Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Thu, 06 Dec 2018 21:40:54 The ER-Golgi v-SNARE Bet1p is required for cross-linking a-agglutinin to the cell wall in yeast Pearl Kipnis, Naomi Thomas, Rafael Ovalle and Peter N. Lipke Correspondence Peter Lipke lipke@genectr.hunter.cuny.edu Dept of Biological Sciences and the Center for Gene Structure and Function, Hunter College of the City University of New York, 695 Park Ave, New York, NY 10021, USA Received 25 March 2004 Revised 28 June 2004 Accepted 13 July 2004 In Saccharomyces cerevisiae, glycosylphosphatidylinositol (GPI)-anchored cell wall mannoproteins, including a-agglutinin, are secreted to the cell surface through vesicular transport pathways. At the cell surface the GPI anchors are cleaved within the glycan, then transglycosylated to form a covalent cross-link to 1,6-b-glucan. Among mutants that were temperature-sensitive for growth and for ability to cross-link the mannoprotein a-agglutinin to the cell wall, one strain was complemented by BET1, which encodes an ER-Golgi v-SNARE. Temperature-sensitive mutations in BET1 caused aberrations in cell wall structure, including excretion of a-agglutinin into the medium, sensitivity to lysis with Zymolyase and hypersensitivity to Calcofluor White. At restrictive temperatures, bet1 mutations block secretion of invertase and other proteins, but a-agglutinin was excreted into the extracellular medium. In wild-type parental or bet1 cells, secretion of a-agglutinin also continued after protein synthesis was blocked with cycloheximide. This secretion was due to continued export of a significant amount of a-agglutinin from compartments distal to the BET1-dependent secretion step. Thus, in bet1 cells the ER-Golgi block allowed secretion to continue, but prevented cell wall incorporation of the a-agglutinin. Therefore, a mutation early in the secretion pathway caused aberrant cell wall synthesis by preventing localization of key components required in wall cross-links. INTRODUCTION Cell walls in Saccharomyces cerevisiae are composed of an outer layer of mannoproteins and an inner layer of 1,3-b-glucans, 1,6-b-glucans and a small amount of chitin. The four components of the cell wall are linked together through the highly branched 1,6-b-glucans (Kapteyn et al., 1996, 1999; Kollar et al., 1995, 1997; Lipke & Ovalle, 1998). Of these components, mannoproteins are synthesized in the endoplasmic reticulum (ER) and secreted through the secretory pathway, being glycosylated in the ER and the Golgi. 1,3-b-glucan and chitin are synthesized at the cell surface, apparently in membrane-bound complexes that utilize cytoplasmic UDP-sugar donors to form a poly- saccharide chain that is extruded through the membrane into the exoplasmic space (Orlean, 1997). The site and mode of synthesis of the 1,6-b-glucan cross-linking polysaccha- ride are unknown, but there is evidence favouring initial synthesis either in the ER (Shahinian & Bussey, 2000) or at the plasma membrane (Montijn et al., 1999). a-Agglutinin is a wall mannoprotein that facilitates mating by mediating specific and kinetically irreversible adhesion of mating type a cells to cells of mating type a (Lipke et al., 1987; Zhao et al., 2001). This adhesin is a member of a large class of wall glycoproteins that are synthesized with glycosyl- phosphatidylinositol (GPI) anchors and are subsequently transported to the cell surface via the secretory pathway (De Groot et al., 2003; Gaynor et al., 1999; Hamada et al., 1998; Lu et al., 1994, 1995; Wojciechowicz et al., 1993). From the Golgi, such proteins are secreted to the exoplasmic face of the cell membrane. In a set of reactions that have not been characterized, the GPI glycan is cleaved, then the remnant attached to the glycoprotein is transglycosylated to 1,6-b-glucan so that the mannoprotein is covalently integrated into the wall complex (Kollar et al., 1995, 1997; Lipke & Ovalle, 1998; Lu et al., 1995). GPI anchors are essential for wall localization of this class of mannoprotein. Mutations that delete the GPI anchor signal of a-agglutinin or other GPI wall proteins result in secretion of the unanchored protein to the cell surface and excretion into the media (De Groot et al., 2003; Tsukahara et al., 2003; Vossen et al., 1997; Wojciechowicz et al., 1993). Anchorage of a-agglutinin to the cell wall is also defective in kre mutants, which have defects in synthesis of 1,6-b- glucan (Lu et al., 1995). The mechanisms responsible for the extracellular assembly and modifications of the cell wall are poorly understood. GPI-defective mutants are growth-impaired (Costello & Abbreviations: CFW, Calcofluor White; ER, endoplasmic reticulum; GPI, glycosylphosphatidylinositol. 0002-7189 G 2004 SGM Printed in Great Britain 3219 Microbiology (2004), 150, 3219–3228 DOI 10.1099/mic.0.27189-0