Our laboratory has continued to study the yeast cell wall and the septum formed at cytokinesis as models for morphogenesis. In previous work on the cell wall, the GTP-binding protein Rho1p had been identified as the regulator of beta-1,3-glucan synthase, the enzyme that catalyzes the formation of the major structural component of the cell wall. Subsequently, we isolated rho1 mutants specifically defective in this regulatory function, but not in other cellular roles of the protein. By use of the mutants it was possible to show that incorporation of two other components of the cell wall, beta-1,6-glucan and mannoproteins, was dependent on the synthesis of beta-1,3-glucan. This suggested that beta-1,3-glucan was made before the two other components. This notion was confirmed when it was found that in the presence of YW3548, a specific inhibitor of mannoprotein incorporation in the wall, both beta-1,3- and beta-1,6-glucan are normally synthesized and attached to each other. It was concluded that the order of assembly in the wall is beta-1,3-glucan, beta-1,6-glucan, mannoprotein. Last comes chitin, which is incorporated after septation, during maturation of the daughter cell. These findings indicate that Rho1p, by turning on and off beta-1,3-glucan synthesis, also does the same with the whole cell wall. As for septum formation, our previous work had shown that, in this process, invagination of an actomyosin ring and deposition of chitin in the invagination are dependent on each other, an indication that these two events are closely interconnected. To further study this connection, fusions between myosin 1 (Myo1p) or chitin synthase 2 (Chs2p) and different fluorescent proteins were constructed. Time-lapse photography of cells containing those proteins showed that contraction of the actomyosin ring and deposition of the chitin primary septum were simultaneous. Chs2p arrived at the mother-bud neck just before septation and was carried away to the vacuole by endocytic vesicles immediately after the event. The close coordination between contractile ring and primary septum suggested that the septation apparatus might function autonomously. To examine this possibility, we revisited our old observation on mutants in septins, proteins forming a ring at the mother-bud neck, which is supposed to act as scaffold for proteins involved in cytokinesis. Those mutants showed structures similar to septa but mislocalized at different positions of the cell cortex. By the use of fusion proteins of Chs2p, Myo1p and septins with fluorescent proteins, we showed that in the mutants all three proteins could be found together at the aberrant localizations and that Myo1p-GFP and Chs3p-GFP patches behaved as observed in normal septation. We concluded that the septation apparatus, with the septin ring as a scaffold, can behave as an autonomous system in budding yeast. In a related study, we had carried out a genetic screen for proteins showing synthetic lethality with CHS3, which codes for the chitin synthase 3 putative catalytic subunit. Chs3p is responsible for a chitin ring at the mother-bud neck and for chitin interspersed in the cell wall. Two genes isolated in the screen coded for Cla4p, a protein kinase of the PAK type, and for Cdc11p, a septin. Mutants in both genes showed similar septin defects, suggesting that Cla4p is required for normal septin organization. Inhibition of Chs3p with nikkomycin Z in the cla4 mutant resulted in widening at the neck, followed by bud elongation and death of the cell. A similar widening was observed in cdc11 mutants in the presence of nikkomycin Z. From these and other results it was concluded that the chitin ring generated under Chs3p action and the septin ring cooperate in preventing growth at the neck. Failure of both systems leads to neck growth and eventually to death of the cell. This is the first experimental evidence for a physiological function of the chitin ring.
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