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

Adipose tissue is a metabolically responsive endocrine organ that secretes a myriad of adipokines. Antidiabetic drugs such as peroxisome proliferator-activated receptor (PPAR) ␥ agonists target adipose tissue gene expression and correct hyperglycemia via whole-body insulin sensitization. The mechanism by which altered gene expression in adipose tissue affects liver and muscle insulin sensitivity (and thus glucose homeostasis) is not fully understood. One possible mechanism involves the alteration in adipokine secretion, in particular the up-regulation of secreted factors that increase whole-body insulin sensitivity. Here, we report the use of transcriptional profiling to identify genes encoding for secreted proteins the expression of which is regulated by PPAR␥ agonists. Of the 379 genes robustly regulated by two structurally distinct PPAR␥ agonists in the epididymal white adipose tissue (EWAT) of db/db mice, 33 encoded for known secreted proteins, one of which was FGF21. Although FGF21 was recently reported to be up-regulated in cultured adipocytes by PPAR␥ agonists and in liver by PPAR␣ agonists and induction of ketotic states, we demonstrate that the protein is transcriptionally up-regulated in adipose tissue in vivo by PPAR␥ agonist treatment and under a variety of physiological conditions, including fasting and high fat diet feeding. In addition, we found that circulating levels of FGF21 protein were increased upon treatment with PPAR␥ agonists and under ketogenic states. These results suggest a role for FGF21 in mediating the antidiabetic activities of PPAR␥ agonists.

We demonstrate here that yeast killer viruses, previously thought to be transmitted only by cytoplasmic mixing during division, mating, or other induced forms of cell fusion, are capable of extracellular transmission. Viral particles from standard K1 and K2 killer strains were used to inoculate sensitive cells of Saccharomyces cerevisiae, rendered competent by spheroplasting, lithium acetate treatment, or by natural mating. Viruses with double-stranded RNA (dsRNA) genomes are widely distributed in nature, found in a wide variety of hosts-namely, animals (1), plants (2), insects (3), fungi (4), and bacteria (5). They are associated with the production of toxins in fungi, with hypovirulence of plant pathogens, including the chestnut blight fungus, which has virtually eliminated the American chestnut, and with rotaviral infections, the major cause of childhood diarrhea. The killer phenomenon in Saccharomyces cerevisiae is unique among eukaryotic dsRNA viral systems in the detail with which interactions between viral and host components have been explored and in the actual number ofhost components known to be involved (6).Type MATERIALS AND METHODS Yeast Strains and Media. Strain GG100-14D (a his3 ura3-50 trpl phoS pho3) contains L1A and L1Bc and was derived from the mating of two nonkiller strains, DB4 (a ura3-50 pho3 phoS) and DB13-1A (a his3-532 trpl gal2 suc2). The tester strains K7.S1 (a arg9) and S6 (a/a) lack M1 dsRNA and are sensitive to yeast killer toxin. The virus donor strains K7 (a arg9)s and Y110 (a/a) contain L1A M1 and L2A L2BC M2, respectively, and are standard K1 and K2 killers. Strain K23.A (a ade2) is a killer strain containing L1A L1BC and M1, used here and in previous work as a control for dsRNA and virus protein determinations. YEPD, methylene blue, and minimal media are described elsewhere (9, 10). YEPD pH 6.5 medium is YEPD medium adjusted to a final pH of 6.5. Buffered YEPD pH 6.5 medium is YEPD medium buffered with 0.2 M Na2HPO4/citrate to pH 6.5.Preparation of Viral Particles. Virus preparations were made from cells of strains K7 and Y110 grown in YEPD medium by Zymolyase/glass bead cell disruption, clarification by centrifugation, and purification of virus on 10-14% (wt/vol) sucrose gradients as described in detail elsewhere (7) with the following exceptions. Cells were not treated with 2-mercaptoethanol but rather were harvested, washed in SEKS buffer (1 M sorbitol/0.1 M EDTA/0.1 M Na2SO3/0.8 M KCl), resuspended in the same buffer, and treated with Zymolyase-20T (Seikagaku Kogyo) at 0.25 mg/g of wet cells. Virus used for inoculating spheroplasts and LiOAc-treated cells was suspended in STC buffer (10 mM Tris-HCl, pH 7.5/1.2 M sorbitol/10 mM CaCl2). Virus preparations used for inoculating mating cells were suspended in PKE buffer (30 mM NaPO4, pH 7.6/150 mM KCI/10 mM EDTA). In both cases virus preparations were used within 1 day and were kept at 4°C. Virus preparations stored refrigerated for 3 weeks became noninfectious.Abbreviations: dsRNA, double-stranded RNA; ScV, Saccharomyc...

Echinocandins and nikkomycins are antibiotics that inhibit the synthesis of the essential cell wall polysaccharide polymers 1,3-␤-glucan and chitin, respectively. Some 40 echinocandin-resistant Saccharomyces cerevisiae mutants were isolated and assigned to five complementation groups. Four complementation groups contained mutants with 38 recessive mutations. The fifth complementation group comprised mutants with one dominant mutation, etg1-3 (strain MS10), and one semidominant mutation, etg1-4 (strain MS14).