. Work was continued on the formation of the chitin-beta(1-6)glucan linkage in the yeast cell wall by Crh1p and Crh2p, the first two proteins identified as necessary for a cross-link in a fungal cell wall. Defects of other cell wall mutants are aggravated by mutations in the Crh proteins, showing their importance for cell wall maintenance. Stress in the form of higher temperature increases the formation of the chitin-beta(1-6)glucan linkage. This increase is coupled for Crh1p to an enhanced expression of the protein. Both proteins redistribute at higher temperature, from the mother-daughter neck to a random localization in the cell cortex, the locale of the chitin-beta(1-6)glucan linkage formation.? ? The Saccharomyces cerevisiae cell wall is composed of beta(1-3)glucan, beta(1-6)glucan, chitin and mannoproteins. These components are connected to one another, giving rise to a tightly woven network, which can withstand large changes in internal turgor pressure and protect the cell from mechanical injury. Almost nothing is known about the mechanisms of synthesis of the cross-links between the cell wall components. Chitin, a minor but essential constituent of the cell wall, is bound in part to beta(1-3)glucan and in part to beta(1-6)glucan. Recently, I developed a new procedure to assess the amount of chitin that is free or bound to each of the two glucans. By using this methodology, I was able to show that the chitin present at the mother?bud neck is mainly bound to beta(1-3)glucan, whereas that dispersed in the cell wall is predominantly linked to beta(1-6)glucan. In a collaboration with Javier Arroyo of the Complutense University in Madrid, Spain, and with the use of the recently devised procedure for the study of cell wall cross-links, we were able to show that Crh1p and Crh2p, two putative translycosylases, were required, in a redundant fashion, for the attachment of chitin to beta(1-6)glucan. This work was started in the previous period and continued in the present one.? Mutants in Crh1p or Crh2p were partially defective in the amount of chitin bound to beta(1-6)glucan and a double mutant totally lacked the cross-link. From the amount of chitin-beta(1-6)glucan remaining in the single mutants, it appears that under normal conditions Crh2p is the major contributor to the linkage. Because chitin attaches to side chains of beta(1-6)glucan, an alternative explanation for the function of the Crh proteins could have been that they are involved in the formation of the branches, thus creating the chitin acceptor. However, beta(1-6)glucan was as branched in a double deletion mutant of Crh1p and Crh2p as in wild type, confirming that the Crh proteins act as transglycosylases, ferrying over chitin made by chitin synthase 3 to the glucan.? Certain mutants, such as fks1 and gas1, defective in the major structural component of the cell wall, beta(1-3)glucan, show an increased content of chitin and of mannoproteins linked to chitin through beta(1-6)glucan, which has been interpreted as a cell repair mechanism. We found that mutation of Crh1p and Crh2p in those strains aggravates their defects, supporting a physiological function of the chitin-beta(1-6)glucan linkage in cell wall maintenance.? The previous finding that Crh1p expression is regulated during the cell cycle, the fact that CRH1 is transcriptionally induced in many cell wall stress conditions and the general knowledge that cell wall synthesis is strictly regulated by the cell integrity pathway encouraged us to study the effect of stress, here applied as an increase in temperature, on the synthesis of the chitin-beta(1-6)glucan linkage. We found a substantial increase in the proportion of that linkage at 38?C, compared to 30?C, both in wild type and in single Crh mutants, showing that both Crh proteins contribute to the augmented synthesis. We also confirmed that the expression of Crh1p, but not that of Crh2p, is increased by temperature and that the cell integrity pathway is strictly required for that increase and for the contribution of Crh1p to the enhancement in chitin-beta(1-6)glucan formation at 38?C.? It remained to be seen what was the underlying mechanism for the increased participation of Crh2p in the formation of the cross-link at 38?C, because the expression of this protein is not enhanced by temperature. We decided to study the distribution of the proteins at different temperatures by fusing them to a suitable epitope or to green fluorescent protein. In this way, we observed an extensive redistribution of both proteins at 38?C, from a main localization at the mother-bud neck to a widespread distribution at the cell cortex, where our previous studies had shown the chitin-beta(1-6glucan complex to be present. These results nicely fit in with a similar redistribution of chitin synthase 3 with temperature, observed in another laboratory. Thus, it seems that under stress both the enzyme responsible for chitin formation and those for its transfer to beta(1-6)glucan move to the same cellular localization, where they are possibly activated to make their products. This change in localization appears to be sufficient for the enhanced contribution of Crh2p to the synthesis of the cross-link, whereas Crh1p also needs an increase in expression.? In conclusion, we have identified the first two proteins involved in fungal cell wall cross-links and studied their physiological function and regulation. A paper on these studies has been submitted.
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