Three galactose inducible promoters for use in C. neoformans var. grubii.

Cryptococcus neoformans is the causative agent of cryptococcal meningoencephalitis, most frequently occurring in immunocompromised individuals. There are three varieties of C. neoformans, var. grubii, var. neoformans, and var. gatti. Worldwide var. grubii is the most prevalent clinical isolate. However, few tools for the study of essential genes in var. grubii exist. Here we describe three endogenous inducible promoters for use in the study of this important opportunistic pathogen. We identified eight potential homologs of S. cerevisiae galactose genes in var. grubii. We found that GAL1, GAL7, and UGE2 were regulated by glucose and galactose and can be used successfully during mating. Our analysis indicated these promoters should prove to be excellent tools for analysis of genes in var. grubii.

Down-regulation of Pkc1-mediated signaling by the deubiquitinating enzyme Ubp3.

Regulated ubiquitination and degradation of signaling proteins have emerged as key mechanisms for modulating the strength and duration of signaling pathways. The reversible nature of the ubiquitination process as well as the large number and diversity of the deubiquitinating enzymes raise the possibility that signaling pathways might be modulated by specific deubiquitinating enzyme(s). Here we provide evidence that in the yeast Saccharomyces cerevisiae, the Pkc1-mediated signaling pathway that controls the cell wall integrity is negatively regulated by the deubiquitinating enzyme Ubp3. Disruption of the UBP3 gene leads to an enhanced activation of the cell wall integrity pathway MAPK Slt2 when cells are challenged with a variety of pathway activation agents such as pheromone and Congo red. The ubp3 deletion mutants accumulate high levels of Pkc1, suggesting potential regulation of Pkc1 by Ubp3. Consistent with this, Pkc1 and Ubp3 interact in vivo, and the stability of Pkc1 is markedly increased in the ubp3 deletion mutants. Moreover, disruption of the PKC1 gene, but not the genes that encode components downstream of Pkc1, completely suppresses other phenotypes displayed by the ubp3 deletion mutants such as hyperactivation of the pheromone-responsive MAPK Fus3 (Wang, Y., and Dohlman, H. G. (2002) J. Biol. Chem. 277, 15766-15772). These findings demonstrate that Ubp3 can regulate Pkc1 by facilitating its destruction and provide the initial evidence that Pkc1 plays a positive role in modulating the parallel pheromone-signaling pathway.