1-Mig1 dynamics in single yeast cells. Mig1 binds to promoters of target genes and mediates glucose repression. Mig1 is predominantly located in the nucleus when glucose is abundant. Upon glucose depletion, Mig1 is phosphorylated by the yeast AMP-activated kinase Snf1 and exported into the cytoplasm. We used a three-channel microfluidic device to establish a high degree of control over the glucose concentration exposed to cells. Following regimes of glucose up- and downshifts, we observed a very rapid response reaching a new steady state within less than 1 min, different glucose threshold concentrations depending on glucose up- or downshifts, a graded profile with increased cell-to-cell variation at threshold glucose concentrations, and biphasic behavior with a transient translocation of Mig1 upon the shift from high to intermediate glucose concentrations. Fluorescence loss in photobleaching and fluorescence recovery after photobleaching data demonstrate that Mig1 shuttles constantly between the nucleus and cytoplasm, although with different rates, depending on the presence of glucose. Taken together, our data suggest that the Snf1-Mig1 system has the ability to monitor glucose concentration changes as well as absolute glucose levels. The sensitivity over a wide range of glucose levels and different glucose concentration-dependent response profiles are likely determined by the close integration of signaling with the metabolism and may provide for a highly flexible and fast adaptation to an altered nutritional status.

Keywords: AMP-activated Kinase (AMPK); Dynamic Control; Glucose Metabolism; Nuclear Translocation; Signal Transduction; Yeast Physiology.

FIGURE 1.

Schematic representation of the yeast Snf1/Mig1 signaling pathway. Shown is the Snf1/Mig1 pathway in the presence of high and low glucose levels in the growth medium. At high glucose levels, hexokinase PII (Hxk2) phosphorylates glucose, and glycolysis is fully active. The Glc7 phosphatase keeps Snf1 and Mig1 dephosphorylated, and, hence, Mig1 is located in the nucleus and, in complex with Cyc8 and Tup1, represses gene expression. At low glucose levels, hexokinase PI (Hxk1) and glucokinase (Glk1) also participate in glucose phosphorylation, but glycolysis is running at lower rate. ADP/AMP protect the SNF1 complex from dephosphorylation, and SNF1 phosphorylates Mig1, mediating its dissociation from Cyc8 and Tup1 and its nuclear exit. Snf1 is phosphorylated by three upstream kinases (Sak1, Tos3, and Elm1). Only Sak1 is shown. Glc-6-P, glucose 6-phosphate; Hxt, hexose transporter.

FIGURE 2.

Single cell data of Mig1 nuclear-cytoplasmic shuttling upon repetitive environmental changes. A, ratio of the mean Mig1-GFP intensity in the nucleus relative to the whole cell obtained from 34 cells plotted over time. Traces for all cells as well as the average are shown. Cells were grown in 4% glucose until mid-log phase and then exposed to three cycles of repeated shifts between 4 and 0.1% glucose. The new conditions were established within 2 s. Each shift lasted for 600 s, resulting in a 3600-s experiment. Images were taken 30 s before the shift, at the shift, and every 60 s after the shift. B, glucose shift regime. conc., concentration. C, images of the same two cells at different times of the experiment.

FIGURE 3.

Mig1 nuclear accumulation following a glucose upshift over time. A, ratio of the mean Mig1-GFP intensity in the nucleus relative to the whole cell obtained from 24–64 single cells plotted over time. Cells were grown in 3% ethanol until mid-log phase, arrayed in the microfluidic device, kept in the same medium, and then shifted to glucose at a final concentration of 4, 2, 0.5, 0.2, 0.1, 0.05, 0.025, 0.01, 0.005, or 0%. The microfluidic flow was shifted at time 0 s, and images were taken 30 s before the shift, at the shift, at 30 s, every 60 s for 420 s, every 120 s for 360 s, and every 180 s for 360 s after the shift. B, mean of the Mig1-GFP intensity ratio for all cells shown in A. Glucose concentrations are represented with different colors, and the size of the circles represents mean ± S.D.

FIGURE 4.

Mig1 nuclear exit following a glucose downshift over time. A, ratio of the mean Mig1-GFP intensity in the nucleus relative to the whole cell obtained from 45–78 individual single cells plotted over time. Cells were grown in 4% glucose until mid-log phase, arrayed in the microfluidic device, kept in the same medium, and then shifted to glucose at a final concentration of 4, 2, 1.5, 1, 0.5, 0.2, 0.1, or 0%. The microfluidic flow was shifted at time 0 s, and images were taken 30 s before the shift, at the shift, at 60 s, every 60 s for 420 s, every 120 s for 360 s, and every 180 s for 360 s after the shift. B, mean of the Mig1-GFP intensity ratio for all cells shown in A. Glucose concentrations are represented with different colors, and the size of the circles represents mean ± S.D. Initial values cannot be compared directly between experiments because different cell arrays where used and the equipment had to be readjusted between experiments.

FIGURE 5.

Mig1 and Snf1 phosphorylation correlate with transient Mig1 relocalization. A, the Snf1 phosphorylation status correlates with Mig1 subcellular localization. A snf1Δ mutant expressing pSNF1-HA was grown to mid-log phase in selective medium supplied with 4% glucose, and then the culture was diluted to 1% glucose. Samples were taken before and at the indicated time points after glucose shift and analyzed by immunoblotting using simultaneously anti-Thr¹⁷² and anti-HA antibodies. Western blot analysis data were quantified relative to total Snf1. Because different blots cannot be compared directly, a cubic spline interpolation of the ratio between phosphorylated Snf1 and total Snf1 over time from five independent experiments was performed in R-3.0.0. These data show that the overall trend in these five experiments is highly reproducible. B, representative blot of phosphorylated Snf1 relative to total Snf1 (Experiment 2, red line in A). G, glucose; E, ethanol. C, Mig1 phosphorylation correlates with its subcellular localization. Cells of a mig1Δ mutant expressing pMIG1-HA was treated as in A. Samples were taken before and at the indicated time points after glucose shift and analyzed by immunoblotting using anti-HA antibody.

FIGURE 6.

Mig1 shuttles between the cytosol and nucleus under all conditions. A, FRAP of Mig1-GFP under different glucose concentrations. Cells expressing Mig1-GFP and Nrd1-mCherry were grown to mid-log phase in 0, 1, or 4% glucose. Nuclei of individual cells were bleached, and the fluorescence recovery was recorded over time in those nuclei. The average of 20–25 cells normalized against the final recovered intensity for each glucose concentration (○) and the fit curve (solid lines) are represented over time. B, single versus double exponential fit. The average of 20 cells normalized against the final fluorescence recovery (○) together with the single (blue line) and double exponential (red line) fit curves plotted over time are shown. C, single cell FRAP curves from 20 cells grown in 4% glucose plotted over time. The mean of these cells is represented by a bold line. D, FLIP of Mig1-GFP under different glucose concentrations. Cells expressing Mig1-GFP and Nrd1-mCherry were grown to mid-log phase in 0, 1, or 4% glucose. In either case, a small region in the cytoplasm of individual cells was bleached, and the loss of fluorescence from the nucleus was recorded over time. The average of 13–24 cells normalized with respect to the initial intensity for each glucose concentration (○) and the fit curve (solid line) are represented over time. Error bars represent mean ± S.E.

FIGURE 7.

Average results of the FRAP and FLIP fitting parameters. The average of 20–25 cells of fast half-time (A), slow half-time (B), and fast fraction (C) of Mig1-GFP fluorescence recovery obtained from double exponential fits applied to 0, 1, and 4% glucose (Fig. 6A). D, average of 13–24 cells of half-time of Mig1-GFP fluorescence loss obtained from single exponential fits applied to 0, 1, and 4% glucose (Fig. 6D). Error bars represent mean ± S.E.

FIGURE 8.

Interpretation of FRAP and FLIP data. A, and B, simple dynamic systems displaying either single or double exponential behavior. All reactions are assumed to have first-order kinetics. C–F, schematic of the pools of phosphorylated and unphosphorylated cytosolic and nuclear Mig1. The size of the circles illustrates the pool size at the start of each experiment. Pools affected by photobleaching at the start of an experiment are indicated by a black and white diagonal pattern, and phosphorylated pools are indicated with a small sphere with the letter P in the top right corner. All reactions are assumed to have first-order kinetics. C, FRAP for glucose-grown cells. After bleaching of nuclear Mig1, we first observe the rapid nuclear entry of unphosphorylated cytosolic Mig1, followed by relocalization of cytosolic phosphorylated Mig1 via a slower dephosphorylation step. This mechanism results in double exponential dynamics of the measured recovery of nuclear Mig1. D, FRAP for ethanol-grown cells. This is similar to the repressing (glucose) scenario but with different initial pool sizes. In this case, there is a relatively large amount of cytosolic Mig1. E, FLIP for glucose-grown cells. Nuclear unphosphoylated Mig1 is relocated to the cytosol via the initially empty pool of nuclear phosphorylated Mig1 (○). If the pool of nuclear phosphorylated Mig1 is initially empty and if the phosphorylation is significantly slower than the export reaction, a single exponential behavior is observed with a time constant that is larger compared with the derepressing scenario (as observed). Alternatively, the nuclear unphosphorylated Mig1 is directly exported to the cytosol (dashed reaction), also rendering single exponential behavior. F, FLIP for ethanol-grown cells. Nuclear Mig1 is assumed to be predominantly phosphorylated and is exported to the cytosol, resulting in single exponential dynamics.