Multiple functional domains of the yeast l,3-beta-glucan synthase subunit Fks1p revealed by quantitative phenotypic analysis of temperature-sensitive mutants

Abstract

The main filamentous structural component of the cell wall of the yeast Saccharomyces cerevisiae is 1,3-beta-glucan, which is synthesized by a plasma membrane-localized enzyme called 1,3-beta-glucan synthase (GS). Here we analyzed the quantitative cell morphology and biochemical properties of 10 different temperature-sensitive mutants of FKS1, a putative catalytic subunit of GS. To untangle their pleiotropic phenotypes, the mutants were classified into three functional groups. In the first group, mutants fail to synthesize 1,3-beta-glucan at the proper subcellular location, although GS activity is normal in vitro. In the second group, mutants have normal 1,3-beta-glucan content but are defective in polarized growth and endocytosis. In the third group, mutations in the putative catalytic domain of Fks1p result in a loss of the catalytic activity of GS. The differences among the three groups suggest that Fks1p consists of multiple domains that are required for cell wall construction and cellular morphogenesis.

Figure 1.—

Cluster analysis of the fks1 mutants based on similarities in cell morphology. (A) Three classes detected by cluster analysis. Dissimilarity indicates a positive angle (0°–180°) between the vectors of the 501 parameter values. Blue values indicate the AU P-value (calculated with the multiscale bootstrap technique) in the dendrogram. Orange rectangles indicate robustly clustered mutant classes with an AU P-value >0.95. Red and green boxes indicate modified R_i′ statistics reflecting morphological differences between cells (see materials and methods). Positive and negative values of the modified R_i′ statistic are depicted in red and green, respectively. Class indicates a mutant group that shows similar morphological features. (B) Coherently changed parameters in each class. The red and green boxes indicate significantly increased and decreased parameters at a significance level of 0.01, respectively (Ohnuki et al. 2007).

Figure 2.—

In vitro GS activity of membrane fractions isolated from fks1 cells. Membrane fractions were isolated from cells cultured at 23°, and GS activity was assayed at 23° (left). Membrane fractions were isolated from the cells cultured at 23° and shifted to 37° for 2 hr, and GS activity was assayed at 30° (right). The data represent the means and standard deviations of at least four experiments.

Figure 3.—

Scatter plot of in vitro GS activity against fks1 mutation sites. Small circles indicate mutation sites of fks1 mutants. Circles enclosed by dotted lines indicate the classes detected and shown in Figure 1. The red arrow indicates the GS activity value of Fks1p (WT). The top parts and the bottom parts of white boxes indicate predicted exoplasmic and cytoplasmic regions, respectively. Black bars indicate predicted transmembrane regions. The small gray box indicates the putative catalytic domain.

Figure 4.—

Scatter plot of parameter A114 (proportion of cells with delocalized actin patches in unbudded cells) against in vitro GS activity. Small circles indicate values of different fks1 strains. Circles enclosed by dotted lines indicate the classes detected in Figure 1. The squared correlation coefficient (R²) of a linear regression model was 0.858 (P < 0.001).

Figure 5.—

In vivo 1,3-β-glucan synthesis in fks1 mutants. (A) Incorporation of [¹⁴C]glucose into 1,3-β-glucan of fks1 mutants. Cells were incubated at 25° (solid bars) or shifted to 34° for 2 hr (open bars), labeled with [¹⁴C]glucose for 2 hr, and incorporation of glucose into 1,3-β-glucan was measured. Data represent the means and standard deviations from at least three experiments. (B) Cell wall staining in fks1 mutants. WT, fks1-1082, fks1-1163, and fks1-1154 were incubated at 37° for 4 hr. 1,3-β-glucan, mannoproteins, and chitin were stained with aniline blue, FITC-Con A, and calcofluor white, respectively. All cells were stained without fixation.

Figure 6.—

Immunoelectron microscopic observation of fks1 cells. (a) WT, (b) fks1-1163, and (c) fks1-1154 were shifted from 25° to 37° for 4 hr, prepared for electron microscopy by freeze-substituted fixation methods, and immunolabeled using an anti-l,3-β-glucan antibody. Bar, 1 μm.

Figure 7.—

Endocytosis in fks1 mutants. (a) WT, (b) fks1-1082, (c) fks1-1163, and (d) fks1-1154 were incubated at 37° for 4 hr in YPD media and further incubated in YPD containing Lucifer yellow at 37° for 30 min. Cells were washed and observed with fluorescence microscopy.

Figure 8.—

Functional map of Fks1p. A black curved line, circles on the line, and a curved dotted line indicate Fks1p, fks1 mutation sites, and the putative catalytic domain, respectively. The gray hatched box indicates the plasma membrane. The class I mutations induce defects in in vivo glucan syntheses. The class II mutations induce defects in polarized cell wall remodeling and in endocytosis. The class III mutations induce defects in glucan synthesis and in endocytosis.