Std1 and Mth1 proteins interact with the glucose sensors to control glucose-regulated gene expression in Saccharomyces cerevisiae

Abstract

The Std1 protein modulates the expression of glucose-regulated genes, but its exact molecular role in this process is unclear. A two-hybrid screen for Std1-interacting proteins identified the hydrophilic C-terminal domains of the glucose sensors, Snf3 and Rgt2. The homologue of Std1, Mth1, behaves differently from Std1 in this assay by interacting with Snf3 but not Rgt2. Genetic interactions between STD1, MTH1, SNF3, and RGT2 suggest that the glucose signaling is mediated, at least in part, through interactions of the products of these four genes. Mutations in MTH1 can suppress the raffinose growth defect of a snf3 mutant as well as the glucose fermentation defect present in cells lacking both glucose sensors (snf3 rgt2). Genetic suppression by mutations in MTH1 is likely to be due to the increased and unregulated expression of hexose transporter genes. In media lacking glucose or with low levels of glucose, the hexose transporter genes are subject to repression by a mechanism that requires the Std1 and Mth1 proteins. An additional mechanism for glucose sensing must exist since a strain lacking all four genes (snf3 rgt2 std1 mth1) is still able to regulate SUC2 gene expression in response to changes in glucose concentration. Finally, studies with green fluorescent protein fusions indicate that Std1 is localized to the cell periphery and the cell nucleus, supporting the idea that it may transduce signals from the plasma membrane to the nucleus.

FIG. 1

Two-hybrid interactions between Std1, Mth1, and the C-terminal domains of the glucose sensors. S. cerevisiae Y153 was transformed with the indicated two-hybrid fusion constructs. Positive two-hybrid interactions are indicated by the growth of cells on medium lacking histidine and containing 25 mM 3-aminotriazole (3-AT).

FIG. 2

Effects of mutations in SNF3 and RGT2 on cell growth. Serial dilutions of wild-type cells and cells with various null alleles as indicated were spotted onto agar plates containing YEPD, YEPD containing antimycin (Ent), SC-glycerol, or SC-raffinose containing antimycin as indicated. Cells were grown for 4 days (except as indicated) and photographed. The strains used were MSY465 (wild type) MSY449 (snf3), MSY403 (rgt2), and MSY441 (snf3 rgt2).

FIG. 3

Suppression of snf3 and snf3 rgt2 phenotypes by mutation of STD1 and MTH1. Serial dilutions of wild-type cells and cells with various null alleles as indicated were spotted onto agar plates containing YEPD, YEPD plus antimycin (ant), SC-glycerol, or SC-raffinose plus antimycin as indicated. The strains used: (A) MSY465 (wild type), MSY449 (snf3), MSY451 (snf3 std1) MSY453 (snf3 mth1), MSY455 (snf3 std1 mth1), and MSY471 (std1 mth1); (B) MSY465 (wild type), MSY441 (snf3 rgt2), MSY443 (snf3 rgt2 std1), MSY445 (snf3 rgt2 mth1), and MSY447 (snf3 rgt2 std1 mth1).

FIG. 4

Invertase expression in cells with mutations in SNF3, RGT2, STD1, and MTH1. Quantitative invertase assays were performed on cells grown under repressing (R) and derepressing (D) conditions (25) as indicated. At least three independent colonies of each strain were assayed, and the error bars represent 1 standard error. The strains used for panels A and B were the same as those used for Fig. 3A and B, respectively.

FIG. 5

HXT gene expression in cells with mutations in SNF3, RGT2, STD1, and MTH1. Cultures were grown in SC medium containing 3% glycerol and 2% ethanol and lacking uracil. Cells were collected and resuspended in the same medium containing either no glucose (−), 0.1% glucose (L), or 6% glucose (H). After 4 h in this medium, cells were harvested and protein extracts were assayed for β-galactosidase activity. Extracts from three independent transformants of each culture were assayed, and the mean value is plotted; the error bars represent 1 standard error. All bar graphs are drawn to the same scale, allowing direct comparisons between the different panels. The strains used were MSY465 (wild type), MSY441 (snf3 rgt2), MSY445 (snf3 rgt2 mth1), MSY443 (snf3 mth1 std1), MSY460 (mth1), MSY467 (std1), MSY471 (std1 mth1), and MSY447 (snf3 rgt2 std1 mth1).

FIG. 6

Snf3 inhibits Std1-mediated gene induction. (A) Invertase activity was measured in repressed cells (25) transformed with 2μm plasmids containing either no insert (v) or complete genomic copies of STD1 or SNF3 as indicated. Invertase activity from three independent transformants was measured, and the mean value is plotted; the error bars represent one standard error. The strains used were MSY401 (wild type) and MSY192 (std1 mth1). (B) Western blot analysis of Std1-3HA. Wild-type cells (MSY401) were transformed with the 2μm plasmids containing either no insert, the SNF3 gene, or an epitope-tagged STD1 gene. Protein extracts were prepared, and the level of the Std1-3HA protein (15 μg per lane) was detected with monoclonal antibody directed against the HA epitope.

FIG. 7

Std1 and Mth1 act through distinct pathways that are Snf1 dependent and Snf1 independent, respectively. (A) Cultures containing the HXT1-lacZ reporter and the indicated 2μm plasmid were grown in SC medium containing 6% glucose lacking uracil and leucine. Cells from mid-logarithmic-phase cultures were collected, and protein extracts were assayed for β-galactosidase activity. Extracts from three independent transformants of each culture were assayed, and the mean value is plotted; the error bars represent 1 standard error. The strains used in this experiment, MSY465 (wild type) and FY1193 (snf1Δ10), were transformed with plasmid YEP351 (Vec), p6A5 (STD1), or pMT51 (MTH1). (B) Cultures containing the HXT4-lacZ reporter were grown in SC medium containing 6% glucose lacking uracil. Cells from mid-logarithmic-phase cultures were collected, and protein extracts were assayed for β-galactosidase activity. Extracts from three independent transformants of each culture were assayed, and the mean value is plotted; the error bars represent 1 standard error. The strains used were MSY465 (wild type), MSY469 (mth1), FY1193 (snf1), and MSY479 (mth1 snf1).

FIG. 8

MTH1 expression is subject to glucose repression. (A) Western blot of protein extracts (25 μg per lane) from cells bearing centromere plasmids encoding either Std1-3HA or Mth1-3HA as indicated. Wild-type cells (MSY401) were grown under repressing (R) and derepressing (D) conditions (25). Arrows indicate the mobility of the full-length proteins. (B) Northern blot of total cellular RNA (15 μg per lane) extracted from wild-type cells (MSY401) under repressing (R) and derepressing (D) conditions or from wild-type cells bearing a 2μm MTH1 plasmid (2μ), as indicated. The blot was first probed with ³²P-labeled DNA complementary to MTH1 and then stripped and reprobed with [³²P]DNA complementary to yeast actin mRNA (ACT1).

FIG. 9

Std1 is localized in cell nucleus and at the plasma membrane. Wild-type (A to E) or snf3 rgt2 (F) cells were analyzed by fluorescence microscopy. Cells expressed either unfused GFP from the GAL1 promoter (20) (A), histone-GFP fusion (34) (B), Snf3-GFP fusion (C), or Std1-GFP fusion (D to F). Fluorescence images were collected of a single Std1-GFP-expressing cell (G to I) that had been fixed with formaldehyde and stained with Hoechst dye. (G) Std1-GFP fluorescence; (I) Hoechst dye fluorescence; (H) composite image of both showing colocalization of the Hoechst and GFP fluorescence. (J) A single Std1-GFP-expressing cell that showed both nuclear and punctate cytoplasmic fluorescence was analyzed sequentially at six different focal planes.

FIG. 10

Model for the glucose signal transduction in yeast. Arrows indicate activation, and lines with perpendicular bars indicate repression. Proteins are represented by ovals, and genes are represented by rectangles. Filled circles represent glucose.