Homologous Subunits of 1,3-Beta-Glucan Synthase Are Important for Spore Wall Assembly in Saccharomyces cerevisiae

Abstract

During sporulation in Saccharomyces cerevisiae, the four haploid nuclei are encapsulated within multilayered spore walls. Glucan, the major constituent of the spore wall, is synthesized by 1,3-␤-glucan synthase, which is composed of a putative catalytic subunit encoded by FKS1 and FKS2. Although another homolog, encoded by FKS3, was identified by homology searching, its function is unknown. In this report, we show that FKS2 and FKS3 are required for spore wall assembly. The ascospores of fks2 and fks3 mutants were enveloped by an abnormal spore wall with reduced resistance to diethyl ether, elevated temperatures, and ethanol. However, deletion of the FKS1 gene did not result in a defective spore wall. The construction of fusion genes that expressed Fks1p and Fks2p under the control of the FKS2 promoter revealed that asci transformed with FKS2p-driven Fks1p and Fks2p were resistant to elevated temperatures, which suggests that the expression of FKS2 plays an important role in spore wall assembly. The expression of FKS1p-driven Fks3p during vegetative growth did not affect 1,3-␤-glucan synthase activity in vitro but effectively suppressed the growth defect of the temperature-sensitive fks1 mutant by stabilizing Rho1p, which is a regulatory subunit of glucan synthase. Based on these results, we propose that FKS2 encodes the primary 1,3-␤-glucan synthase in sporulation and that FKS3 is required for normal spore wall formation because it affects the upstream regulation of 1,3-␤-glucan synthase.Sporulation in the budding yeast Saccharomyces cerevisiae provides a model system for studying the developmental processes of many eukaryotic cells. Sporulation in the a/␣ diploid cells is triggered by carbon starvation and is followed by meiosis and the formation of asci that contain four haploid spores encapsulated within a spore wall (25, 33). The spore wall, which consists of four distinct layers, has been shown to play a central role in protecting the cell from environmental damage. The inner two layers consist of ␤-glucan and mannan (4), components that are similar to those found in the vegetative cell wall. In contrast, the outer layer consists of chitosan, a polymer of ␤-1,4-linked glucosamine, and the outermost layer consists of dityrosine, both of which are specific to the spore wall (3,4,5,6,43). Previous investigations of spore wall formation have focused on the specific components of the spore wall, such as chitosan and dityrosine. The mechanism of assembly of spore walls, including the synthesis of the inner two layers, remains unclear. We speculate that ␤-glucan is also largely responsible for spore resistance to environmental damage, since glucan is the major constituent of the spore wall (4) and provides rigidity to the cell wall during vegetative growth (36).In yeast, glucan is constituted predominantly by 1,3-␤-glucan, which is synthesized by 1,3-␤-glucan synthase (GS), which in turn consists of a catalytic and a regulatory subunit. Two genes for the putative catalytic subunit of GS in budding yeast have been...

Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

“…Fks2 is important in sporulation because fks2D fks2D diploids have a severe defect in this process (Mazur et al 1995;Huang et al 2005;Ishihara et al 2007). Homozygous fks3D fks3D diploids also form abnormal spores, indicating a role for Fks3 in ascopore wall formation, although Fks3's role in sporulation does not overlap with Fks2's.…”

Sporulation in the Budding Yeast Saccharomyces cerevisiae

“…But the function of FKS3 may not be directly related to that of FKS1 and FKS2, as overexpression of neither FKS1 nor FKS2 can rescue the fks3D defects (Ishihara et al 2007). Expression of FKS3 in vegetative cells can rescue an fks1 temperature-sensitive allele, but it does so by influencing upstream regulators of FKS1 rather than by providing glucan synthase activity (Ishihara et al 2007). Thus, the precise role of Fks3 in assembly of the b-glucan layer remains to be determined.…”

“…Simultaneous deletion of FKS1 and FKS2 is lethal (Inoue et al 1995;Mazur et al 1995). During sporulation, Fks2 is primarily responsible for synthesis of the b-glucan layer, due largely to its higher expression (Mazur et al 1995;Ishihara et al 2007). …”

“…Fks3, also plays a role in spore wall assembly, as fks3D mutants display spore wall defects (Ishihara et al 2007;Suda et al 2009). But the function of FKS3 may not be directly related to that of FKS1 and FKS2, as overexpression of neither FKS1 nor FKS2 can rescue the fks3D defects (Ishihara et al 2007).…”

Regulation of Cell Wall Biogenesis in Saccharomyces cerevisiae: The Cell Wall Integrity Signaling Pathway

“…The Rho1 interaction site on Fks1 and Fks2, which has not yet been identified, may give clues as to the manner by which this GTPase activates the enzyme. A third gene encoding a homolog of FKS1/2, called FKS3, is important for spore wall formation, but appears to function as a positive regulator of GS activity rather than as a catalytic subunit, possibly by stabilizing Rho1 (Ishihara et al 2007). …”