The Yeast Cell Wall and Septum as Paradigms of Cell Growth and Morphogenesis (2001)

CSII, CSIII, chitin synthase I, II, and III, respectively endoplasmic reticulum. Roman numerals are used to designate the enzymes because, in addition to the putative catalytic moieties, i.e. Chs1p, Chs2p and Chs3p, they may contain activators (such as Chs4p for CSIII) and perhaps proteins needed to guide the newly formed polysaccharide through the plasma membrane Fungal cells, like their bacterial and plant counterparts but unlike animal cells, are encased in a sturdy shell, the cell wall, which is essential for their survival. Fungal cells have a very high turgor pressure, and even a minor chink in the cell wall structure can lead to bursting and death (1Cabib E. Sburlati A. Bowers B. Silverman S.J. J. Cell Biol. 1989; 108: 1665-1672Crossref PubMed Scopus (138) Google Scholar). Yet, because the cell wall is external to the cell, it must grow with it and endure all the changes that the cell undergoes during the division cycle. To ensure the continuous integrity of the cell wall without interfering with its plasticity and constant growth, elaborate control mechanisms must be operating, which need to be strictly coordinated with those governing the cell cycle. There are two major reasons for studying the fungal cell wall and its specialized structure for cell division, the septum. 1) Of theoretical interest is that the cell wall, because it imparts shape to the cell in a constantly changing pattern and because of its relatively simple composition, serves as a good model for morphogenesis at the molecular level. 2) Another reason, of an applied nature, is that cell wall synthesis is an attractive target for antifungal agents because it is unique to the pathogen. Because of the versatility of budding yeast for biochemical and genetic studies, the most comprehensive body of work on fungal cell walls has been carried out with this organism. Therefore, this review will deal specifically with Saccharomyces cerevisiae. The yeast cell wall consists mainly of polysaccharides made up of three sugars, glucose, mannose, and N-acetylglucosamine. One of the glucose polysaccharides, β(1→3)glucan, is the major structural component of the wall. Another, β(1→6)glucan, is relatively minor in amount but very important for cross-linking (see below). The mannose polysaccharides are linked to proteins to form a mannoprotein layer mainly localized at the external surface and acting as a filter for large molecular weight materials (2Zlotnik H. Fernández M.P. Bowers B. Cabib E. J. Bacteriol. 1984; 159: 1018-1026Crossref PubMed Google Scholar). Finally, the small amount of GlcNAc present in the wall (1–3%) is in the β(1→4)-linked linear polysaccharide chitin, which is mostly concentrated in the septal region although some is dispersed throughout the wall. Despite its small quantity, chitin is essential for yeast survival (3Shaw J.A. Mol P.C. Bowers B. Silverman S.J. Valdivieso M.H. Durán A. Cabib E. J. Cell Biol. 1991; 114: 111-123Crossref PubMed Scopus (361) Google Scholar), probably because of its central role in septation. An early indication that the yeast cell wall components are attached to each other by covalent linkages was that mannoproteins, insoluble in native walls, became soluble in water after digestion of the walls with an endo-β(1→3)-glucanase (2Zlotnik H. Fernández M.P. Bowers B. Cabib E. J. Bacteriol. 1984; 159: 1018-1026Crossref PubMed Google Scholar). This suggested a direct or indirect attachment between β(1→3)glucan and mannoproteins. The first fully characterized linkage between cell wall components was the β(1→4) bond between the reducing terminal GlcNAc of a chitin chain and the nonreducing terminal glucose of β(1→3)glucan (Fig.1 A) (4Kollár R. Petráková E. Ashwell G. Robbins P.W. Cabib E. J. Biol. Chem. 1995; 270: 1170-1178Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Other studies revealed an association between mannoproteins, β(1→6)glucan and β(1→3)glucan (5Kapteyn J.C. Montijn R.C. Vink E. de la Cruz J. Llobell A. Shimoi H. Lipke P. Klis F.M. Glycobiology. 1996; 6: 337-345Crossref PubMed Scopus (223) Google Scholar). Finally, the linkages of a complex including those three components plus chitin were identified and partially characterized (Fig. 1 B) (6Kollár R. Reinhold B.B. Petráková E. Yeh H.J.C. Ashwell G. Drgonová J. Kapteyn J.C. Klis F.M. Cabib E. J. Biol. Chem. 1997; 272: 17762-17775Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar). In this complex, β(1→6)glucan occupies a central position. To it, chitin and β(1→3)glucan are directly attached by glycosidic linkages, whereas mannoprotein is joined to the polysaccharide through the lipidless remnant of a glycosylphosphatidylinositol anchor (Fig.1 B). These anchors have been identified as signals for the incorporation of proteins in the cell wall after transit through the plasma membrane (for review, see Ref. 7Lipke P.N. Ovalle R. J. Bacteriol. 1998; 180: 3735-3740Crossref PubMed Google Scholar). In addition to the complex shown in Fig. 1 B, another was isolated that lacked the mannoprotein. Together with the previous identification of a chitin-β(1→3)glucan compound, this finding suggested the notion of a “flexible building block” (6Kollár R. Reinhold B.B. Petráková E. Yeh H.J.C. Ashwell G. Drgonová J. Kapteyn J.C. Klis F.M. Cabib E. J. Biol. Chem. 1997; 272: 17762-17775Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar) or “module” (7Lipke P.N. Ovalle R. J. Bacteriol. 1998; 180: 3735-3740Crossref PubMed Google Scholar) for cell wall construction. These modules, of somewhat variable structure, would form a lattice all around the cell, thus generating an essentially continuous envelope. The modules may be joined to each other through hydrogen bonds (Fig. 1 C). It is also possible that the β(1→3)glucan branches joined to the main chain by β(1→6) linkages are longer than formerly thought, and this would provide the means for a practically endless network of the branched polysaccharide around the cell (Fig. 1 D). Other linkages are also present in the cell wall. A family of proteins called Pir (proteins with internalrepeats) seems to be attached directly to β(1→3)glucan through an alkali-labile linkage of as yet unresolved nature (8Mrsa V. Seidl T. Gentzsch M. Tanner W. Yeast. 1997; 13: 1145-1154Crossref PubMed Scopus (190) Google Scholar, 9Kapteyn J.C. Van Egmont P. Slevi E. Van Den Ende H. Makarow M. Klis F.M. Mol. Microbiol. 1999; 31: 1835-1844Crossref PubMed Scopus (141) Google Scholar). In addition, some proteins are released from the wall with sulfhydryl compounds (Fig. 1, C and D) (10Moukadiri I. Jaafar L. Zueco J. J. Bacteriol. 1999; 181: 4741-4745Crossref PubMed Google Scholar). Obviously, the cross-links must contribute to the mechanical strength of the wall. They could also be important in the loosening and remodeling of the wall, such as at bud emergence. Some critical linkages could be cut temporarily by appropriate enzymes to allow for wall expansion, while the remainder of the network would keep the wall together. The growth of the cell wall must accompany that of the cell that it encloses. In particular, new cell wall must be generated at the emergence of a new bud; its synthesis continues throughout the growth of the bud and stops at maturation. To study the temporal and spatial regulatory controls that direct this process, β(1→3)glucan, the major structural component of the yeast cell wall, has been used as a model. The enzymatic system that catalyzes the synthesis of β(1→3)glucan from UDP-glucose is bound to the plasma membrane (for review, see Ref. 11Cabib E. Drgonová J. Drgon T. Annu. Rev. Biochem. 1998; 67: 307-333Crossref PubMed Scopus (156) Google Scholar). An early finding was that GTP is a potent activator of β(1→3)glucan synthase at micromolar concentration (11Cabib E. Drgonová J. Drgon T. Annu. Rev. Biochem. 1998; 67: 307-333Crossref PubMed Scopus (156) Google Scholar). Later studies with yeast and other fungi led to the stepwise solubilization of two fractions from the membrane, both required simultaneously for glucan synthesis. One contains a small (26 kDa) GTP-binding protein; the other harbors the catalytic component(s) of the synthase (11Cabib E. Drgonová J. Drgon T. Annu. Rev. Biochem. 1998; 67: 307-333Crossref PubMed Scopus (156) Google Scholar). The GTP-binding protein was identified as Rho1p (12Drgonová J. Drgon T. Tanaka K. Kollár R. Chen G.-C. Ford R.A. Chan C.S.M. Takai Y. Cabib E. Science. 1996; 272: 277-279Crossref PubMed Scopus (302) Google Scholar,13Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (394) Google Scholar), whereas a large hydrophobic protein, Fks1p, was found in the catalytic fraction (14Douglas C.M. Foor F. Marrinan J.A. Morin N. Nielsen J.B. Dahl A.M. Mazur P. Baginski N. Li W. El-Sherbeini M. Klemas J.A. Mandala S.M. Frommer B.M. Kurtz M.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12907-12911Crossref PubMed Scopus (338) Google Scholar). Properties of mutants and experiments with specific inhibitors indicate that Fks1p or its close homolog Fks2p is necessary for β(1→3)glucan synthesis in vivo (14Douglas C.M. Foor F. Marrinan J.A. Morin N. Nielsen J.B. Dahl A.M. Mazur P. Baginski N. Li W. El-Sherbeini M. Klemas J.A. Mandala S.M. Frommer B.M. Kurtz M.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12907-12911Crossref PubMed Scopus (338) Google Scholar). There is no proof that Fks1p or Fks2p contains the catalytic site for glucan synthesis, but they are the only present candidates for such a function. Their participation in the synthetic reaction is supported by the coimmunoprecipitation of Fks1p and Rho1p (13Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (394) Google Scholar). The discovery that a GTP-binding protein was the regulator of β(1→3)glucan synthase went a long way to explain how the synthesis of the polysaccharide product can be started or shut off (11Cabib E. Drgonová J. Drgon T. Annu. Rev. Biochem. 1998; 67: 307-333Crossref PubMed Scopus (156) Google Scholar). GTP-binding proteins can act as molecular switches because they are active when bound to GTP and inactive when bound to GDP. The conversion from active to inactive form is stimulated by GTPase-activating proteins, whereas the opposite shift is helped by GTP-GDP exchange factors. Two proteins, Rom1p and Rom2p, have been identified as GTP-GDP exchange factors for Rho1p (15Ozaki K. Tanaka K. Imamura H. Hihara T. Kameyama T. Nonaka H. Hirano H. Matsuura Y. Takai Y. EMBO J. 1996; 15: 2196-2207Crossref PubMed Scopus (183) Google Scholar), whereas Bem2p and Sac7p are potential, albeit yet unproven, GTPase-activating proteins for Rho1p (11Cabib E. Drgonová J. Drgon T. Annu. Rev. Biochem. 1998; 67: 307-333Crossref PubMed Scopus (156) Google Scholar). The shift of Rho1p between the active and inactive state provides a rationale for the switching on and off of β(1→3)glucan and cell wall synthesis. As pointed out below, Rho1p has other functions in addition to regulation of β(1→3)glucan synthase. We recently found that rho1 mutants with a temperature-sensitive defect specific for the glucan synthesis function die at the nonpermissive temperature. 1D.-H. Roh and E. Cabib, unpublished results. Although for some timerho1 mutants incorporate other components in the cell wall, these components cannot be anchored to β(1→3)glucan. These results underline the importance of β(1→3)glucan for the preservation of cell wall architecture. The mechanism of biosynthesis of β(1→6)glucan, a central component of the wall cross-linked modules, has been studied by a genetic approach. Bussey and co-workers (reviewed in Ref. 16Shahinian S. Bussey H. Mol. Microbiol. 2000; 35: 477-489Crossref PubMed Scopus (143) Google Scholar) took advantage of the fact that β(1→6)glucan is a receptor for the K1 killer toxin to isolate mutants resistant to the toxin because of their diminished β(1→6)glucan content. 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The cell wall and up about of the yeast cell the cell a deal of its and of its synthetic to these cell wall and is of cell growth and it must be strictly in and to accompany the other that are of the cell cycle. studying those regulatory mechanisms can how the cell proteins, and with a the study of yeast cell wall and has a of about the new for antifungal compounds have been The main have been chitin and β(1→3)glucan synthesis, for which such as the and for chitin and the for have The glucan synthesis inhibitors are study of the fungal cell wall may be to to both new and We for a critical of the