Nikkomycin Z inhibits chitin synthase in vitro but does not exhibit antifungal activity against many pathogens. Assays of chitin synthase isozymes and growth assays with isozyme mutants were used to demonstrate that nikkomycin Z is a selective inhibitor of chitin synthase 3. (20,25).In vitro effects of nikkomycin Z. The availability of mutant strains that express or overexpress single chitin synthase activities makes it possible to obtain extracts containing individual chitin synthase enzymes. We prepared digitonin-treated cell extracts from wild-type S288C cells (14) (Chsl is the predominant chitin synthase activity found in extracts of wild-type cells treated with trypsin) and cells that contain high-copy-number CHS2 (SSY563-9B or ECY36-3D [YEp352-CHS2]) (Table 1). For a 250-mg cell pellet (an original culture volume of -100 ml is usually sufficient), 750 ,u1 of 1% digitonin-25 mM MES (morpholineethanesulfonic acid; pH 6.3) was added and the cells were shaken at 30°C for 15 min. They were centrifuged at -12,000 X g for 5 min, and the pellet was washed with 2.7 ml of 25 mM MES, pH 6.3. The pellet was resuspended in a total volume of 750 ,u1 of 25 mM MES, pH 6.3-33% glycerol.Membranes from strain ECY36-3C were prepared for Chs3 assays by a modification of the method of Orlean (20). Cells were washed once in 50 mM Tris-HCl, pH 8.0-5 mM MgCl2, and after membranes were prepared, they were resuspended in the same buffer containing 33% glycerol.Enzyme assays were performed to measure the in vitro sensitivity of the three chitin synthases to nikkomycin Z and/or polyoxin D (both from Calbiochem). The cells were first
The Saccharomyces cerevisiae SRKI gene, when expressed on a low-copy shuttle vector, partially suppresses the phenotype associated with elevated levels of cyclic AMP-dependent protein kinase activity and suppresses the temperature-sensitive cell cycle arrest of the insi mutant. SRK1 is located on chromosome IV, 3 centimorgans from gcn2. A mutant carrying a deletion mutation in srkl is viable. SRKI encodes a 140-kDa protein with homology to the dis3+ protein from Schizosaccharomyces pombe. The ability of SRKI to alleviate partially the defects caused by high levels of cyclic AMP-dependent protein kinase and the similarity of its encoded protein to dis3' suggest that SRK1 may have a role in protein phosphatase function.SRKI suppresses traits associated with hyperactivity of cAMP-dependent protein kinase. Saccharomyces cerevisiae strains with mutations in the low-Km cyclic AMP (cAMP) phosphodiesterase (pde2lsraS) gene fail to properly regulate cAMP levels in the presence of exogenous cAMP and lose viability as a consequence (27). This property was used to clone the PDE2 gene from a yeast genomic library constructed in the yeast shuttle vector YCp5O (provided by M. Rose, Princeton University) (27). In addition to plasmids containing the bona fide PDE2 gene, we recovered another plasmid, pW31 (Fig. 1), which protected the pde2 strain from loss of viability in the presence of exogenous cAMP but failed to influence the low glycogen levels associated with pde2. To determine whether pW31 could complement other mutations in the cAMP-dependent protein kinase pathway, we transformed yeast strains (2) which contain a HIS3 disruption in the gene encoding the regulatory subunit of cAMP-dependent protein kinase (bcyllsral) (6, 25) with pW31 and tested Ura+ transformants for traits associated with bcyl. bcyl ::HIS3 transformants containing pW31 were more resistant to a 52°C heat shock than were those bcyl: :H153 transformants containing only the YCp5O vector (Fig. 2B) and were also able to grow on maltose (Fig. 2C)-or ethanol (Fig. 2D)-containing medium. pW31 did not fully complement bcyl; the pW31 transformants of EG286-1-1OC were still sensitive to nitrogen starvation (Fig. 2E), although some beyl strains that we tested did show partial resistance to starvation. beyl strains containing the SRKI plasmids were unable to grow on acetate-or glycerol-containing medium (data not shown), and glycogen levels remained low, as judged by the inability of iodine vapors to stain the transformants (Fig. 2F). When present on a 2,um multicopy vector, the genomic DNA present in pW31 did not complement bcyl to any greater extent than on YCp5O (data not shown).
Five sequences were isolated by selection for multiple copy plasmids that conferred resistance to laminarinase, an enzyme that specifically degrades cell wall (1-3) glucan linkages. Strains carrying three of these plasmids showed alterations in cell wall glucan labelling. One of these plasmids carried PBS2, a previously identified, non-essential gene which produces a variety of phenotypes and encodes a mitogen-activated protein kinase kinase analogue (Boguslawski and Polazzi, 1987). Cells carrying PBS2 at multiple copy show a small decrease in cell wall (1-6) glucans. Measurements of (1-3) glucan synthase activity in multi-copy PBS2 cells showed an approximate 30-45% increase in enzyme specific activity while a pbs2 disruption strain showed a decrease in glucan synthase activity of approximately 45% relative to control. A pbs2 disruption strain was laminarinase super-sensitive and supersensitive to K 1 killer toxin while a strain carrying PBS2 at multiple copy was resistant to killer toxin. A second plasmid carried a portion of the MHP1 gene which has been reported to encode a microtubule-interacting protein (Irminger-Finger et al., 1996). The MHP1 gene product is a predicted 1398 amino acid protein and only approximately 80% of the amino portion of this protein is required for laminarinase resistance. Cells carrying the amino portion of MHP1 at multiple copy show a decrease in high molecular weight cell wall (1-6) glucans and were killer toxin resistant while a disruption strain was viable and killer toxin super-sensitive. Cells carrying this plasmid showed decreased levels of high molecular weight (1-6) glucans and increased glucan synthase activity. The laminarinase resistance conferred by the third plasmid mapped to the previously uncharacterized YCL051W open reading frame and this gene was therefore named LRE1 (laminarinase resistance). The LRE1 gene encodes a non-essential 604 amino acid hydrophilic protein. Unexpectedly, cells carrying LRE1 at multiple copy show no alteration in cell wall glucans or glucan synthase activity. Subcloning experiments demonstrated that the production of these cell wall effects requires the presence of both LRE1 and YCL052C (PBN1), a second open reading frame present on the original plasmid. Cells carrying multiple copies of PBN1 alone show no significant alterations in cell wall glucans or glucan synthase activity, indicating that these effects require the presence of multiple copies of both genes.
screen for cell wall acting antifungal agents. The compound shows moderate activity in both greenhouse tests against plant disease caused by pathogenic fungi and in in vitro tests
We have isolated a snf1/ccr1 mutant of Saccharomyces cerevisiae which loses viability upon starvation and fails to accumulate glycogen in response to abrupt depletion of phosphate or glucose. A snf1 null mutant is sensitive to heat stress and starvation and fails to accumulate glycogen during growth in rich medium. The phenotypes of the snf1 mutants are those commonly associated with an overactivation of the adenylate cyclase pathway. Mutations in adenylate cyclase or RAS2 which decrease the level of cAMP in the cell moderate the snf1 phenotype. In contrast, a mutation in RAS2 (RAS2val19) which increases the level of cAMP or a mutation in the regulatory subunit (BCY1) of cAMP-dependent protein kinase which results in unregulated cAMP-dependent protein kinase activity accentuates the snf1 phenotype. However, the action of SNF1 in the stress response appears at least partly independent of cAMP-dependent protein kinase because a snf1 phenotype is observed in a strain that lacks all three of the genes that encode the catalytic subunits of cAMP-dependent protein kinase. SNF1 therefore acts at least in part through a cAMP-independent pathway.
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