Suppressors of an adenylate cyclase deletion in the fission yeast Schizosaccharomyces pombe

Abstract

Schizosaccharomyces pombe utilizes two opposing signaling pathways to sense and respond to its nutritional environment. Glucose detection triggers a cyclic AMP signal to activate protein kinase A (PKA), while glucose or nitrogen starvation activates the Spc1/Sty1 stress-activated protein kinase (SAPK). One process controlled by these pathways is fbp1+ transcription, which is glucose repressed. In this study, we isolated strains carrying mutations that reduce high-level fbp1+ transcription conferred by the loss of adenylate cyclase (git2delta), including both wis1- (SAPK kinase) and spc1- (SAPK) mutants. While characterizing the git2delta suppressor strains, we found that the git2delta parental strains are KCl sensitive, though not osmotically sensitive. Of 102 git2delta suppressor strains, 17 strains display KCl-resistant growth and comprise a single linkage group, carrying mutations in the cgs1+ PKA regulatory subunit gene. Surprisingly, some of these mutants are mostly wild type for mating and stationary-phase viability, unlike the previously characterized cgs1-1 mutant, while showing a significant defect in fbp1-lacZ expression. Thus, certain cgs1- mutant alleles dramatically affect some PKA-regulated processes while having little effect on others. We demonstrate that the PKA and SAPK pathways regulate both cgs1+ and pka1+ transcription, providing a mechanism for cross talk between these two antagonistically acting pathways and feedback regulation of the PKA pathway. Finally, strains defective in both the PKA and SAPK pathways display transcriptional regulation of cgs1+ and pka1+, suggesting the presence of a third glucose-responsive signaling pathway.

FIG. 1.

Phenotypic analysis of adenylate cyclase deletion and nft⁻ suppressor strains. Approximately 10⁵ cells of strains FWP101 (wild type [w.t.]), FWP190 (git2Δ), JSP10 (git2Δ cgs1-10), JSP12 (git2Δ spc1-12), and JSP29 (git2Δ wis1-29) were spotted to the indicated growth medium and photographed after 3 days of growth (5 days for the YEA plus gluconate plate).

FIG. 2.

Microscopic examination of cells after 24 h of growth in the presence of 1 M KCl. Strains FWP101 (wild type), FWP190 (git2Δ), JSP10 (git2Δ cgs1-10), JSP12 (git2Δ spc1-12), and JSP29 (git2Δ wis1-29) were grown to exponential phase in YEL liquid medium and then subcultured for 24 h in YEL in the presence or absence of 1 M KCl. Cells were stained with 1 μg of Hoechst 33342 stain/ml to stain the DNA prior to photographing.

FIG. 3.

Survival of S. pombe strains in the presence of 1 M KCl. Strains FWP101 (wild type), FWP190 (git2Δ), CHP742 (git2Δ cgs1-1), JSP10 (git2Δ cgs1-10), JSP12 (git2Δ spc1-12), and JSP29 (git2Δ wis1-29) were grown to exponential phase in YEL liquid medium and subcultured at 2 × 10⁷ cells/ml in YEL plus 1 M KCl. After 24, 48, and 72 h, cells were washed with YEL medium and adjusted to 2 × 10⁷ cells/ml along with four 10-fold serial dilutions. Five microliters of each culture was spotted to a YEA plate and grown for 3 days before photographing.

FIG. 4.

Characterization of mutants for salt-sensitive versus osmotically sensitive growth. Strains FWP101 (wild type), FWP190 (git2Δ), JSP10 (git2Δ cgs1-10), JSP12 (git2Δ spc1-12), and JSP29 (git2Δ wis1-29) were grown in YEL liquid medium to exponential phase and adjusted to 10⁷ cells/ml. Five fivefold serial dilutions were carried out, and 4 μl of each culture was spotted onto YEA, YEA plus 2 M sorbitol, and YEA plus 1 M KCl plates. The plates were photographed after 3 days of growth.

FIG. 5.

PCR analysis of cgs1⁺ transcriptional start site. The schematic shows exons 1 and 2 of cgs1⁺ (open boxes), the intervening intron, the relative locations to which the three forward (F1, F2, and F3) and one reverse (R1) PCR primers bind, and the site of the nucleotide altered by the cgs1-10 mutation. PCR was carried out with two forward primers and one reverse primer, as indicated above the lanes. Template DNA was either genomic DNA from strain 972 (lanes labeled G) or plasmid DNA from the SPLE1 cDNA library (lanes labeled c). The PCR products were subjected to electrophoresis in a 1.5% agarose gel, with a 100-bp ladder (lanes labeled M) as a size standard.

FIG. 6.

Stationary-phase viability assay. Strains FWP101 (wild type), FWP190 (git2Δ), CHP742 (git2Δ cgs1-1), JSP10 (git2Δ cgs1-10), JSP12 (git2Δ spc1-12), and JSP29 (git2Δ wis1-29) were grown to exponential phase in YEL liquid medium and subcultured at 10⁷ cells/ml in YEL liquid medium and grown for 9 days, after which cultures were adjusted to 8 × 10⁷ cells/ml. Five fivefold serial dilutions were carried out, and 4 μl of each culture was spotted onto YEA. The plates were photographed after 3 days of growth.

FIG. 7.

Transcription of cgs1⁺ and pka1⁺ in strains defective in PKA or SAPK signaling. Strains FWP101 (wild type), FWP190 (git2Δ), JSP10 (git2Δ cgs1-10), JSP12 (git2Δ spc1-12), and JSP29 (git2Δ wis1-29) were grown to exponential phase in YE5S liquid medium containing either 8% glucose (lanes labeled R) or 0.1% glucose with 3% glycerol (lanes labeled D). The RNA was isolated and analyzed as described in Materials and Methods.