Differential Expression and Function of Two Homologous Subunits of Yeast 1,3-β-D-Glucan Synthase

Abstract

1,3-␤-D-Glucan is a major structural polymer of yeast and fungal cell walls and is synthesized from UDP-glucose by the multisubunit enzyme 1,3-␤-D-glucan synthase. Previous work has shown that the FKS1 gene encodes a 215-kDa integral membrane protein (Fks1p) which mediates sensitivity to the echinocandin class of antifungal glucan synthase inhibitors and is a subunit of this enzyme. We have cloned and sequenced FKS2, a homolog of FKS1 encoding a 217-kDa integral membrane protein (Fks2p) which is 88% identical to Fks1p. The residual glucan synthase activity present in strains with deletions of fks1 is (i) immunodepleted by antibodies prepared against FKS2 peptides, demonstrating that Fks2p is also a component of the enzyme, and (ii) more sensitive to the echinocandin L-733,560, explaining the increased sensitivity of fks1 null mutants to this drug. Simultaneous disruption of FKS1 and FKS2 is lethal, suggesting that Fks1p and Fks2p are alternative subunits with essential overlapping function. Analysis of FKS1 and FKS2 expression reveals that transcription of FKS1 is regulated in the cell cycle and predominates during growth on glucose, while FKS2 is expressed in the absence of glucose. FKS2 is essential for sporulation, a process which occurs during nutritional starvation. FKS2 is induced by the addition of Ca 2؉ to the growth medium, and this induction is completely dependent on the Ca 2؉ /calmodulin-dependent phosphoprotein phosphatase calcineurin. We have previously shown that growth of fks1 null mutants is highly sensitive to the calcineurin inhibitors FK506 and cyclosporin A. Expression of FKS2 from the heterologous ADH1 promoter results in FK506-resistant growth. Thus, the sensitivity of fks1 mutants to these drugs can be explained by the calcineurin-dependent transcription of FKS2. Moreover, FKS2 is also highly induced in response to pheromone in a calcineurin-dependent manner, suggesting that FKS2 may also play a role in the remodeling of the cell wall during the mating process.The cell wall of Saccharomyces cerevisiae is essential for the integrity and shape of the cell and is a highly dynamic structure the composition and architecture of which vary widely depending upon the composition of the growth medium and the stage of the cell cycle (41). In addition, when haploid cells encounter pheromone of the opposite mating type, the cells transiently arrest in the G 1 phase of the cell cycle and develop an elongated projection requiring new cell wall synthesis (12). Furthermore, diploid cells which are nutritionally starved undergo meiosis and sporulation, a process requiring the formation of new cell wall around the developing spores (reviewed in reference 42).An important component of each of these cell wall types is the glucose polymer 1,3-␤-D-glucan (10, 38, 41). 1,3-␤-D-Glucan synthase (UDP-glucose:1,3-␤-D-glucan 3-␤-D-glucosyltransferase; EC 2.4.1.34) is a membrane enzyme activated by GTP which has been fractionated into soluble (GTP-binding) and membrane-bound (catalytic) components (39, 53). Members of...

Loss of Function ofKRE5Suppresses Temperature Sensitivity of Mutants Lacking Mitochondrial Anionic Lipids

“…␤-1,3-Glucan is synthesized by ␤-1,3-glucan synthase (GS) localized on the plasma membrane (Qadota et al, 1996). GS is composed of a catalytic subunit encoded by the two homologous genes FKS1 and FKS2 (Inoue et al, 1995;Mazur et al, 1995), and a regulatory subunit, the small GTPase, Rho1p (Drgonova et al, 1996;Qadota et al, 1996). The Rho-type GTPase is generally regulated by switching between a GDPbound inactive state and a GTP-bound active state (Wei et al, 1997;Ihara et al, 1998).…”

The putative flavin carrier family FlcA-C is important forAspergillus fumigatusvirulence

“…[24][25][26][27][28] In all these fungal pathogens, calcineurin is important for growth, morphology, state transitions, cation homeostasis, stress responses, and cell membrane and cell wall integrity pathways. 1,2,[29][30][31][32][33] Aspergillus fumigatus is an opportunistic fungal pathogen and the most important species causing pulmonary fungal infections. 15,[34][35][36][37][38] Although A. fumigatus is able to cause many clinical forms, the most important is the invasive pulmonary aspergillosis (IPA), that has mortality rates as high as 80% in immunosuppressed patients.…”

Differential regulation of cell wall biogenesis during growth and development in yeast

“…Although the inner two layers appear to have a general composition similar to that of the vegetative wall, the details are different. For example, Fks2p appears to be the glucan synthase used during sporulation (Mazur et al, 1995), and micro-array studies suggest that the cell wall protein population differs, with all the genes for the Pir proteins downregulated, as well as SED1, SPI1, CRH1 and CRH2, and genes such as TIR2 and YOR214c upregulated. Several members of the glycosyl transferase GAS family are downregulated to make place for others, and almost all genes in the BGL family are downregulated, whereas two sporulation-specific glucanases become expressed (Chu et al, 1998, and references therein).…”

“…Pkc1p, which is activated by Rho1p and in turn activates the Slt2-MAP kinase pathway, is also localized to sites of active growth, in a manner dependent on Rho1p and partially dependent on the actin cytoskeleton (Andrews & Stark, 2000). Unfortunately, the outputs of the Slt2-MAP kinase cascade are not fully understood, although it is clear that this cascade is involved in transcriptional regulation of several genes involved in cell wall biosynthesis (Igual et al, 1996 ;Jung & Levin, 1999), including an alternative 1,3-β-glucan synthase catalytic subunit, FKS2, which is transcribed in response to cell wall stress (Zhao et al, 1998) as well as during growth on carbon sources other than glucose (Mazur et al, 1995). Most likely, more direct effectors, i.e.…”