Redundant Regulation of Cdk1 Tyrosine Dephosphorylation in Saccharomyces cerevisiae.

Cdk1 activity drives both mitotic entry and the metaphase-to-anaphase transition in all eukaryotes. The kinase Wee1 and the phosphatase Cdc25 regulate the mitotic activity of Cdk1 by the reversible phosphorylation of a conserved tyrosine residue. Mutation of cdc25 in Schizosaccharomyces pombe blocks Cdk1 dephosphorylation and causes cell cycle arrest. In contrast, deletion of MIH1, the cdc25 homolog in Saccharomyces cerevisiae, is viable. Although Cdk1-Y19 phosphorylation is elevated during mitosis in mih1∆ cells, Cdk1 is dephosphorylated as cells progress into G1, suggesting that additional phosphatases regulate Cdk1 dephosphorylation. Here we show that the phosphatase Ptp1 also regulates Cdk1 dephosphorylation in vivo and can directly dephosphorylate Cdk1 in vitro. Using a novel in vivo phosphatase assay, we also show that PP2A bound to Rts1, the budding yeast B56-regulatory subunit, regulates dephosphorylation of Cdk1 independently of a function regulating Swe1, Mih1, or Ptp1, suggesting that PP2A(Rts1) either directly dephosphorylates Cdk1-Y19 or regulates an unidentified phosphatase.

A Wee1 checkpoint inhibits anaphase onset.

Cdk1 drives both mitotic entry and the metaphase-to-anaphase transition. Past work has shown that Wee1 inhibition of Cdk1 blocks mitotic entry. Here we show that the budding yeast Wee1 kinase, Swe1, also restrains the metaphase-to-anaphase transition by preventing Cdk1 phosphorylation and activation of the mitotic form of the anaphase-promoting complex/cyclosome (APC(Cdc20)). Deletion of SWE1 or its opposing phosphatase MIH1 (the budding yeast cdc25(+)) altered the timing of anaphase onset, and activation of the Swe1-dependent morphogenesis checkpoint or overexpression of Swe1 blocked cells in metaphase with reduced APC activity in vivo and in vitro. The morphogenesis checkpoint also depended on Cdc55, a regulatory subunit of protein phosphatase 2A (PP2A). cdc55Δ checkpoint defects were rescued by mutating 12 Cdk1 phosphorylation sites on the APC, demonstrating that the APC is a target of this checkpoint. These data suggest a model in which stepwise activation of Cdk1 and inhibition of PP2A(Cdc55) triggers anaphase onset.

Biofilm matrix regulation by Candida albicans Zap1.

A biofilm is a surface-associated population of microorganisms embedded in a matrix of extracellular polymeric substances. Biofilms are a major natural growth form of microorganisms and the cause of pervasive device-associated infection. This report focuses on the biofilm matrix of Candida albicans, the major fungal pathogen of humans. We report here that the C. albicans zinc-response transcription factor Zap1 is a negative regulator of a major matrix component, soluble beta-1,3 glucan, in both in vitro and in vivo biofilm models. To understand the mechanistic relationship between Zap1 and matrix, we identified Zap1 target genes through expression profiling and full genome chromatin immunoprecipitation. On the basis of these results, we designed additional experiments showing that two glucoamylases, Gca1 and Gca2, have positive roles in matrix production and may function through hydrolysis of insoluble beta-1,3 glucan chains. We also show that a group of alcohol dehydrogenases Adh5, Csh1, and Ifd6 have roles in matrix production: Adh5 acts positively, and Csh1 and Ifd6, negatively. We propose that these alcohol dehydrogenases generate quorum-sensing aryl and acyl alcohols that in turn govern multiple events in biofilm maturation. Our findings define a novel regulatory circuit and its mechanism of control of a process central to infection.

Candida albicans transcription factor Rim101 mediates pathogenic interactions through cell wall functions.

pH-responsive transcription factors of the Rim101/PacC family govern virulence in many fungal pathogens. These family members control expression of target genes with diverse functions in growth, morphology and environmental adaptation, so the mechanistic relationship between Rim101/PacC and infection is unclear. We have focused on Rim101 from Candida albicans, which we find to be required for virulence in an oropharyngeal candidiasis model. Rim101 affects the yeast-hypha morphological transition, a major virulence requirement in disseminated infection models. However, virulence in the oropharyngeal candidiasis model is independent of the yeast-hypha transition because it is unaffected by an nrg1 mutation, which prevents formation of yeast cells. Here we have identified Rim101 target genes in an nrg1Delta/Delta mutant background and surveyed function using an overexpression-rescue approach. Increased expression of Rim101 target genes ALS3, CHT2, PGA7/RBT6, SKN1 or ZRT1 can partially restore pathogenic interaction of a rim101Delta/Delta mutant with oral epithelial cells. Four of these five genes govern cell wall structure. Our results indicate that Rim101-dependent cell wall alteration contributes to C. albicans pathogenic interactions with oral epithelial cells, independently of cell morphology.

Regulation of the Candida albicans cell wall damage response by transcription factor Sko1 and PAS kinase Psk1.

The environmental niche of each fungus places distinct functional demands on the cell wall. Hence cell wall regulatory pathways may be highly divergent. We have pursued this hypothesis through analysis of Candida albicans transcription factor mutants that are hypersensitive to caspofungin, an inhibitor of beta-1,3-glucan synthase. We report here that mutations in SKO1 cause this phenotype. C. albicans Sko1 undergoes Hog1-dependent phosphorylation after osmotic stress, like its Saccharomyces cerevisiae orthologues, thus arguing that this Hog1-Sko1 relationship is conserved. However, Sko1 has a distinct role in the response to cell wall inhibition because 1) sko1 mutants are much more sensitive to caspofungin than hog1 mutants; 2) Sko1 does not undergo detectable phosphorylation in response to caspofungin; 3) SKO1 transcript levels are induced by caspofungin in both wild-type and hog1 mutant strains; and 4) sko1 mutants are defective in expression of caspofungin-inducible genes that are not induced by osmotic stress. Upstream Sko1 regulators were identified from a panel of caspofungin-hypersensitive protein kinase-defective mutants. Our results show that protein kinase Psk1 is required for expression of SKO1 and of Sko1-dependent genes in response to caspofungin. Thus Psk1 and Sko1 lie in a newly described signal transduction pathway.

Development of a Brassica seed cDNA microarray.

Brassica species represent several important crops including canola (Brassica napus). Understanding of genetic elements that contribute to seed-associated functions will impact future improvements in the canola crop. Brassica species share a very close taxonomic and molecular relationship with Arabidopsis thaliana. However, there are several subtle but distinct seed-associated agronomic characteristics that differ among the oil seed crop species. To address these, we have generated 67,535 ESTs predominately from Brassica seeds, analyzed these sequences, and identified 10,642 unigenes for the preparation of a targeted seed cDNA array. A set of 10,642 PCR primer pairs was designed and corresponding amplicons were produced for spotting, along with relevant controls. Critical quality control tests produced satisfactory results for use of this microarray in biological experiments. The microarray was also tested with specific RNA targets from embryos, germinating seeds, and leaf tissues. The hybridizations, signal intensities, and overall quality of these slides were consistent and reproducible. Additionally, there are 429 ESTs represented on the array that show no homology with any A. thaliana annotated gene or any gene in the Brassica genome databases or other plant databases; however, all of these probes hybridized to B. napus transcripts, indicating that the array also will be useful in defining expression patterns for genes so far unique to Brassica species.