The Saccharomyces cerevisiae FKS1 (ETG1) gene encodes an integral membrane protein which is a subunit of 1,3-beta-D-glucan synthase.

Abstract

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

Cell Wall β-(1,6)-Glucan of Saccharomyces cerevisiae

“…Similar to the nonradioactive 23.6 min peak, there was complete degradation of 14 C-radiolabeled 23.6 min fraction upon endo-␤-(1,6)-glucanase treatment (Fig. 8) with a pattern similar to the one obtained after the degradation of 23.6 min polymer obtained from the S. cerevisiae cell wall.…”

“…Following sample injection, a gradient run (flow rate 1 ml/min) was performed as follows: 0 -2 min, isocratic step (98% A ϩ 2% B), 2-15 min 98% A ϩ 2% B to 65% A ϩ 35% B, 15-22 min 65% A ϩ 35% B to 57% A ϩ 43% B, 22-23 min 57% A ϩ 43% B to 100% B, and 23-25 min 100% B. Samples were detected on a pulsed electrochemical detector (nonradiolabeled digests) or using a radiometric detector (Packard Radiomatic Flo-one, equipped with a 500-l liquid type cell) for the 14 C-radiolabeled samples. Specific detection of the 14 C-radiolabeled compounds was performed at 156 keV with a scintillant at a flow rate of 2.0 ml/min.…”

Mechanisms of cytokinesis in budding yeast

“…The glucans are synthesized by two 1,3-b-D-glucan synthases (GSs), each of which consists of a putative catalytic subunit and a regulatory subunit. The catalytic subunits for the two GSs are Fks1/Gcs1 and Fks2/Gcs2, respectively [Douglas et al, 1994;Inoue et al, 1995;Mazur and Baginsky, 1996], whereas the regulatory subunit for both GSs is Rho1 [Mazur and Baginsky, 1996;Qadota et al, 1996]. Fks1 plays a major role in cell wall synthesis during vegetative growth, whereas Fks2 mainly functions during nutritional starvation, mating, and sporulation [Levin, 2005;Park and Bi, 2007].…”

Round Shape Enlargement of the Yeast Spheroplast of Saccharomyces cerevisiae by HM-1 Toxin.

“…The exact nature of the receptor has not been clarified, but cellular b-1,3-glucan synthase, a large protein spanning the membrane sixteen times, 26,27) could possibly be an HM-1 receptor. HM-1 perhaps interacts with b-1,3-glucan synthase in the membrane of spheroplasts and continues to inhibit the synthesis of glucan, an important component of cell walls, resulting in perfectly round spheroplasts with smooth surfaces.…”