Architecture of the Yeast Cell Wall

doi:10.1074/jbc.272.28.17762

Architecture of the Yeast Cell Wall

β(1→6)-GLUCAN INTERCONNECTS MANNOPROTEIN, β(1→3)-GLUCAN, AND CHITIN*

From the ^‡Laboratory of Biochemistry and Metabolism, the ^‖Laboratory of Bioorganic Chemistry, and the ^**Laboratory of Cell Biochemistry and Biology, NIDDK, Bethesda, Maryland 20892, the ^§Mass Spectrometry Resource, Boston University School of Medicine, Boston, Massachusetts 02118, the ^¶Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, and the ^‡Institute of Molecular Cell Biology, University of Amsterdam, 1098 SM Amsterdam, The Netherlands

Abstract

In a previous study (Kollár, R., Petráková, E., Ashwell, G., Robbins, P. W., and Cabib, E. (1995) J. Biol. Chem. 270, 1170–1178), the linkage region between chitin and β(1→3)-glucan was solubilized and isolated in the form of oligosaccharides, after digestion of yeast cell walls with β(1→3)-glucanase, reduction with borotritide, and subsequent incubation with chitinase. In addition to the oligosaccharides, the solubilized fraction contained tritium-labeled high molecular weight material. We have now investigated the nature of this material and found that it represents areas in which all four structural components of the cell wall, β(1→3)-glucan, β(1→6)-glucan, chitin, and mannoprotein are linked together. Mannoprotein, with a protein moiety about 100 kDa in apparent size, is attached to β(1→6)-glucan through a remnant of a glycosylphosphatidylinositol anchor containing five α-linked mannosyl residues. The β(1→6)-glucan has some β(1→3)-linked branches, and it is to these branches that the reducing terminus of chitin chains appears to be attached in a β(1→4) or β(1→2) linkage. Finally, the reducing end of β(1→6)-glucan is connected to the nonreducing terminal glucose of β(1→3)-glucan through a linkage that remains to be established. A fraction of the isolated material has three of the main components but lacks mannoprotein. From these results and previous findings on the linkage between mannoproteins and β(1→6)-glucan, it is concluded that the latter polysaccharide has a central role in the organization of the yeast cell wall. The possible mechanism of synthesis and physiological significance of the cross-links is discussed.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§§ To whom correspondence should be addressed: Bldg. 10, Rm. 9N-115, National Institutes of Health, 10 Center Dr., Bethesda, MD 20892.
↵1 The abbreviations used are: HPLC, high performance liquid chromatography; HPAEC, high performance anion exchange chromatography; ConA, concanavalin A; ConA⁺, cell wall fraction that binds to ConA-Sepharose; ConA⁻, cell wall fraction that does not bind to ConA-Sepharose; fraction M, high molecular weight material remaining after β(1→6)-glucanase digestion of ConA⁺; endo-H, endo-β-N-acetylglucosaminidase H; GPI, glycosylphosphatidylinositol; ES-MS, electrospray mass spectrometry; CID, collision-induced decomposition; ER, endoplasmic reticulum; PVDF, polyvinylidene difluoride; Manp, mannopyranosyl.
↵2 In the course of this study it was found that the commercial preparation of Pronase used contained endo-β(1→6)-glucanase activity. Therefore, Pronase could not be used in most experiments. Here, however, the contaminating activity was beneficial, because it allowed us to isolate mannan free of β(1→6)-glucan by collecting the void volume fraction in gel columns.
- Received April 3, 1997.