JOURNAL OF BACTERIOLOGY, 0021-9193/98/$04.00+0 Mar. 1998, p. 1418–1424 Vol. 180, No. 6 Copyright © 1998, American Society for Microbiology Loss of the Plasma Membrane-Bound Protein Gas1p in Saccharomyces cerevisiae Results in the Release of 1,3-Glucan into the Medium and Induces a Compensation Mechanism To Ensure Cell Wall Integrity ARTHUR F. J. RAM, 1 † JOHAN C. KAPTEYN, 1 ROY C. MONTIJN, 1 L. HELEEN P. CARO, 1 JEROEN E. DOUWES, 2 WALTER BAGINSKY, 3 PAUL MAZUR, 3 HERMAN VAN DEN ENDE, 1 AND FRANS M. KLIS 1 * Institute of Molecular Cell Biology, BioCentrum Amsterdam, University of Amsterdam, 1098 SM Amsterdam, 1 and Department of Epidemiology and Public Health, Agricultural University, Wageningen, 2 The Netherlands, and Merck Research Laboratories, Rahway, New Jersey 07065 3 Received 25 September 1997/Accepted 12 January 1998 Deletion of GAS1/GGP1/CWH52 results in a lower -glucan content of the cell wall and swollen, more spherical cells (L. Popolo, M. Vai, E. Gatti, S. Porello, P. Bonfante, R. Balestrini, and L. Alberghina, J. Bacteriol. 175:1879–1885, 1993; A. F. J. Ram, S. S. C. Brekelmans, L. J. W. M. Oehlen, and F. M. Klis, FEBS Lett. 358:165–170, 1995). We show here that gas1 cells release 1,3-glucan into the medium. Western analysis of the medium proteins with 1,3-glucan- and 1,6-glucan-specific antibodies showed further that at least some of the released 1,3-glucan was linked to protein as part of a 1,3-glucan–1,6-glucan–protein complex. These data indicate that Gas1p might play a role in the retention of 1,3-glucan and/or -glucosylated proteins. Interestingly, the defective incorporation of 1,3-glucan in the cell wall was accompanied by an increase in chitin and mannan content in the cell wall, an enhanced expression of cell wall protein 1 (Cwp1p), and an increase in 1,3-glucan synthase activity, probably caused by the induced expression of Fks2p. It is proposed that the cell wall weakening caused by the loss of Gas1p induces a set of compensatory reactions to ensure cell integrity. The cell wall of Saccharomyces cerevisiae is a supramolecular structure that determines the shape of the cell and is respon- sible for its mechanical strength. It consists of four main com- ponents that are synthesized and modified either by plasma membrane-bound complexes (chitin and 1,3-glucan) or by enzymes in the secretory pathway (cell wall mannoproteins and possibly also 1,6-glucan) (for reviews, see references 29 and 39). An important component of the cell wall is the glucose polymer 1,3-glucan. The 1,3-glucan synthase activity is lo- calized at the inner side of the plasma membrane and activated by GTP-bound Rho1p (10, 36, 44). Multiple approaches have led to the identification of a putative membrane-bound subunit of a 1,3-glucan synthase complex. The gene FKS1 (8, 11) was also cloned as CND1 (15), CWH53 (46), ETG1 (7), GSC1 (21), and PBR1 (3), and it encodes a large protein of 215 kDa with multiple transmembrane helices. Loss of the gene resulted in a dramatic reduction in 1,3-glucan synthase activity (8, 21, 41), as well as a reduction in 1,3-glucan content (3, 46). An alter- native subunit of the 1,3-glucan synthase complex was cloned by homology to FKS1. The homolog FKS2/GSC2 is 88% iden- tical to FKS1. Disruption of either FKS1 or FKS2 yields viable cells, but simultaneous disruption is lethal (21, 35), indicating that they have overlapping functions. Transcription of FKS1 is cell cycle regulated (35, 46) and predominates during growth on glucose (35). FKS2 is expressed in the absence of glucose and is induced by the addition of Ca 2+ . Disruption of FKS1 induces the expression of FKS2, which is responsible for the residual glucan synthase activity in an fks1 strain (35). 1,3-Glucans are synthesized as linear molecules that are extruded into the periplasmic space. In a mature cell wall, however, most of the 1,3-glucan is branched (34), covalently linked to chitin (30), or covalently linked to 1,6-glucan and 1,6-glucan-containing cell wall mannoproteins (13, 27, 37). The formation of branch points and cross-links presumably takes place outside the plasma membrane by transglucosyla- tion reactions catalyzed by extracellular enzymes. The formation of a rigid cell wall requires proper cross- linking of the cell components. We therefore anticipated that mutations leading to defects in cross-linking would affect cell wall integrity. To search for such genes, we carried out a genetic screen for mutants hypersensitive to Calcofluor White (45). Calcofluor White is known to interfere with the extracel- lular assembly of cell wall components. One of the isolated mutants, cwh52, was shown to be identical to GAS1/GGP1 (46). This gene encodes an abundant 125-kDa glycoprotein anchored to the external face of the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor (38, 52). We will refer to this gene as GAS1 for the remainder of this paper. The function of Gas1p is unknown. Deletion of GAS1 was not lethal but resulted in an apparently lower -glucan content of the cell wall (46) and a more spherical morphology (42). In view of the localization of Gas1p at the extracellular side of the plasma membrane, these data suggest a possible role for Gas1p * Corresponding author. Mailing address: Fungal Cell Wall Group, Institute of Molecular Cell Biology, BioCentrum Amsterdam, Univer- sity of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Nether- lands. Phone: (31) 20 5257834. Fax: (31) 20 5257934. E-mail: klis@bio .uva.nl. † Present address: Institute of Molecular Plant Sciences, Aspergillus Group, Clusius Laboratories, 2333 AL Leiden, The Netherlands. 1418