Characterization of alcohol-induced filamentous growth in Saccharomyces cerevisiae - PubMed (original) (raw)

Characterization of alcohol-induced filamentous growth in Saccharomyces cerevisiae

M C Lorenz et al. Mol Biol Cell. 2000 Jan.

Free PMC article

Abstract

Diploid cells of the budding yeast Saccharomyces cerevisiae starved for nitrogen differentiate into a filamentous growth form. Poor carbon sources such as starches can also stimulate filamentation, whereas haploid cells undergo a similar invasive growth response in rich medium. Previous work has demonstrated a role for various alcohols, by-products of amino acid metabolism, in altering cellular morphology. We found that several alcohols, notably isoamyl alcohol and 1-butanol, stimulate filamentous growth in haploid cells in which this differentiation is normally repressed. Butanol also induces cell elongation and changes in budding pattern, leading to a pseudohyphal morphology, even in liquid medium. The filamentous colony morphology and cell elongation require elements of the pheromone-responsive MAPK cascade and TEC1, whereas components of the nutrient-sensing machinery, such as MEP2, GPA2, and GPR1, do not affect this phenomenon. A screen for 1-butanol-insensitive mutants identified additional proteins that regulate polarized growth (BUD8, BEM1, BEM4, and FIG1), mitochondrial function (MSM1, MRP21, and HMI1), and a transcriptional regulator (CHD1). Furthermore, we have also found that ethanol stimulates hyperfilamentation in diploid cells, again in a MAPK-dependent manner. Together, these results suggest that yeast may sense a combination of nutrient limitation and metabolic by-products to regulate differentiation.

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Figures

Figure 1

Figure 1

Several alcohols induce haploid filamentation. Wild-type strains MLY41 (MATa) and MLY61 (MATa/α) were incubated on solid SLAD medium containing 1% (vol/vol) of the indicated alcohol. The strains were grown at 30°C for 4 d on parafilm-seated plates before being photographed at ×25 magnification. The structures of the less common alcohols are shown below the panels.

Figure 2

Figure 2

A time course of butanol-induced morphological changes in liquid medium. Strains MLY41 (wild-type MATa), MLY61 (wild-type MATa/α), and 27-06 (Δste12 MATa) were grown overnight in liquid YPD medium, diluted into 5 ml of YPD with (+) or without (−) 1% (vol/vol) butanol in 15-ml sealed, screw-cap tubes, and grown at 30°C for the indicated times. Cells were spotted to microscope slides and photographed at ×400 magnification. The images at the 30-h time point are presented at half the magnification of the other panels to show the multicellular asters that develop in the haploid wild-type strain.

Figure 3

Figure 3

Effect of strain background on butanol-induced morphological changes. (A) Haploid strains MLY41 (Σ1278b), 10556-24D (W303), and FY69 (S288c) were incubated in liquid YPD with (+) or without (−) butanol and analyzed after 12 h for morphological abnormalities, as in Figure 2. (B) The same strains as in A were incubated on solid SLAD medium for 4 d at 30°C, scraped off the agar with a toothpick, and analyzed microscopically, as in Figure 2.

Figure 4

Figure 4

Aberrant morphologies induced in W303 after butanol exposure. Strain 10556-24D, harboring the URA3 vector YEplac195 and the LEU2 vector YEplac181, was incubated on solid SLAD medium with or without 1% butanol for 4 d at 30°C. Cells were scraped off the plate and resuspended in 10 μl of water on a microscope slide. (A) Typical cell morphology of this strain on medium lacking butanol. (B–I) Different morphologies of this W303 strain on SLAD + butanol.

Figure 5

Figure 5

Reporter gene activity after butanol exposure. MATa and MATa/α strains, both wild type (MLY41, MLY61) and Δste12 (MLY216a, MLY216a/α), were transformed with an empty URA3 vector (YEplac195) and a LEU2 vector containing either the FUS1-lacZ reporter (pJB207; top panels) or the FRE-lacZ reporter (pIL30-LEU2; bottom panels). The strains were then grown in YNB medium overnight (in duplicate) and split three ways; to the three cultures were added nothing, 1% (vol/vol) butanol, or 10 μM α-factor. β-Galactosidase activity was assayed after 4 or 24 h at 30°C in rolling cultures.

Figure 6

Figure 6

Butanol stimulates filamentation in glucose-poor medium. The MATa strain MLY41 (top panel) and the MATa/α strain MLY61 (bottom panel) were incubated on YNB (2% glucose, 37 mM ammonium), SLAD (2% glucose, 50 μM ammonium), or medium with 37 mM ammonium and either 0.2% or 0.02% glucose, either with (+) or without (−) 1% (vol/vol) butanol. Strains were grown for 4 d at 30°C.

Figure 7

Figure 7

Genetic analysis of the butanol-induced phenomenon. Strains MLY41 (wild-type MATa), MLY61 (wild-type MATa/α), 27-06 (Δste12 MATa), MLY183a (_Δtec1 MAT_α), MLY132a (Δgpa2 MATa), MLY232a (Δgpr1 MATa), and MLY108a (Δmep2 MATa) were incubated on SLAD medium with or without 1% (vol/vol) butanol. After 4 d at 30°C, colonies were photographed at ×25 magnification.

Figure 8

Figure 8

Genetic analysis of the ethanol-induced phenomenon. Strains MLY41 (wild-type MATa), MLY61 (wild-type MATa/α), HLY352 (Δste12/Δste12 MATa/α), MLY183a/α (Δtec1/Δtec1 MATa/α), MLY108a/α (Δmep2/Δmep2 MATa/α), MLY132a/α (Δgpa2/Δgpa2 MATa/α), and MLY232a/α (Δgpr1/Δgpr1 MATa/α) were incubated on SLAD medium with or without 1% (vol/vol) ethanol. After 2 d at 30°C, colonies were photographed at ×25 magnification.

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