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Pheromone-regulated genes required for yeast mating differentiation.

Erdman S, Lin L, Malczynski M, Snyder M - J. Cell Biol. (1998)

Bottom Line: Mutations in four novel genes, FIG1, FIG2, KAR5/ FIG3, and FIG4 were found to cause mating defects.Fig4p is a member of a family of eukaryotic proteins that contain a domain homologous to the yeast Sac1p.Our results indicate that a variety of novel genes are expressed specifically during mating differentiation to mediate proper cell morphogenesis, cell fusion, and other steps of the mating process.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Yale University, New Haven, Connecticut 06520-8103, USA.

ABSTRACT
Yeast cells mate by an inducible pathway that involves agglutination, mating projection formation, cell fusion, and nuclear fusion. To obtain insight into the mating differentiation of Saccharomyces cerevisiae, we carried out a large-scale transposon tagging screen to identify genes whose expression is regulated by mating pheromone. 91,200 transformants containing random lacZ insertions were screened for beta-galactosidase (beta-gal) expression in the presence and absence of alpha factor, and 189 strains containing pheromone-regulated lacZ insertions were identified. Transposon insertion alleles corresponding to 20 genes that are novel or had not previously been known to be pheromone regulated were examined for effects on the mating process. Mutations in four novel genes, FIG1, FIG2, KAR5/ FIG3, and FIG4 were found to cause mating defects. Three of the proteins encoded by these genes, Fig1p, Fig2p, and Fig4p, are dispensible for cell polarization in uniform concentrations of mating pheromone, but are required for normal cell polarization in mating mixtures, conditions that involve cell-cell communication. Fig1p and Fig2p are also important for cell fusion and conjugation bridge shape, respectively. The fourth protein, Kar5p/Fig3p, is required for nuclear fusion. Fig1p and Fig2p are likely to act at the cell surface as Fig1:: beta-gal and Fig2::beta-gal fusion proteins localize to the periphery of mating cells. Fig4p is a member of a family of eukaryotic proteins that contain a domain homologous to the yeast Sac1p. Our results indicate that a variety of novel genes are expressed specifically during mating differentiation to mediate proper cell morphogenesis, cell fusion, and other steps of the mating process.

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Mating projection  formation by MATa wild-type and figΔ cells in the  presence of isotropic mating  pheromone. Cells shown  were treated for 2 h with  α-factor mating pheromone  at a concentration of 5 μg/ml.  figΔ cells treated with reduced concentrations of pheromone and/or examined at  additional timepoints (i.e., 1,  4, 6 h after treatment) also  appeared normal in the shape,  number, and rate of appearance of mating projections  relative to wild-type cells.
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Figure 3: Mating projection formation by MATa wild-type and figΔ cells in the presence of isotropic mating pheromone. Cells shown were treated for 2 h with α-factor mating pheromone at a concentration of 5 μg/ml. figΔ cells treated with reduced concentrations of pheromone and/or examined at additional timepoints (i.e., 1, 4, 6 h after treatment) also appeared normal in the shape, number, and rate of appearance of mating projections relative to wild-type cells.

Mentions: As observed with the transposon insertion alleles, fig1Δ, fig2Δ, kar5Δ/fig3Δ, and fig4Δ mutants appeared normal for cell cycle arrest and recovery, pheromone sensitivity, and projection formation at all pheromone concentrations tested (Fig. 3 for mating projection results; refer to Materials and Methods). However, the figΔ strains each exhibited quantitative mating defects, and the severity of the defect differed depending upon the mating condition (Table IV). At 30°C, absence of Fig1p, Kar5/Fig3p, or Fig4p results in a bilateral mating defect that reduces mating efficiency 2.5-, 77.4-, and 2.9-fold, respectively, relative to that of a wild-type strain. In contrast, loss of Fig2p reproducibly increases the mating efficiency 3.2–7.2-fold in both unilateral and bilateral matings. Increased mating efficiency through the loss of a gene product in otherwise wild-type cells is a novel phenotype for a gene that functions in mating. The increased mating efficiency for fig2Δ strains is likely because of their enhanced agglutination relative to wild-type cells (see below). The mating phenotypes of the fig1Δ, fig2Δ, and kar5Δ/fig3Δ strains were the same as their respective transposon insertion mutants. We also tested the relative mating efficiencies of fig1Δ, fig2Δ, and fig4Δ mutants using mating conditions that concentrate cells on filters (Sprague, 1991). Under these conditions, the relative mating efficiencies of fig 1Δ and fig4Δ were similar to those observed by liquid conditions. The increased mating efficiency of fig2Δ strains was no longer observed; instead we observed a 6.6-fold decrease in mating efficiency relative to wild-type strains. We presume that in contrast to liquid mating conditions that require cells to agglutinate to mate efficiently (Kurjan, 1993), the close packing of cells caused by collection on filters reduces or eliminates the need for agglutination in the filter-mating assays. As noted below, the increased mating efficiency of fig2Δ strains in liquid assays is likely due to the hyperagglutination activity of these cells; this activity is no longer expected to be important in filter-mating assays.


Pheromone-regulated genes required for yeast mating differentiation.

Erdman S, Lin L, Malczynski M, Snyder M - J. Cell Biol. (1998)

Mating projection  formation by MATa wild-type and figΔ cells in the  presence of isotropic mating  pheromone. Cells shown  were treated for 2 h with  α-factor mating pheromone  at a concentration of 5 μg/ml.  figΔ cells treated with reduced concentrations of pheromone and/or examined at  additional timepoints (i.e., 1,  4, 6 h after treatment) also  appeared normal in the shape,  number, and rate of appearance of mating projections  relative to wild-type cells.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140177&req=5

Figure 3: Mating projection formation by MATa wild-type and figΔ cells in the presence of isotropic mating pheromone. Cells shown were treated for 2 h with α-factor mating pheromone at a concentration of 5 μg/ml. figΔ cells treated with reduced concentrations of pheromone and/or examined at additional timepoints (i.e., 1, 4, 6 h after treatment) also appeared normal in the shape, number, and rate of appearance of mating projections relative to wild-type cells.
Mentions: As observed with the transposon insertion alleles, fig1Δ, fig2Δ, kar5Δ/fig3Δ, and fig4Δ mutants appeared normal for cell cycle arrest and recovery, pheromone sensitivity, and projection formation at all pheromone concentrations tested (Fig. 3 for mating projection results; refer to Materials and Methods). However, the figΔ strains each exhibited quantitative mating defects, and the severity of the defect differed depending upon the mating condition (Table IV). At 30°C, absence of Fig1p, Kar5/Fig3p, or Fig4p results in a bilateral mating defect that reduces mating efficiency 2.5-, 77.4-, and 2.9-fold, respectively, relative to that of a wild-type strain. In contrast, loss of Fig2p reproducibly increases the mating efficiency 3.2–7.2-fold in both unilateral and bilateral matings. Increased mating efficiency through the loss of a gene product in otherwise wild-type cells is a novel phenotype for a gene that functions in mating. The increased mating efficiency for fig2Δ strains is likely because of their enhanced agglutination relative to wild-type cells (see below). The mating phenotypes of the fig1Δ, fig2Δ, and kar5Δ/fig3Δ strains were the same as their respective transposon insertion mutants. We also tested the relative mating efficiencies of fig1Δ, fig2Δ, and fig4Δ mutants using mating conditions that concentrate cells on filters (Sprague, 1991). Under these conditions, the relative mating efficiencies of fig 1Δ and fig4Δ were similar to those observed by liquid conditions. The increased mating efficiency of fig2Δ strains was no longer observed; instead we observed a 6.6-fold decrease in mating efficiency relative to wild-type strains. We presume that in contrast to liquid mating conditions that require cells to agglutinate to mate efficiently (Kurjan, 1993), the close packing of cells caused by collection on filters reduces or eliminates the need for agglutination in the filter-mating assays. As noted below, the increased mating efficiency of fig2Δ strains in liquid assays is likely due to the hyperagglutination activity of these cells; this activity is no longer expected to be important in filter-mating assays.

Bottom Line: Mutations in four novel genes, FIG1, FIG2, KAR5/ FIG3, and FIG4 were found to cause mating defects.Fig4p is a member of a family of eukaryotic proteins that contain a domain homologous to the yeast Sac1p.Our results indicate that a variety of novel genes are expressed specifically during mating differentiation to mediate proper cell morphogenesis, cell fusion, and other steps of the mating process.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Yale University, New Haven, Connecticut 06520-8103, USA.

ABSTRACT
Yeast cells mate by an inducible pathway that involves agglutination, mating projection formation, cell fusion, and nuclear fusion. To obtain insight into the mating differentiation of Saccharomyces cerevisiae, we carried out a large-scale transposon tagging screen to identify genes whose expression is regulated by mating pheromone. 91,200 transformants containing random lacZ insertions were screened for beta-galactosidase (beta-gal) expression in the presence and absence of alpha factor, and 189 strains containing pheromone-regulated lacZ insertions were identified. Transposon insertion alleles corresponding to 20 genes that are novel or had not previously been known to be pheromone regulated were examined for effects on the mating process. Mutations in four novel genes, FIG1, FIG2, KAR5/ FIG3, and FIG4 were found to cause mating defects. Three of the proteins encoded by these genes, Fig1p, Fig2p, and Fig4p, are dispensible for cell polarization in uniform concentrations of mating pheromone, but are required for normal cell polarization in mating mixtures, conditions that involve cell-cell communication. Fig1p and Fig2p are also important for cell fusion and conjugation bridge shape, respectively. The fourth protein, Kar5p/Fig3p, is required for nuclear fusion. Fig1p and Fig2p are likely to act at the cell surface as Fig1:: beta-gal and Fig2::beta-gal fusion proteins localize to the periphery of mating cells. Fig4p is a member of a family of eukaryotic proteins that contain a domain homologous to the yeast Sac1p. Our results indicate that a variety of novel genes are expressed specifically during mating differentiation to mediate proper cell morphogenesis, cell fusion, and other steps of the mating process.

Show MeSH
Related in: MedlinePlus