The Ras/cAMP pathway and the CDK-like kinase Ime2 regulate the MAPK Smk1 and spore morphogenesis in Saccharomyces cerevisiae

Abstract

Meiotic development (sporulation) in the yeast Saccharomyces cerevisiae is induced by nutritional deprivation. Smk1 is a meiosis-specific MAP kinase homolog that controls spore morphogenesis after the meiotic divisions have taken place. In this study, recessive mutants that suppress the sporulation defect of a smk1-2 temperature-sensitive hypomorph were isolated. The suppressors are partial function alleles of CDC25 and CYR1, which encode the Ras GDP/GTP exchange factor and adenyl cyclase, respectively, and MDS3, which encodes a kelch-domain protein previously implicated in Ras/cAMP signaling. Deletion of PMD1, which encodes a Mds3 paralog, also suppressed the smk1-2 phenotype, and a mds3-Delta pmd1-Delta double mutant was a more potent suppressor than either single mutant. The mds3-Delta, pmd1-Delta, and mds3-Delta pmd1-Delta mutants also exhibited mitotic Ras/cAMP phenotypes in the same rank order. The effect of Ras/cAMP pathway mutations on the smk1-2 phenotype required the presence of low levels of glucose. Ime2 is a meiosis-specific CDK-like kinase that is inhibited by low levels of glucose via its carboxy-terminal regulatory domain. IME2-DeltaC241, which removes the carboxy-terminal domain of Ime2, exacerbated the smk1-2 spore formation phenotype and prevented cyr1 mutations from suppressing smk1-2. Inhibition of Ime2 in meiotic cells shortly after Smk1 is expressed revealed that Ime2 promotes phosphorylation of Smk1's activation loop. These findings demonstrate that nutrients can negatively regulate Smk1 through the Ras/cAMP pathway and that Ime2 is a key activator of Smk1 signaling.

Figure 1.—

Isolation of extragenic suppressors of smk1 (ess mutants). (A) Strategy for homozygosis and scoring suppressor mutations of the smk1-2 sporulation phenotype (see text for details). (B) Flourescence assay of suppressor strains. The indicated strains were grown on YEPD medium at 30° and sporulated for 4 days at the permissive temperature (26°) or the nonpermissive temperature (34°) on a nitrocellulose filter that was then processed for fluorescence visualization.

Figure 2.—

ess mutants hyperaccumulate starch. The indicated haploid strains were grown overnight on YEPD medium, stained with iodine vapors, and photographed. The intensity of black staining correlates with the relative level of starch, a diagnostic phenotype of mitotic yeast strains that are deficient in PKA.

Figure 3.—

Mutations in Ras/cAMP pathway components and IME2 modify the smk1-2 fluorescence phenotype. (A) Deletion of MDS3 and/or PMD1 suppress the sporulation phenotype of smk1-2. Homozygous diploid strains of the indicated genotype were grown on YEPD, sporulated at the smk1-2 permissive temperature (26°) or nonpermissive temperature (34°) for 4 days, and processed using the fluorescence assay. (B) The C-terminal domain of Ime2 is required for cyr1-ess to suppress smk1-2. Diploid cells of the indicated genotype were assayed as described in A. All strains are homozygous except for the IME2-ΔC241/IME2 strains as indicated.

Figure 4.—

IME2-ΔC241 exacerbates the smk1-2 sporulation phenotype. The indicated strains were pregrown in YEPD, sporulated, stained with DAPI, and photographed using phase-contrast microscopy. All strains are homozygous except for the IME2-ΔC241/IME2 strain in F as indicated. Note the predominance of asci containing four refractile spores formed in the smk1-2 strain at the semipermissive temperature (arrows in C), the asci containing two refractile spores and two nuclei that are not contained in spore walls in the IME2-ΔC241/IME2-ΔC241 strain (arrows in D), and asci containing four meiotic products and the absence of refractile spores in the homozygous IME2-ΔC241/IME2-ΔC241 smk1-2/smk1-2 strain (arrows in E).

Figure 5.—

Ime2 promotes Smk1 activation. (A) An ime2-as1 SMK1-HA homozygous diploid was pregrown in YEPA and transferred to sporulation medium to induce rapid sporulation. At 4.5 hr, Bn-PP1 in DMSO (+) or DMSO alone (−) was added and cells were incubated at 30°. Cells were withdrawn at the indicated times, and total cellular extracts were prepared and analyzed by Western immunoblot using an HA antibody to detect Smk1-HA (−Smk1). The same filter was subsequently analyzed using a PSTAIRE antibody, which detects a closely spaced doublet consisting of Cdc28 and Pho85 to control for total protein (−con). (B) Same as in A, except that Bn-PP1 was added at 5.5 hr when Smk1 protein levels were higher, and cellular extracts analyzed at 6.5 or 8.5 hr as indicated. The mnd2-Δ strain was also spo11-Δ to prevent checkpoint-mediated arrest due to the unregulated activation of Ama1. The phosphorylated (active) form of Smk1-HA is indicated with a circled P.

Figure 6.—

A model for signaling interactions that connect the Ras/cAMP pathway, Ime2, Smk1, and the transcriptional program of sporulation. The early (Ime1-inducible), and middle (Ndt80-inducible) phases of the transcriptional program of meiosis are indicated by horizontal lines. High levels of glucose lead to high PKA activity that inhibits IME1 and prevents meiotic initiation (top). The regulation of the Smk1 pathway by PKA occurs only in the presence of intermediate levels of glucose that are insufficient to prevent meiotic initiation (middle). In the intermediate state PKA is shown to negatively regulate Smk1 directly, through Ime2, or through both mechanisms as discussed in the text (dotted lines). Ime2 is shown to activate the Smk1 pathway through Ndt80-dependent and Ndt80-independent mechanisms. The Ndt80-independent mechanism is largely based on the relatively rapid kinetics of Smk1 inhibition by Bn-PP1 in the ime2-as1 strain and therefore should be considered provisional (dotted lines). Transition to a low PKA state (bottom) is predicted to relieve inhibition and increase Smk1 activity through the pathways indicated.