The Hog1 SAPK controls the Rtg1/Rtg3 transcriptional complex activity by multiple regulatory mechanisms

Abstract

Cells modulate expression of nuclear genes in response to alterations in mitochondrial function, a response termed retrograde (RTG) regulation. In budding yeast, the RTG pathway relies on Rtg1 and Rtg3 basic helix-loop-helix leucine Zipper transcription factors. Exposure of yeast to external hyperosmolarity activates the Hog1 stress-activated protein kinase (SAPK), which is a key player in the regulation of gene expression upon stress. Several transcription factors, including Sko1, Hot1, the redundant Msn2 and Msn4, and Smp1, have been shown to be directly controlled by the Hog1 SAPK. The mechanisms by which Hog1 regulates their activity differ from one to another. In this paper, we show that Rtg1 and Rtg3 transcription factors are new targets of the Hog1 SAPK. In response to osmostress, RTG-dependent genes are induced in a Hog1-dependent manner, and Hog1 is required for Rtg1/3 complex nuclear accumulation. In addition, Hog1 activity regulates Rtg1/3 binding to chromatin and transcriptional activity. Therefore Hog1 modulates Rtg1/3 complex activity by multiple mechanisms in response to stress. Overall our data suggest that Hog1, through activation of the RTG pathway, contributes to ensure mitochondrial function as part of the Hog1-mediated osmoadaptive response.

FIGURE 1:

The Rtg1 and Rtg3 transcription factors are essential for adaptation to osmostress. (A) Mutations in RTG1 and RTG3 genes render cells osmosensitive. Wild-type and the indicated mutant strains were grown to logarithmic phase and diluted to 0.05 OD₆₆₀. Tenfold serial dilution series were spotted on plates with or without NaCl or sorbitol at the indicated concentrations. Growth was scored after 2–5 d. (B) Rtg1, Rtg3, and the Hog1 SAPK are required for the induction of RTG target genes in response to osmostress. Indicated yeast strains were grown on MD-Gln and treated with 0.4 M NaCl at the indicated times. Total RNA was analyzed by Northern blot analysis for transcript levels of CIT2, PYC1, and DLD3, with RDN18 as a loading control.

FIGURE 2:

In vivo binding of Hog1 to Rtg1 and Rtg3. Strains expressing Rtg1-HA or Rtg3-HA from the endogenous locus were transformed with a plasmid expressing GST or GST-Hog1 under the P_TEF1 promoter. Cells were grown in MD-Gln, and samples were taken before (−) and after (+)treatment with 0.4 M NaCl for 10 min. Protein extracts were treated or not with 200 U/ml of recombinant DNase I for 5 h at 4ºC. GST-Hog1 was pulled-down by glutathione-Sepharose 4B, and the presence of Rtg1-HA (A) and Rtg3-HA (B) was probed by immunoblotting using anti–HA specific monoclonal antibody (top). Total extracts are presented in the middle panel. The amount of precipitated GST proteins was detected using anti–GST specific monoclonal antibody (bottom).

FIGURE 3:

Occupancy of Rtg1, Hog1, and RNA Pol II at RTG-dependent promoters. (A) Binding of Rtg1/3 complex to CIT2 and DLD3 promoters in response to osmostress is dependent on Hog1. Wild-type and hog1Δ strains carrying HA-tagged Rtg1 were grown in MD-Gln and subjected to osmostress (0.4 M NaCl) for the indicated times. Proteins were immunoprecipitated with anti-HA monoclonal antibody, and binding to the promoter regions of CIT2 and DLD3 loci was analyzed by PCR. Results are shown as the fold induction of treated relative to nontreated (time zero) samples normalized to TEL2. Data represent mean ± SD of three independent experiments. (B) Recruitment of Hog1 to CIT2 and DLD3 promoters in response to osmostress is dependent on Rtg1. Wild-type and rtg1Δ strains were analyzed by ChIP as described for (A). (C) Rtg1 is required for recruitment of RNA Pol II at CIT2 and DLD3 promoters in response to osmostress. Wild-type and rtg1Δ strains were analyzed by ChIP as described for (A), using anti-Rpb1 antibody.

FIGURE 4:

Role of Hog1 in regulating the subcellular localization of Rtg1/3 complex upon osmostress. (A) Rtg1/3 complex translocates into the nucleus upon osmostress in a Hog1-dependent manner. Wild-type and hog1Δ strains with plasmids carrying GFP-tagged Rtg1 or Rtg3 expressed under their own promoters were grown in MD-Gln to 0.5–0.7 OD₆₆₀ and stressed or not with 0.4 M NaCl for 5 min before being used for fluorescence microscopy analysis. Cells expressing Rtg1-GFP were treated with 1 μg/ml of rapamycin (Rap) for 15 min. (B) Rtg1/3 nuclear translocation is not dependent on Hog1 catalytic activity. hog1Δ strains expressing Rtg1-GFP or Rtg3-GFP with an empty monocopy vector or carrying wild-type Hog1 or Hog1^KNN were treated as in (A). (C) Subcellular localization of the Rtg1/3 complex depends on the subcellular localization of Hog1. hog1Δ strains expressing Rtg1-GFP or Rtg3-GFP with a monocopy vector carrying wild-type Hog1, Hog1^TAYA, or a vector carrying NLS-containing Hog1 (Hog1^NLS) were treated as in (A).

FIGURE 5:

Hog1 catalytic activity is required for Rtg1/3 chromatin binding and RTG-dependent gene expression. (A) Association of Rtg1/3 complex to CIT2 and DLD3 promoters in response to osmostress is dependent on Hog1 catalytic activity. hog1Δ cells expressing HA-tagged Rtg1 with an empty monocopy vector or carrying full-length Hog1 or Hog1^KNN were grown in MD-Gln and treated with 0.4 M NaCl at the indicated times. Proteins were immunoprecipitated with anti-HA monoclonal antibody, and binding to the promoter regions of CIT2 and DLD3 loci was analyzed by PCR. Results are shown as the fold induction of treated relative to nontreated (time zero) samples normalized to TEL2. Data represent mean ± SD of three independent experiments. (B) The Hog1 SAPK activity is required for the induction of RTG-dependent genes in response to osmostress. The same strains and growth conditions as in (A) were used. Total RNA was analyzed by Northern blot analysis for transcript levels of CIT2 and DLD3, with RDN18 as a loading control.

FIGURE 6:

Rtg3 is phosphorylated by the Hog1 SAPK. (A) Hog1 phosphorylates Rtg3 in vitro. Left, GST-tagged full-length Rtg3 and fragments Rtg3-M1 (aa 1–210), Rtg3-M2 (aa 1–183), and Rtg3-M3 (aa 211–486) were purified from E. coli and assayed with in vitro activated Hog1. Phosphorylated proteins were detected by autoradiography, and proteins were detected by Coomassie Blue staining. Arrows indicate the positions of Rtg3 fragments. Right, mutations of Rtg3 Thr-197, Ser-222, Ser-227, Thr-249, and Ser-376 to Ala (Rtg3^5M) abolish in vitro phosphorylation by Hog1. (B) Rtg3 is phosphorylated upon osmostress. Left, rtg1Δ cells expressing Rtg3-HA were grown to 0.6–1 OD₆₆₀, and samples were taken before (−) or 5 min after (+) addition of NaCl to a final concentration of 0.4 M. Protein extracts were treated (+) or not (−) with 10 U of AP. Rtg3-HA mobility was assessed by immunoblotting using anti-HA monoclonal antibody. Middle, rtg1Δhog1Δ cells expressing Rtg3-HA and carrying a mutant allele of HOG1 (HOG1as) were preincubated or not for 10 min with 5 μM of 1NM-PP1 prior to osmostress at the same conditions as in the left panel. Right, mutations of Rtg3 Thr-197, Ser-222, Ser-227, Thr-249, and Ser-376 to Ala (Rtg3^5M) abolish phosphorylation by osmostress. rtg1Δrtg3Δ strains were transformed with HA-tagged Rtg3 wild-type and the Rtg3 nonphosphorylatable mutant (Rtg3^5M) and processed as in the left panel. (C) Hog1 phosphorylation sites in Rtg3 are required for transcriptional activity. rtg3Δ strain was transformed with plasmids expressing wild-type Rtg3 or the Rtg3 nonphosphorylatable mutant (Rtg3^5M), grown in MD-Gln to 0.5–0.7 OD₆₆₀, and subjected to osmotic stress (0.4 M NaCl) for the indicated length of time. Total RNA was assayed by Northern blot analysis for transcript levels of CIT2 and DLD3, with RDN18 as a loading control.

FIGURE 7:

The Hog1 SAPK directly modulates the Rtg1/3 complex upon osmostress by a triple mechanism: (1) regulating the nuclear accumulation of the heterodimeric complex (no catalytic activity of Hog1 is required); (2) stimulating the association of the complex to the responsive promoters (Hog1 catalytic activity is required, but not Rtg3 phosphorylation); and (3) controlling Rtg3 transcriptional activity (Hog1 catalytic activity and Rtg3 phosphorylation are required).