In previous work it was found that Rho1p, a GTP-binding protein from yeast, has at least three different functions, all essential for viability. Rho1p is a regulator of the biosynthesis of beta(1-3)glucan, the major structural component of the yeast cell wall; it regulates protein kinase C, which controls a protein kinase cascade required for cell integrity; finally, it is necessary for progression in the G1 stage of the cell cycle and for cell polarization. A study on the last of these three topics was completed in this period and published in the Journal of Cell Biology. Since the main interest of our laboratory is in cell wall synthesis, it was desirable to separate the function of Rho1p in glucan synthesis from the other two. With that purpose, the RHO1 gene was subjected to in vitro mutagenesis and a number of temperature-sensitive rho1 mutants were isolated. These mutants are now scrutinized to determine the performance of different functions of Rho1p at the nonpermissive temperature. One product of this study was the isolation of mutants with extremely low activity of glucan synthase. With these mutants, it was possible to establish that Rho1p is essential for synthesis of beta(1-3)glucan in vivo.One of the main subjects under study in this laboratory has been the formation of the chitinous primary septum of yeast, that grows to separate mother and daughter cell at cytokinesis. Little is known about the spatial and temporal regulation of the enzyme responsible for the synthesis of the septum central disk, chitin synthase II (ChsII). A genetic screen has been set up for the identification of proteins required for ChsII function. This screen takes advantage of the previous finding that a simultaneous defect in ChsII and in chitin synthase III (ChsIII, gene CHS3) is lethal; therefore, cells deleted for CHS3, but carrying that gene on a plasmid will be unable to lose the plasmid if ChsII function is seriously impaired. Such a strain is mutagenized and the population is screened for cells that cannot lose the CHS3-containing plasmid by scoring colonies for a color change that depends on the product of the ADE3 gene (appropriate mutations in ADE3 and/or ADE2 had also been engineered in the test strain). The mutants with the expected phenotype are subjected to a battery of tests to screen out false positives. Those that survive these tests are transformed with a yeast genomic library, to identify the genes coding for proteins presumably necessary for ChsII function. So far, the screen has yielded genes coding for two protein kinases that were previously implicated in the control of the yeast cell cycle. The study continues, to determine whether the action of the kinases on ChsII is direct or indirect and to isolate further genes that participate in septum formation. - yeast cell wall morphogenesis chitin glucan
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