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...

In Saccharomyces cerevisiae, mutations in FKSJ confer hypersensitivity to the immunosuppressants FK506 and cyclosporin A, while mutations in ETGI confer resistance to the cell-wall-active echinocandins (inhibitors of 1,3-J3D-glucan synthase) and, in some cases, concomitant hypersensitivity to the chitin synthase inhibitor nikkomycin Z.The FKS1 and ETGI genes were cloned by complementation of these phenotypes and were found to be identical. The immunosuppressants FK506 and cyclosporin A (CsA) also have antifungal activity. Although vegetative growth of yeast is not potently inhibited by these drugs, recovery from mating factor arrest is (8). The drugs inhibit yeast recovery and T-cell activation by similar mechanisms. Each binds to an intracellular receptor (FKBP12 for FK506 and cyclophilin for CsA), and the receptor-drug complex inhibits the Ca2+/ calmodulin-dependent protein phosphatase calcineurin (9, 10). We previously described a mutation (Jksl-l) which results in calcineurin-dependent growth and hypersensitivity to FK506 (FKs) and CsA (11). We cloned the hypersensitivity locus (FKSJ) to help identify targets of calcineurin.t To our surprise FKS1 and ETGI are identical.:MATERIALS AND METHODS Microbiological Methods and Strains. YPAD and drop-out (DO) media and procedures for mating, sporulation, tetrad analysis, transformation, gene disruption, and determination of antibiotic sensitivity have been described (6,12). Meiotic progeny of diploid YFK016 (12) were mated to produce the yeast a/a diploid YFK419 (homozygous for ade2-101 his3-A200 leu2-Al lys2-801 trpl-Al, and ura3-52). R560-1C (MATa ade2-1 canl his3-11,15 leu2-3,112 trpl-l ura3-1 etgl-l) and MS14 (MATa etgl4) are spontaneous L-733,560-resistant (EchR) mutants derived from W303-1A (6) and X2180-1A (7), respectively. EchR mutants are resistant to drug on uracil DO medium at 8 ,g/ml, whereas the wild type is sensitive at 0.25 ,ug/ml. Heterozygous (etgl-l/+) strains exhibited intermediate resistance (Echl phenotype) and were resistant at 1 pg/ml but sensitive at 4 ug/ml.Cloning. A plasmid (pFF119) complementing Jksl-l was selected from a yeast genomic library of strain GRF88 (13) on uracil DO medium containing FK506 at 1 pg/ml. A library of genomic DNA (provided by S. Parent) from strain YFK093 (12) was constructed as described (14) by partial Sau3A1 digestion, partial fill-in of overhangs, and insertion of the fragments into the partially filled-in Sal I site of plasmid YEp24. The YFK093 library was introduced into strain R560-1C by the spheroplast transformation method, uracil

Elongation factor 2 (EF2) is an essential protein catalyzing ribosomal translocation during protein synthesis and is highly conserved in all eukaryotes. It is largely interchangeable in translation systems reconstituted from such divergent organisms as human, wheat, and fungi. We have identified the sordarins as selective inhibitors of fungal protein synthesis acting via a specific interaction with EF2 despite the high degree of amino acid sequence homology exhibited by EF2s from various eukaryotes. In vitro reconstitution assays using purified components from human, yeast, and plant cells demonstrate that sordarin sensitivity is dependent on fungal EF2. Genetic analysis of sordarin-resistant mutants of Saccharomyces cerevisiae shows that resistance to the inhibitor is linked to the genes EFT1 and EFT2 that encode EF2. Sordarin blocks ribosomal translocation by stabilizing the fungal EF2-ribosome complex in a manner similar to that of fusidic acid. The fungal specificity of the sordarins, along with a detailed understanding of its mechanism of action, make EF2 an attractive antifungal target. These findings are of particular significance due to the need for new antifungal agents.The elongation phase of translation in fungi requires the soluble elongation factors EF1␣, EF2, and EF3. EF1␣ and EF2 are members of the GTPase superfamily of proteins and are characterized by common structural motifs and their ability to alternate between conformational states in response to binding GDP or GTP. These proteins are required for translation in all eukaryotes, while EF3 is unique to fungi and essential for fungal protein synthesis (1). EF2 catalyzes the translocation of the ribosome along messenger RNA, presumably by stimulating a gross rearrangement of the ribosome that results in peptidyl-tRNA transfer and the movement of mRNA by one codon. The protein sequence of EF2 has been highly conserved throughout evolution, with Saccharomyces cerevisiae EF2 sharing 66% identity and 85% homology to human EF2. Despite this high degree of similarity, a class of tetracyclic diterpene glycoside natural products, the sordarins, has now been identified as selective inhibitors of EF2 function in fungal protein synthesis. Sordarin, produced by species of the fungal genus Sordaria, was described as an antifungal agent in 1970 (2, 3), but the mode of action of this family has not been examined until now. In this report, we show that sordarins specifically bind to the S. cerevisiae EF2-ribosome complex and block protein synthesis by inhibiting the release of EF2 from the posttranslocational ribosome. Our observations show that it is possible to inhibit fungal EF2 specifically, which may provide an opportunity for developing antifungal agents with a unique and selective mechanism of action. EXPERIMENTAL PROCEDURESSordarin was isolated essentially as described for Sordaria arenosa (2). Reticulocyte and wheat germ lysates were obtained from Promega.Assays-IC 50 values were determined from growth inhibition assays in which cells were inoculated a...

The structurally unrelated immunosuppressants FK506 and cyclosporin A (CsA) act similarly, inhibiting a Ca(2+)-dependent signal required for interleukin-2 transcription and T-cell activation. Each drug binds to its cytosolic receptor, FKBP-12 and cyclophilin, respectively, and the drug-receptor complexes inhibit the Ca2+/calmodulin-dependent protein phosphatase, calcineurin. In yeast, calcineurin has been implicated in recovery from alpha-mating factor arrest. Here we show that FK506 bound to yeast FKBP-12 appears to form a complex with yeast calcineurin. Moreover, recovery from mating factor arrest is highly sensitive to FK506 or CsA, and this sensitivity requires the presence of FKBP-12 or cyclophilin, respectively. These results define a key physiological target of an FK506- and CsA-sensitive signal pathway in yeast, suggest a high degree of mechanistic conservation with mammalian cells, and indicate that further examination of the yeast system should provide insight into the same process in T cells.

Saccharomyces cerevisiae has two highly homologous genes, FKS1 and FKS2, which encode interchangeable putative catalytic subunits of 1,3-␤-glucan synthase (GS), an enzyme that synthesizes an essential polymer of the fungal cell wall. To determine if GS in Aspergillus species is similar, an FKS homolog, fksA, was cloned from Aspergillus nidulans by cross-hybridization, and the corresponding protein was purified. Sequence analysis revealed a 5,716-nucleotide coding region interrupted by two 56-bp introns. The fksA gene encodes a predicted peptide of 229 kDa, FksAp, that shows a remarkable degree of conservation in size, charge, amino acid identity, and predicted membrane topology with the S. cerevisiae FKS proteins (Fksps). FksAp exhibits 64 and 65% identity to Fks1p and Fks2p, respectively, and 79% similarity. Hydropathy analysis of FksAp suggests an integral membrane protein with 16 transmembrane helices that coincide with the transmembrane helices of the Saccharomyces Fksps. The sizes of the nontransmembrane domains are strikingly similar to those of Fks1p. The region of FksAp most homologous to the Saccharomyces FKS polypeptides is a large hydrophilic domain of 578 amino acids that is predicted to be cytoplasmic. This domain is 86% identical to the corresponding region of Fks1p and is a good candidate for the location of the catalytic site. Antibodies raised against a peptide derived from the FksAp sequence recognize a protein of ϳ200 kDa in crude membranes and detergent-solubilized active extracts. This protein is enriched ϳ300-fold in GS purified by product entrapment. Purified anti-FksAp immunoglobulin G immunodepletes nearly all of the GS activity in crude or purified extracts when Staphylococcus aureus cells are used to precipitate the antibodies, although it does not inhibit enzymatic activity when added to extracts. The purified GS is inhibited by echinocandins with a sensitivity equal to that displayed by whole cells. Thus, the product of fksA is important for the activity of highly purified preparations of GS, either as the catalytic subunit itself or as an associated copurifying subunit that mediates susceptibility of enzymatic activity to echinocandin inhibition.