New methods for the determination of polysaccharide cross-links in the yeast cell wall and their impact on the understanding of cross-link formation. The fungal cell wall is necessary for survival of the cells, which would burst in its absence, because of internal turgor pressure. For this reason, the cell wall is an obvious target for antifungal agents. The cell wall owes its resilience to the cross-links between its components, polysaccharides and mannoproteins. For several years, I have studied the mechanism of formation of the cross-links between chitin, an N-acetylglucosamine polymer, and the glucose polysaccharides beta(1-3) and beta(1-6)glucan in the yeast cell wall. These studies required the creation of procedures for the quantitative determination of the different forms of chitin, either free or attached to one or the other of the glucans. I devised such a procedure, based on solubilization of the cell wall components by carboxymethylation, preceded or not by digestion with specific glucanases and followed by chromatography on a size column. Because chitin was specifically labeled with 14-C in vivo, it was possible to track it throughout the procedure and determine the percentage of its different forms by comparison of the chromatographic profiles (1). By the use of this method, it was found that yeast strains lacking both of the putative transglycosylases Crh1p and Crh2pl had no chitin linked to beta(1-6)glucan, although an apparently normal amount of chitin was attached to beta(1-3)glucan (2). Further work confirmed that the linkage between chitin and glucan was created by transfer of part of a nascent chitin chain to the glucose polymer (3). In parallel to this work, I was trying to develop some simpler procedure for cell wall analysis, because the one outlined above is quite long and cumbersome. To this end, I tried to exploit the affinity, due to hydrogen bonding, between chains of beta(1-3)-linked glucan. I found that a gel of curdlan, a bacterial beta(1-3)glucan, could adsorb 14C-labeled carboxymethylated yeast beta(1-3)glucan, whereas similarly labeled beta(1-6)glucan remained unbound. This constituted the base for a new analytical procedure: when 14C-labeled cell walls solubilized by carboxymethylation were added to curdlan gel, the chitin-beta(1-3)glucan and the chitin-beta(1-6)glucan-beta(1-3)glucan complexes were adsorbed by the gel, whereas the free chitin remained in the supernatant. If the cell walls were treated with beta(1-6)glucanase before carboxymethylation, both the free chitin and that previously bound to beta(1-6)glucan, now also free, were left unbound. Thus, by measuring radioactivity in the different fractions and making appropriate subtractions, it was possible to ascertain the percentage of chitin that was originally free or bound to one or the other of the two glucans. The results for a wild type strain were similar to those of the carboxymethylation-chromatography method. However, with the mutant crh1delta crh2delta all the chitin appeared to be free, in contrast with the results with the previous procedure. To resolve this apparent contradiction, I devised a third method, based on a different principle, for the analysis of the cross-links. Here, I took advantage of the fact that, whereas chitin is totally insoluble in aqueous solvents, its deacetylated product, chitosan, is soluble in dilute acetic acid solutions. All the chitin in cell walls, labeled in vivo with 14C-glucosamine, was converted into chitosan by autoclaving in 50% sodium hydroxide. The free chitin (now chitosan) was extracted with acetic acid. Digestion with beta(1-6)glucanase, followed by acetic acid extraction, solubilized the chiosan bound to beta(1-6)glucan;repetition of this operation with the use of beta(1-3)glucanase finally yielded the chitosan linked to beta(1-3)glucan. Again, the results for a wild type strain were similar to those of the carboxymethylation-chromatography method, but in the double mutant crh1delta crh2delta all the chitin seemed to be free. The coincidence in the results of the two new procedures prompted a reinvestigation of the carboxymethylation-chromatography method. Use of a recombinant beta(1-3)glucanase in that method in place of the commercial preparation (zymolyase) previously employed, yielded results in line with those of the other two methods. This indicated the presence of a contaminating enzymatic activity in the zymolyase, which turned out to be a small amount of chitinase. The chitinase was eliminated from the zymolyase by passage through a chitin column. Use of the purified zymolyase in the carboxymethylation-chromatography method now showed that all the chitin was free in the crh1delta crh2delta strain, in agreement with the other two procedures. The conclusion is that Crh1p and Crh2p are responsible for attachment of chitin to both beta(1-6) and beta(1-3)glucan, i.e. we know now the mechanism for the formation of all chitin cross-links. Another consequence of this work is that two new procedures, much simpler and faster than the carboxymethylation-chromatography method, are now available for studies of fungal cell wall structure. A paper embodying these findings is in press in Eukaryotic Cell. 1. Cabib, E. and Durn, A. (2005) Synthase III-dependent chitin is bound to different acceptors depending on location on the cell wall of budding yeast. J. Biol. Chem. 280, 9170-9179. 2. Cabib, E., Blanco, N., Grau, C., Rodrguez-Pea, J.M., and J. Arroyo (2007) Crh1p and Crh2p are required for the cross-linking of chitin to beta(1-6)glucan in the Saccharomyces cerevisiae cell wall. Mol. Microbiol. 63, 921-935. 3. Cabib, E., Farkas, V., Kosik, O., Blanco, N., Arroyo, J., and McPhie, P. (2008) Assembly of the yeast cell wall. Crh1p and Crh2p act as transglycosylases in vivo and in vitro. J. Biol. Chem. 283, 29859-29872.