ANALYSIS OF FISSION YEAST CELL WALL

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Purified endoglucanases have been used to determine the composition of Schizosaccharomyces pombe cell wall. This structure has been traditionally studied after isolating its components (mannoproteins, alpha-1,3 glucan, beta-1,3glucan and a branched beta-glucan) with hot alkali. Instead, we sequentially removed the polysaccharides by digesting with endo beta-1,3 glucanase and with a novel endo alpha-1,3 glucanase (mutanase). This method uses sequential enzymatic hydrolysis, which is more gentle than hot alkali treatment. The method is reproducible and allows for the major components of the cell wall to be more specifically and reproducibly separated into defined fractions than previous methods. After this gentle isolation, we observed that a branched beta-glucan is much more abundant than previously described. By scaling-up the new protocol we prepared large amounts of the highly branched glucan and determined its structural features by chemical, MALDI-TOF MS, 13C and 1H high field NMR analyses. These analyses have allowed us to clearly define a highly branched cell wall polymer that is resistant to known glucanases. Finally, to demonstrate its application, we have determined the cell wall composition of known mutant strains.