Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. Oct 1995; 15(10): 5671–5681.
PMCID: PMC230817

Differential expression and function of two homologous subunits of yeast 1,3-beta-D-glucan synthase.

Abstract

1,3-beta-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-beta-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 Ca2+ to the growth medium, and this induction is completely dependent on the Ca2+/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.

Full Text

The Full Text of this article is available as a PDF (621K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. [PubMed]
  • Ammerer G. Expression of genes in yeast using the ADCI promoter. Methods Enzymol. 1983;101:192–201. [PubMed]
  • Andrews BJ, Herskowitz I. Identification of a DNA binding factor involved in cell-cycle control of the yeast HO gene. Cell. 1989 Apr 7;57(1):21–29. [PubMed]
  • Andrews BJ, Moore L. Mutational analysis of a DNA sequence involved in linking gene expression to the cell cycle. Biochem Cell Biol. 1992 Oct-Nov;70(10-11):1073–1080. [PubMed]
  • Aperia A, Ibarra F, Svensson LB, Klee C, Greengard P. Calcineurin mediates alpha-adrenergic stimulation of Na+,K(+)-ATPase activity in renal tubule cells. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7394–7397. [PMC free article] [PubMed]
  • Appeltauer U, Achstetter T. Hormone-induced expression of the CHS1 gene from Saccharomyces cerevisiae. Eur J Biochem. 1989 Apr 15;181(1):243–247. [PubMed]
  • Breeden L, Nasmyth K. Cell cycle control of the yeast HO gene: cis- and trans-acting regulators. Cell. 1987 Feb 13;48(3):389–397. [PubMed]
  • Breuder T, Hemenway CS, Movva NR, Cardenas ME, Heitman J. Calcineurin is essential in cyclosporin A- and FK506-sensitive yeast strains. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5372–5376. [PMC free article] [PubMed]
  • Brizuela L, Chrebet G, Bostian KA, Parent SA. Antifungal properties of the immunosuppressant FK-506: identification of an FK-506-responsive yeast gene distinct from FKB1. Mol Cell Biol. 1991 Sep;11(9):4616–4626. [PMC free article] [PubMed]
  • Cabib E, Bowers B, Sburlati A, Silverman SJ. Fungal cell wall synthesis: the construction of a biological structure. Microbiol Sci. 1988 Dec;5(12):370–375. [PubMed]
  • Clipstone NA, Crabtree GR. Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation. Nature. 1992 Jun 25;357(6380):695–697. [PubMed]
  • Cross F, Hartwell LH, Jackson C, Konopka JB. Conjugation in Saccharomyces cerevisiae. Annu Rev Cell Biol. 1988;4:429–457. [PubMed]
  • Cross FR, Tinkelenberg AH. A potential positive feedback loop controlling CLN1 and CLN2 gene expression at the start of the yeast cell cycle. Cell. 1991 May 31;65(5):875–883. [PubMed]
  • Cunningham KW, Fink GR. Ca2+ transport in Saccharomyces cerevisiae. J Exp Biol. 1994 Nov;196:157–166. [PubMed]
  • Cyert MS, Kunisawa R, Kaim D, Thorner J. Yeast has homologs (CNA1 and CNA2 gene products) of mammalian calcineurin, a calmodulin-regulated phosphoprotein phosphatase. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7376–7380. [PMC free article] [PubMed]
  • Cyert MS, Thorner J. Regulatory subunit (CNB1 gene product) of yeast Ca2+/calmodulin-dependent phosphoprotein phosphatases is required for adaptation to pheromone. Mol Cell Biol. 1992 Aug;12(8):3460–3469. [PMC free article] [PubMed]
  • Douglas CM, Foor F, Marrinan JA, Morin N, Nielsen JB, Dahl AM, Mazur P, Baginsky W, Li W, el-Sherbeini M, et al. The Saccharomyces cerevisiae FKS1 (ETG1) gene encodes an integral membrane protein which is a subunit of 1,3-beta-D-glucan synthase. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12907–12911. [PMC free article] [PubMed]
  • Douglas CM, Marrinan JA, Li W, Kurtz MB. A Saccharomyces cerevisiae mutant with echinocandin-resistant 1,3-beta-D-glucan synthase. J Bacteriol. 1994 Sep;176(18):5686–5696. [PMC free article] [PubMed]
  • el-Sherbeini M, Clemas JA. Nikkomycin Z supersensitivity of an echinocandin-resistant mutant of Saccharomyces cerevisiae. Antimicrob Agents Chemother. 1995 Jan;39(1):200–207. [PMC free article] [PubMed]
  • Eng WK, Faucette L, McLaughlin MM, Cafferkey R, Koltin Y, Morris RA, Young PR, Johnson RK, Livi GP. The yeast FKS1 gene encodes a novel membrane protein, mutations in which confer FK506 and cyclosporin A hypersensitivity and calcineurin-dependent growth. Gene. 1994 Dec 30;151(1-2):61–71. [PubMed]
  • Foor F, Parent SA, Morin N, Dahl AM, Ramadan N, Chrebet G, Bostian KA, Nielsen JB. Calcineurin mediates inhibition by FK506 and cyclosporin of recovery from alpha-factor arrest in yeast. Nature. 1992 Dec 17;360(6405):682–684. [PubMed]
  • Frantz B, Nordby EC, Bren G, Steffan N, Paya CV, Kincaid RL, Tocci MJ, O'Keefe SJ, O'Neill EA. Calcineurin acts in synergy with PMA to inactivate I kappa B/MAD3, an inhibitor of NF-kappa B. EMBO J. 1994 Feb 15;13(4):861–870. [PMC free article] [PubMed]
  • Garrett-Engele P, Moilanen B, Cyert MS. Calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, is essential in yeast mutants with cell integrity defects and in mutants that lack a functional vacuolar H(+)-ATPase. Mol Cell Biol. 1995 Aug;15(8):4103–4114. [PMC free article] [PubMed]
  • Gewain KM, Occi JL, Foor F, MacNeil DJ. Vectors for generating nested deletions and facilitating subcloning G+C-rich DNA between Escherichia coli and Streptomyces sp. Gene. 1992 Sep 21;119(1):149–150. [PubMed]
  • Gietz RD, Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. [PubMed]
  • Gribskov M, Burgess RR. Sigma factors from E. coli, B. subtilis, phage SP01, and phage T4 are homologous proteins. Nucleic Acids Res. 1986 Aug 26;14(16):6745–6763. [PMC free article] [PubMed]
  • Hagen DC, McCaffrey G, Sprague GF., Jr Pheromone response elements are necessary and sufficient for basal and pheromone-induced transcription of the FUS1 gene of Saccharomyces cerevisiae. Mol Cell Biol. 1991 Jun;11(6):2952–2961. [PMC free article] [PubMed]
  • Halachmi D, Eilam Y. Calcium homeostasis in yeast cells exposed to high concentrations of calcium. Roles of vacuolar H(+)-ATPase and cellular ATP. FEBS Lett. 1993 Jan 18;316(1):73–78. [PubMed]
  • Hartmann E, Rapoport TA, Lodish HF. Predicting the orientation of eukaryotic membrane-spanning proteins. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5786–5790. [PMC free article] [PubMed]
  • Hereford LM, Osley MA, Ludwig TR, 2nd, McLaughlin CS. Cell-cycle regulation of yeast histone mRNA. Cell. 1981 May;24(2):367–375. [PubMed]
  • Hohmann S, Gozalbo D. Structural analysis of the 5' regions of yeast SUC genes revealed analogous palindromes in SUC, MAL and GAL. Mol Gen Genet. 1988 Mar;211(3):446–454. [PubMed]
  • Iida H, Yagawa Y, Anraku Y. Essential role for induced Ca2+ influx followed by [Ca2+]i rise in maintaining viability of yeast cells late in the mating pheromone response pathway. A study of [Ca2+]i in single Saccharomyces cerevisiae cells with imaging of fura-2. J Biol Chem. 1990 Aug 5;265(22):13391–13399. [PubMed]
  • Jain J, McCaffrey PG, Miner Z, Kerppola TK, Lambert JN, Verdine GL, Curran T, Rao A. The T-cell transcription factor NFATp is a substrate for calcineurin and interacts with Fos and Jun. Nature. 1993 Sep 23;365(6444):352–355. [PubMed]
  • Jones JS, Prakash L. Yeast Saccharomyces cerevisiae selectable markers in pUC18 polylinkers. Yeast. 1990 Sep-Oct;6(5):363–366. [PubMed]
  • Kane SM, Roth R. Carbohydrate metabolism during ascospore development in yeast. J Bacteriol. 1974 Apr;118(1):8–14. [PMC free article] [PubMed]
  • Kang MS, Cabib E. Regulation of fungal cell wall growth: a guanine nucleotide-binding, proteinaceous component required for activity of (1----3)-beta-D-glucan synthase. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5808–5812. [PMC free article] [PubMed]
  • Klee CB, Draetta GF, Hubbard MJ. Calcineurin. Adv Enzymol Relat Areas Mol Biol. 1988;61:149–200. [PubMed]
  • Klis FM. Review: cell wall assembly in yeast. Yeast. 1994 Jul;10(7):851–869. [PubMed]
  • Krisak L, Strich R, Winters RS, Hall JP, Mallory MJ, Kreitzer D, Tuan RS, Winter E. SMK1, a developmentally regulated MAP kinase, is required for spore wall assembly in Saccharomyces cerevisiae. Genes Dev. 1994 Sep 15;8(18):2151–2161. [PubMed]
  • Kronstad JW, Holly JA, MacKay VL. A yeast operator overlaps an upstream activation site. Cell. 1987 Jul 31;50(3):369–377. [PubMed]
  • Kuno T, Tanaka H, Mukai H, Chang CD, Hiraga K, Miyakawa T, Tanaka C. cDNA cloning of a calcineurin B homolog in Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1991 Oct 31;180(2):1159–1163. [PubMed]
  • Kurtz MB, Douglas C, Marrinan J, Nollstadt K, Onishi J, Dreikorn S, Milligan J, Mandala S, Thompson J, Balkovec JM, et al. Increased antifungal activity of L-733,560, a water-soluble, semisynthetic pneumocandin, is due to enhanced inhibition of cell wall synthesis. Antimicrob Agents Chemother. 1994 Dec;38(12):2750–2757. [PMC free article] [PubMed]
  • Liu J, Farmer JD, Jr, Lane WS, Friedman J, Weissman I, Schreiber SL. Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell. 1991 Aug 23;66(4):807–815. [PubMed]
  • Liu Y, Ishii S, Tokai M, Tsutsumi H, Ohki O, Akada R, Tanaka K, Tsuchiya E, Fukui S, Miyakawa T. The Saccharomyces cerevisiae genes (CMP1 and CMP2) encoding calmodulin-binding proteins homologous to the catalytic subunit of mammalian protein phosphatase 2B. Mol Gen Genet. 1991 May;227(1):52–59. [PubMed]
  • Luan S, Li W, Rusnak F, Assmann SM, Schreiber SL. Immunosuppressants implicate protein phosphatase regulation of K+ channels in guard cells. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2202–2206. [PMC free article] [PubMed]
  • McCaffrey PG, Luo C, Kerppola TK, Jain J, Badalian TM, Ho AM, Burgeon E, Lane WS, Lambert JN, Curran T, et al. Isolation of the cyclosporin-sensitive T cell transcription factor NFATp. Science. 1993 Oct 29;262(5134):750–754. [PubMed]
  • McIntosh EM, Atkinson T, Storms RK, Smith M. Characterization of a short, cis-acting DNA sequence which conveys cell cycle stage-dependent transcription in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Jan;11(1):329–337. [PMC free article] [PubMed]
  • McIntosh EM, Ord RW, Storms RK. Transcriptional regulation of the cell cycle-dependent thymidylate synthase gene of Saccharomyces cerevisiae. Mol Cell Biol. 1988 Nov;8(11):4616–4624. [PMC free article] [PubMed]
  • Mendoza I, Rubio F, Rodriguez-Navarro A, Pardo JM. The protein phosphatase calcineurin is essential for NaCl tolerance of Saccharomyces cerevisiae. J Biol Chem. 1994 Mar 25;269(12):8792–8796. [PubMed]
  • Mol PC, Park HM, Mullins JT, Cabib E. A GTP-binding protein regulates the activity of (1-->3)-beta-glucan synthase, an enzyme directly involved in yeast cell wall morphogenesis. J Biol Chem. 1994 Dec 9;269(49):31267–31274. [PubMed]
  • Nakajima-Shimada J, Iida H, Tsuji FI, Anraku Y. Monitoring of intracellular calcium in Saccharomyces cerevisiae with an apoaequorin cDNA expression system. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6878–6882. [PMC free article] [PubMed]
  • Nakamura T, Liu Y, Hirata D, Namba H, Harada S, Hirokawa T, Miyakawa T. Protein phosphatase type 2B (calcineurin)-mediated, FK506-sensitive regulation of intracellular ions in yeast is an important determinant for adaptation to high salt stress conditions. EMBO J. 1993 Nov;12(11):4063–4071. [PMC free article] [PubMed]
  • Nakamura T, Tsutsumi H, Mukai H, Kuno T, Miyakawa T. Ca2+/calmodulin-activated protein phosphatase (PP2B) of Saccharomyces cerevisiae. PP2B activity is not essential for growth. FEBS Lett. 1992 Aug 31;309(1):103–106. [PubMed]
  • Nasmyth K. A repetitive DNA sequence that confers cell-cycle START (CDC28)-dependent transcription of the HO gene in yeast. Cell. 1985 Aug;42(1):225–235. [PubMed]
  • Northrop JP, Ho SN, Chen L, Thomas DJ, Timmerman LA, Nolan GP, Admon A, Crabtree GR. NF-AT components define a family of transcription factors targeted in T-cell activation. Nature. 1994 Jun 9;369(6480):497–502. [PubMed]
  • Ogas J, Andrews BJ, Herskowitz I. Transcriptional activation of CLN1, CLN2, and a putative new G1 cyclin (HCS26) by SWI4, a positive regulator of G1-specific transcription. Cell. 1991 Sep 6;66(5):1015–1026. [PubMed]
  • O'Keefe SJ, Tamura J, Kincaid RL, Tocci MJ, O'Neill EA. FK-506- and CsA-sensitive activation of the interleukin-2 promoter by calcineurin. Nature. 1992 Jun 25;357(6380):692–694. [PubMed]
  • Parent SA, Nielsen JB, Morin N, Chrebet G, Ramadan N, Dahl AM, Hsu MJ, Bostian KA, Foor F. Calcineurin-dependent growth of an FK506- and CsA-hypersensitive mutant of Saccharomyces cerevisiae. J Gen Microbiol. 1993 Dec;139(12):2973–2984. [PubMed]
  • Ram AF, Brekelmans SS, Oehlen LJ, Klis FM. Identification of two cell cycle regulated genes affecting the beta 1,3-glucan content of cell walls in Saccharomyces cerevisiae. FEBS Lett. 1995 Jan 23;358(2):165–170. [PubMed]
  • Sarokin L, Carlson M. Short repeated elements in the upstream regulatory region of the SUC2 gene of Saccharomyces cerevisiae. Mol Cell Biol. 1986 Jul;6(7):2324–2333. [PMC free article] [PubMed]
  • Schöler A, Schüller HJ. A carbon source-responsive promoter element necessary for activation of the isocitrate lyase gene ICL1 is common to genes of the gluconeogenic pathway in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1994 Jun;14(6):3613–3622. [PMC free article] [PubMed]
  • Schreiber SL. Immunophilin-sensitive protein phosphatase action in cell signaling pathways. Cell. 1992 Aug 7;70(3):365–368. [PubMed]
  • Schreiber SL, Crabtree GR. The mechanism of action of cyclosporin A and FK506. Immunol Today. 1992 Apr;13(4):136–142. [PubMed]
  • Sigal NH, Dumont FJ. Cyclosporin A, FK-506, and rapamycin: pharmacologic probes of lymphocyte signal transduction. Annu Rev Immunol. 1992;10:519–560. [PubMed]
  • Sipos L, von Heijne G. Predicting the topology of eukaryotic membrane proteins. Eur J Biochem. 1993 May 1;213(3):1333–1340. [PubMed]
  • Stewart AA, Ingebritsen TS, Manalan A, Klee CB, Cohen P. Discovery of a Ca2+- and calmodulin-dependent protein phosphatase: probable identity with calcineurin (CaM-BP80). FEBS Lett. 1982 Jan 11;137(1):80–84. [PubMed]
  • Vai M, Gatti E, Lacanà E, Popolo L, Alberghina L. Isolation and deduced amino acid sequence of the gene encoding gp115, a yeast glycophospholipid-anchored protein containing a serine-rich region. J Biol Chem. 1991 Jul 5;266(19):12242–12248. [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Cited in Books
    Cited in Books
    PubMed Central articles cited in books
  • Compound
    Compound
    PubChem Compound links
  • Gene
    Gene
    Gene links
  • GEO Profiles
    GEO Profiles
    Related GEO records
  • HomoloGene
    HomoloGene
    HomoloGene links
  • MedGen
    MedGen
    Related information in MedGen
  • Nucleotide
    Nucleotide
    Published Nucleotide sequences
  • Pathways + GO
    Pathways + GO
    Pathways, annotations and biological systems (BioSystems) that cite the current article.
  • Protein
    Protein
    Published protein sequences
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...