Ime1 and Ime2 are required for pseudohyphal growth of Saccharomyces cerevisiae on nonfermentable carbon sources

Abstract

Pseudohyphal growth and meiosis are two differentiation responses to nitrogen starvation of diploid Saccharomyces cerevisiae. Nitrogen starvation in the presence of fermentable carbon sources is thought to induce pseudohyphal growth, whereas nitrogen and sugar starvation induces meiosis. In contrast to the genetic background routinely used to study pseudohyphal growth (Σ1278b), nonfermentable carbon sources stimulate pseudohyphal growth in the efficiently sporulating strain SK1. Pseudohyphal SK1 cells can exit pseudohyphal growth to complete meiosis. Two stimulators of meiosis, Ime1 and Ime2, are required for pseudohyphal growth of SK1 cells in the presence of nonfermentable carbon sources. Epistasis analysis suggests that Ime1 and Ime2 act in the same order in pseudohyphal growth as in meiosis. The different behaviors of strains SK1 and Σ1278b are in part attributable to differences in cyclic AMP (cAMP) signaling. In contrast to Σ1278b cells, hyperactivation of cAMP signaling using constitutively active Ras2(G19V) inhibited pseudohyphal growth in SK1 cells. Our data identify the SK1 genetic background as an alternative genetic background for the study of pseudohyphal growth and suggest an overlap between signaling pathways controlling pseudohyphal growth and meiosis. Based on these findings, we propose to include exit from pseudohyphal growth and entry into meiosis in the life cycle of S. cerevisiae.

FIG. 1.

Nonfermentable carbon sources stimulate pseudohyphal growth in WT a/α diploid SK1 strains (AMP 1618 × AMP 1619 transformed with pRS316). Identical results were obtained with another diploid WT strain (AMP 109) (cf. Fig. 1 and 2). Colony morphology (A), agar invasion (B), and ascus formation (C) are shown after growth for 7 days. (D) Stimulation of pseudohyphal growth in respiration-deficient petite cells. The colony morphology after 7 days of growth is shown. (E) Stimulation of pseudohyphal growth by ethanol. (F) Asci formed by pseudohyphal cells contain four viable spores. Asci formed by a WT strain (AMP109) on SLA medium supplemented with 2% KOAc were dissected with a tetrad dissection microscope, and spores were placed onto a YPD plate and allowed to germinate and grow for 2 days. (G) PCR genotyping of the mating type locus reveals a 2:2 segregation pattern for MATa and MATα.

FIG. 2.

Ime1 and the protein kinase activity of Ime2 are required for pseudohyphal growth. The colony morphology of WT (AMP 109) and ime1Δ/ime1Δ (AMP 115) strains (A) and of WT (MSY 135-43 × MSY 136-40) and ime2Δ/ime2Δ (MSY 202-14 × MSY 203-27) strains (B) is shown. Bar graphs show percentages of pseudohyphal colonies. For each strain and carbon source, >200 colonies were classified as pseudohyphal or nonpseudohyphal. Error bars represent standard errors. (C and D) Agar invasion by WT (AMP 109) and ime1Δ/ime1Δ (AMP 115) strains (C) and by WT (MSY 135-12 × MSY 138-17) and ime2Δ/ime2Δ (MSY 203-22 × MSY 206-36) strains (D). Filamentation (E) and agar invasion (F) of WT (KSY 187) and K97R-ime2/K97R-ime2 (KSY 162) strains were scored after 7 days of growth.

FIG. 3.

IME1 acts through IME2 to stimulate pseudohyphal growth. Colony morphology is shown for WT (MSY 135-12 × MSY 138-17) (A), ime1Δ/ime1Δ (AMP 115) (B), and ime2Δ/ime2Δ (MSY 203-22 × MSY 206-36) (C) strains transformed with empty vector (pRS426) or 2μm plasmids expressing IME1 (pHS103) or IME2 (pHS105) from their endogenous promoters. Similar results were obtained with an rme1Δ/rme1Δ strain (MSY 135-12 × MSY 138-17) and an RME1/RME1 strain (AMP 109). For simplicity, only MSY 135-12 × MSY 138-17 is shown in panel A. (D) Agar invasion of the strains in panel A. Filamentation and agar invasion were scored after 7 days of growth.

FIG. 4.

Recruitment of Ime1 to early meiotic gene promoters, including the IME2 promoter, by Ume6 is required for pseudohyphal growth. (A) Pseudohyphal growth is derepressed in a ume6Δ/ume6Δ (MSY 184-55 × MSY 185-65) strain compared to that in a WT strain (MSY 133-34 × MSY 136-40). (B) Abrogation of the interaction between Ume6 and Ime1 in cells expressing the T99N-UME6 allele as the sole source of Ume6 results in a pseudohyphal growth defect. Filamentation and agar invasion phenotypes of ume6Δ/ume6Δ (MSY 186-68 × MSY 188-119) strains carrying empty vector (pRS316) or plasmids expressing WT Ume6 (pRS316-UME6-lexA) or T99N-Ume6 (pRS316-T99N-UME6-lexA) are shown. Filamentation and agar invasion were scored after 7 days of growth.

FIG. 5.

Ime1 and Ime2 act independent of Flo11 expression and the filamentation MAPK cascade. (A) Haploid invasive growth is not defective in ime1Δ and ime2Δ strains. Invasive growth was scored after 3 days of growth on YPD plates. The strains used were upper WT (MSY 135-12), ime2Δ (MSY 203-27), lower WT (MSY 558-38), and ime1Δ (MSY 552-17). (B) Deletion of IME1 does not affect transcription of FLO11. RNA samples isolated from a WT (AMP 109) or ime1Δ/ime1Δ (AMP 115) strain grown to mid-log phase on YPAc and shifted for the indicated times to C-SPO medium were analyzed by Northern blotting. (C and D) The microscopic appearance of microcolonies is shown for WT (AMP 109) andime1Δ/ime1Δ (AMP 115) strains (C) and for WT (MSY 136-40 × MSY 135-12) and ime2Δ/ime2Δ (MSY 202-14 × MSY 203-27) strains (D) grown for 12 to 24 h on SLA plates containing glucose or acetate as a carbon source. The P values for all pairwise strain and medium comparisons are <0.01. (E) Examples of bud site selection in WT (AMP 109) and ime1Δ/ime1Δ (AMP 115) cells grown on SLA acetate plates at 30°C for 18 h. Bud scars were stained with calcofluor white and are false-colored in green. Bud and birth scars stained with FITC-WGA are false-colored in red to reveal the polarity of the cells. Note that calcofluor white does not stain the birth scar and that FITC-WGA does not stain the chitin ring between the mother and its growing bud. (F) Order of budding of mother and daughter cells in WT (AMP 109) and ime1Δ/ime1Δ (AMP 115) strains grown on SLA acetate plates. Abbreviations: D, daughter; M, mother. Uppercase letters represent the cell budding first, and lowercase letters represent the cell budding last. The numbers identify mother-daughter pairs. The cells from which the colonies originated are labeled with an “F.” These cells are spherical and display a random, nonpolar budding pattern.

FIG. 6.

Characterization of the roles of IME1 and IME2 in pseudohyphal growth in the Σ1278b background. (A) Inhibition of pseudohyphal growth by nonfermentable carbon sources in the Σ1278b genetic background. The strain used was MLY 61 a/α. (B) IME1 (MSY 699-01 a/α) and IME2 (MSY 694-51 a/α) are not required for pseudohyphal growth on glucose in Σ1278b cells. In both panels, the colony morphologies after 7 days of growth on the indicated carbon sources are shown.

FIG. 7.

cAMP signaling is hyperactive in Σ1278b cells compared to SK1 cells. (A) Sporulation of a/α diploid SK1 (AMP 109; filled bars) and Σ1278b (MLY 61 a/α; open bars) cells. (B) Induction of heat shock genes in WT cells (AMP 109 and MLY 61 a/α) shifted from 30°C to 39°C for 30 min. (C and D) Accumulation of glycogen (C) and trehalose (D) in WT cells (AMP 109 [filled bars] and MLY 61 a/α [open bars]) shifted from 25°C to 37°C for the indicated times. (E) Expression of constitutively active Ras2^G19V from plasmid pMW2 in a WT a/α diploid SK1 strain (AMP 109) inhibits sporulation. Filled bars, AMP 109 plus pRS316; open bars, AMP 109 plus pMW2. (F) Deletion of GPR1 in a WT a/α diploid Σ1278b strain (MLY 61 a/α) increases sporulation. Filled bars, WT (MLY 61 a/α); open bars, gpr1Δ/gpr1Δ strain (MLY 232 a/α). (G and H) Glycogen accumulation is shown for cells shifted from 25°C to 37°C for the indicated times. (G) AMP 109 transformed with pRS316 (filled bars) or pMW2 expressing Ras2^G19V (open bars). (H) MLY 61 a/α (black bars), MLY 232 a/α (gpr1Δ/gpr1Δ; gray bars), and MLY 187 a/α (ras2Δ/ras2Δ; open bars). (I and J) Trehalose accumulation in the cells shown in graphs G and H. For each measurement, the average and standard error for two replicates are shown.

FIG. 8.

cAMP signaling represses pseudohyphal growth in SK1 cells. (A) Expression of IME1 and FLO11 in WT SK1 (AMP 109) and Σ1278b (MLY 61 a/α) strains grown on rich acetate medium (2% KOAc, 1% yeast extract, 2% peptone) to exponential growth phase. All bands are from the same blot. P values were derived from an unpaired, two-tailed t test (n = 6). (B and C) Addition of 5 mM cAMP to SLA glucose plates (B) or expression of constitutively active Ras2^G19V from plasmid pMW2 (C) inhibits pseudohyphal growth in SK1 cells (AMP 109). Filamentation and agar invasion were scored after 7 days of growth. Bars, 40 μm for glucose and 100 μm for the other carbon sources. (D) Acetate induces expression of FLO11 in a WT SK1 strain (AMP 109). Cells were grown to mid-log phase on YPD or YPAc before isolation of RNA for Northern analysis. The Σ1278b strain was MLY 61 a/α. (E) Comparison of steady-state mRNA levels for IME1, IME2, the EMGs HOP1 and SPO13, and the inositol biosynthetic gene INO1 in an SK1 strain (AMP 1619) grown to exponential growth phase on glucose (lane 1) or acetate (lane 2) or 4, 8, or 12 h after being shifted to sporulation medium (C-SPO medium) (see Materials and Methods).

FIG. 9.

Control of cell differentiation by the early meiotic cascade. (A) Revised life cycle of S. cerevisiae incorporating sporulation of pseudohyphal cells. Haploid a and α cells grow and divide in a nutrient-rich environment. Mating type switching allows a cells to switch to an α mating type and vice versa. a and α cells mate to form an a/α diploid cell when exposed to mating pheromones secreted by their opposite mating types. a/α diploid cells grow and divide in a nutrient-rich environment. Severe starvation triggers sporulation of a/α diploids and formation of an ascus harboring four haploid spores. Moderate starvation triggers pseudohyphal growth, which may allow yeasts to forage for nutrients. Severe starvation of pseudohyphae also induces sporulation. After exposure to nutrients and breakdown of the ascus wall, the spores germinate to form haploid a and α cells. (B) Model summarizing how the early meiotic cascade consisting of Ime1, Ume6, and Ime2 regulates cell differentiation in diploid S. cerevisiae cells. Starvation of a/α diploid cells induces expression of Ime1 and conversion of the transcriptional repressor Ume6 to an activator, leading to induction of early meiotic genes, including IME2. Activation of IME1 and IME2 is a general differentiation signal that promotes both pseudohyphal growth and meiosis. Induction of pseudohyphal growth or meiosis by Ime1 is abolished by a T99N mutation in Ume6. Modulation of the differentiation signal generated by IME1 and IME2 by other, uncharacterized events is expected to govern the choice between pseudohyphal growth and meiosis.