Targeting the MEF2-like transcription factor Smp1 by the stress-activated Hog1 mitogen-activated protein kinase

Abstract

Exposure of Saccharomyces cerevisiae to increases in extracellular osmolarity activates the stress-activated Hog1 mitogen-activated protein kinase (MAPK), which is essential for cell survival upon osmotic stress. Yeast cells respond to osmotic stress by inducing the expression of a very large number of genes, and the Hog1 MAPK plays a critical role in gene transcription upon stress. To understand how Hog1 controls gene expression, we designed a genetic screen to isolate new transcription factors under the control of the MAPK and identified the MEF2-like transcription factor, Smp1, as a target for Hog1. Overexpression of SMP1 induced Hog1-dependent expression of osmoresponsive genes such as STL1, whereas smp1Delta cells were defective in their expression. Consistently, smp1Delta cells displayed reduced viability upon osmotic shock. In vivo coprecipitation and phosphorylation studies showed that Smp1 and Hog1 interact and that Smp1 is phosphorylated upon osmotic stress in a Hog1-dependent manner. Hog1 phosphorylated Smp1 in vitro at the C-terminal region. Phosphorylation of Smp1 by the MAPK is essential for its function, since a mutant allele unable to be phosphorylated by the MAPK displays impaired stress responses. Thus, our data indicate that Smp1 acts downstream of Hog1, controlling a subset of the responses induced by the MAPK. Moreover, Smp1 concentrates in the nucleus during the stationary phase, and the lack of SMP1 results in cells that lose viability in the stationary phase. Localization of Smp1 depends on HOG1, and consistently, hog1Delta cells also lose viability during this growth phase. These data suggest that Smp1 could be mediating a role for the Hog1 MAPK during the stationary phase.

FIG. 1.

Smp1 transcription factor modulates HOG1-mediated STL1 gene expression. (A) Yeast multicopy plasmids capable of inducing STL1-LacZ expression in wild-type cells were isolated, and their dependence on HOG1 was assayed in wild-type cells (YEN2) and hog1Δ cells (YEN7). A representative filter β-galactosidase assay demonstrating induction of STL1-LacZ by several positive clones from the screening is shown. (B) Smp1 modulates expression of STL1-LacZ. Wild-type, hog1Δ, and smp1Δ strains containing the STL1-LacZ reporter system (strains YEN2, YEN7, and YEN48) were transformed with a control plasmid or a multicopy plasmid expressing SMP1. β-Galactosidase activity was assayed in cells that were grown to mid-log phase and that were subjected (open bars) or not subjected (control; filled bars) to hyperosmotic stress (0.4 M NaCl for 35 min). β-Galactosidase activity is given in nanomoles per minute per milligram and is the result of the measurement in quadruplicate of results for two independent transformants.

FIG. 2.

In vivo phosphorylation of Smp1 upon osmotic stress depends on Hog1. (A) GST-SMP1 was expressed under the P_GAL1 promoter in wild-type (TM141) and hog1Δ (TM233) cells. Yeast cells were grown in the presence of galactose for 4 h and subjected (+) or not subjected (−) to a brief osmotic shock (0.4 M NaCl for 10 min), and the extracts were treated (+) or not treated (−) with 10 U of alkaline phosphatase or phosphatase inhibitors (Phos. Inh). Extracts were separated by SDS-PAGE, and GST-tagged Smp1 was detected by the use of polyclonal GST-specific antibodies. The control strain (without GST-SMP1) was the wild-type strain (TM141). (B) Time course of Hog1 and Smp1 phosphorylation upon osmotic stress. Cells expressing GST-SMP1 in a wild-type strain were subjected to osmotic stress (0.4 M NaCl), and cells were collected at different times. Phosphorylation of Smp1 and Hog1 was monitored by Western blotting by using anti-GST antibodies and anti-phospho-p38 MAPK (Thr180/Tyr182) antibodies (New England BioLabs), respectively. The level of phosphorylation was measured from scanned films by using Quantity One software (Bio-Rad).

FIG. 3.

In vivo binding of Hog1 to Smp1. (A) Interaction as shown by two-hybrid analysis of Hog1 and Smp1. The wild-type version and a mutant that lacks the MADS and MEF2 domains (ΔMADS Smp1) fused to the LexA DB were expressed with the full-length HOG1 fused to the GAL4 activator domain. A representative filter β-galactosidase assay demonstrating interactions between Hog1 and Smp1 is shown. Proteins encoded by the control plasmids pLexA-RAS^V12 and pACT-RAF, which are known to interact with each other, are shown for comparison. (B) Smp1 coprecipitates with Hog1. Strain YEN114 (which expresses HA-tagged Hog1 from the wild-type locus) was transformed with a plasmid expressing GST or GST-SMP1 under the P_GAL1 promoter. Cells were grown in the presence of galactose, and samples were taken before (−) or 10 min after (+) the addition of NaCl to a final concentration of 0.4 M. GST proteins were affinity purified through a glutathione-Sepharose matrix; the presence of HA-Hog1 in the precipitates was probed by immunoblotting with anti-HA (indicated by αHA at right), and GST-containing proteins (indicated by αGST at right) were detected by using antibodies against GST. Total, <10% of the input protein; Prec., total amount of Hog1 or GST precipitated.

FIG. 4.

Hog1 phosphorylates the C-terminal domain of Smp1. (A) In vitro-activated Hog1 phosphorylates Smp1. Various Smp1 fragments were tested for their ability to be phosphorylated by an in vitro-activated Hog1. The positions of the Smp1 fragments included in the constructs are indicated in parentheses. Recombinant tagged proteins were purified from E. coli and subjected to phosphorylation by activated Hog1 as described in Materials and Methods. Phosphorylated proteins were resolved by SDS-PAGE and detected by autoradiography. Asterisks indicate the positions of Smp1 proteins detected by Coomassie staining. (B) Mutation of Smp1 Ser348, Ser357, Thr365, and Ser376 to Ala abolishes Hog1 phosphorylation. The wild-type Smp1 fragment (amino acids 275 to 452) and various Smp1 site-directed mutants were tested for Hog1 phosphorylation as described in the legend for panel A. The Smp1 mutants and their corresponding mutations (indicated in parentheses) are as follows: m2 (Ser348 and Ser357 to Ala), m4 (Ser348, Ser357, Thr365, and Ser376 to Ala), and m1 (Thr365 and Ser376 to Ala). After phosphorylation, the proteins were resolved by SDS-PAGE, and phosphorylated proteins were detected by autoradiography (upper panel). GST-tagged Smp1 proteins were detected by Coomassie blue stain (lower panel). The asterisk indicates a contaminant phosphorylation not corresponding to Smp1.

FIG. 5.

Smp1 phosphorylations are required for Smp1 transcriptional activity. (A) Mutation of Ser348, Ser357, Thr365, and Ser376 to Ala abolishes in vivo Hog1 phosphorylation. Full-length Smp1 and the quadruple-mutant protein Smp1-m4 (mutations: Ser348, Ser357, Thr365, and Ser376 to Ala) were tested for Hog1 phosphorylation as described in the legend for Fig. 2A. After phosphorylation, the proteins were resolved by SDS-PAGE and detected by immunoblotting by using anti-GST polyclonal antibodies. (B) Hog1 phosphorylation modulates Smp1 function. A wild-type strain and the smp1Δ mutant strain (YEN44) containing the reporter STL1::lacZ were transformed with a centromeric plasmid expressing wild-type SMP1 or the quadruple-mutant SMP1-m4 (unphosphorylatable by Hog1). The cells were subjected to a brief osmotic shock, and β-galactosidase activity was assayed as described in the legend for Fig. 1B. β-Galactosidase activity is given as fold induction of control versus that of NaCl-treated cells and is the result of the measurement in quadruplicate of results for two independent transformants.

FIG. 6.

Cell survival upon osmotic stress is reduced in hog1Δ and smp1Δ strains. To analyze the role of Smp1 in osmotic stress adaptation, we measured the incorporation of PI, as described in Material and Methods, as a measure of cell viability in wild-type, hog1Δ mutant, and smp1Δ mutant strains. Yeast cells were grown in YPD and subjected (open bars) or not subjected (filled bars) to an osmotic shock (1 M NaCl for 60 min). Viability was determined by the addition of PI and measurement of the percentage of PI-positive cells by using a flow cytometer. The results are the measurements in duplicate from six independent experiments. The data were confirmed by Phloxin B staining and visual microscopy (see Materials and Methods).

FIG. 7.

Smp1 nuclear localization depends on Hog1. The wild-type and the hog1Δ mutant strains were transformed with a plasmid carrying a GFP-tagged Smp1 expressed under its own promoter. The cells were grown and Smp1-GFP was detected by fluorescence microscopy as described in Materials and Methods. (A) Smp1 localizes into the nuclei of wild-type cells in the stationary phase. Cells of the indicated types carrying Smp1-GFP were grown to the stationary phase, and images were taken. Representative images showing the localization of Smp1-GFP are presented. The positions of the nuclei were determined by DAPI staining. (B) Quantitative data were obtained from wild-type or hog1Δ cells growing at the mid-logarithmic (Mid log.) or stationary phase of growth. The data represent the results of three independent experiments which included counting 300 cells each.

FIG. 8.

Hog1 and Smp1 are required for cell viability in stationary phase. Wild-type (•), hog1Δ (▿), smp1Δ (○), or hog1Δ smp1Δ (▾) cells were grown in minimal medium to stationary phase and allowed to grow for the indicated period of time. Viability was determined by adding PI and measuring the percentage of PI-positive cells by using a flow cytometer. The results are the measurements in duplicate from four independent experiments. The data were confirmed by Phloxin B staining and visual microscopy (see Materials and Methods).