Mig1 localization exhibits biphasic behavior which is controlled by both metabolic and regulatory roles of the sugar kinases

Abstract

Glucose, fructose and mannose are the preferred carbon/energy sources for the yeast Saccharomyces cerevisiae. Absence of preferred energy sources activates glucose derepression, which is regulated by the kinase Snf1. Snf1 phosphorylates the transcriptional repressor Mig1, which results in its exit from the nucleus and subsequent derepression of genes. In contrast, Snf1 is inactive when preferred carbon sources are available, which leads to dephosphorylation of Mig1 and its translocation to the nucleus where Mig1 acts as a transcription repressor. Here we revisit the role of the three hexose kinases, Hxk1, Hxk2 and Glk1, in glucose de/repression. We demonstrate that all three sugar kinases initially affect Mig1 nuclear localization upon addition of glucose, fructose and mannose. This initial import of Mig1 into the nucleus was temporary; for continuous nucleocytoplasmic shuttling of Mig1, Hxk2 is required in the presence of glucose and mannose and in the presence of fructose Hxk2 or Hxk1 is required. Our data suggest that Mig1 import following exposure to preferred energy sources is controlled via two different pathways, where (1) the initial import is regulated by signals derived from metabolism and (2) continuous shuttling is regulated by the Hxk2 and Hxk1 proteins. Mig1 nucleocytoplasmic shuttling appears to be important for the maintenance of the repressed state in which Hxk1/2 seems to play an essential role.

Keywords: Glucose repression; Hexokinase; Hexose; Localization; Microfluidic; Mig1; Oscillation; Yeast.

Fig. 1

The carbon catabolite repression pathway. a When hexoses are absent, the Snf1 kinase is active and phosphorylates Mig1, which localizes to the cytoplasm. This enables expression from promoters, such as the SUC2 and HXK1 promoter. b Available hexoses are transported into the cell by hexose transporters and subsequently phosphorylated by Hxk1, Hxk2 and/or Glk1 enzymes. All hexose-phosphates are isomerized to fructose-6-phosphate and enter glycolysis. Snf1 kinase is inhibited through unknown mechanisms, which relieves the inhibitory phosphorylation of Mig1 and allows it to act as a transcriptional repressor of SUC2 and HXK1 expression in the nucleus. Hxk2 takes part in repression of SUC2 in the nucleus

Fig. 2

a Time-lapse images of Mig1-GFP tagged wild type cells in brightfield (overlaid with segmentation mask) and GFP channel. Cells were shifted from ethanol (510 mM) to mannose (220 mM) at 0 min. b Quantification of Mig1 localization from data in a. The Mig1 localization index was calculated as the fraction of cellular Mig1 present in the nucleus (see image analysis section of materials and methods for details). The time-point of maximum Mig1 localization from 0 to 60 min after the media shift for each cell is marked with open triangles. Mig1 localization pulse maxima are indicated by closed triangles. c Metrics extracted from single cell traces in b. The maximum and mean Mig1 localization from 0 to 60 min after the media shift are shown with open symbols. The mean Mig1 localization pulse height for each cell and the mean overall localization of Mig1 from 240 to 480 min after the media shift are shown with closed symbols. d Maximum Mig1 localization from 0 to 60 min and e time-averaged Mig1 localization from 240 to 480 min after shift from ethanol to 220 mM of the indicated hexose in wild type and hexokinase mutants. Each point represents a single cell measurement. Colored horizontal lines indicate the mean, and the 95% confidence interval and one standard deviation are shown in consecutively lighter hues of the respective color. The mean value is given in the top of the graph and significance is indicated by star (p < 0.05, paired T test against wild type condition). Black horizontal lines indicate the basal Mig1 localization level as determined from the time points before the media shift

Fig. 3

Short (a) and long-term (b) response of Mig1 localization after shift from ethanol to 220 mM of the indicated hexose in a hxk1∆hxk2∆ background overexpressing one of the sugar kinases. Each point represents a single cell measurement. Horizontal lines indicate the mean, and the 95% confidence interval and one standard deviation are shown in consecutively lighter hues of the respective color. The mean value is given in the top of the graph and significance is indicated by a star (p < 0.05, paired T test against wild type condition). Black horizontal lines indicate the basal Mig1 localization level as determined from the time points before the media shift

Fig. 4

Expression from the SUC2 (a) or HXK1 (b) promoter after shift from ethanol to 220 mM of the indicated hexose in wild type (WT) and hexokinase mutants. Each point represents a single cell measurement. Horizontal lines indicate the mean, and the 95% confidence interval and one standard deviation are shown in consecutively lighter hues of the respective color. The mean value is given in the top of the graph and significance is indicated by a star (p < 0.05, paired T test against wild type condition). Black horizontal lines indicate the basal fluorescence level as determined from the time points before the media shift