The fungal pathogen Candida albicans switches from a yeast-like to a filamentous mode of growth in response to a variety of environmental conditions. We examined the morphogenetic behavior of C. albicans yeast cells lacking the BCY1 gene, which encodes the regulatory subunit of protein kinase A. We cloned the BCY1 gene and generated a bcy1 tpk2 double mutant strain because a homozygous bcy1 mutant in a wild-type genetic background could not be obtained. In the bcy1 tpk2 mutant, protein kinase A activity (due to the presence of the TPK1 gene) was cyclic AMP independent, indicating that the cells harbored an unregulated phosphotransferase activity. This mutant has constitutive protein kinase A activity and displayed a defective germinative phenotype in N-acetylglucosamine and in serum-containing medium. The subcellular localization of a Tpk1-green fluorescent protein (GFP) fusion protein was examined in wild-type, tpk2 null, and bcy1 tpk2 double mutant strains. The fusion protein was observed to be predominantly nuclear in wild-type and tpk2 strains. This was not the case in the bcy1 tpk2 double mutant, where it appeared dispersed throughout the cell. Coimmunoprecipitation of Bcy1p with the Tpk1-GFP fusion protein demonstrated the interaction of these proteins inside the cell. These results suggest that one of the roles of Bcy1p is to tether the protein kinase A catalytic subunit to the nucleus.Candida albicans is an opportunistic human fungal pathogen of great medical significance in immunocompromised patients (25). This fungus has the capability of switching its mode of growth between budding yeast and hypha or pseudohypha in response to environmental signals. Genetic evidence indicates that the morphogenetic switch to the hyphal mode of growth, though associated with pathogenicity and virulence (20), is necessary but not sufficient to trigger disease (5). The relationship between morphology and pathogenicity has been the focus of intensive research devoted to the study of the developmental programs involved in the dimorphic transition.The remarkable conservation of signal transduction pathways in fungi allowed the identification of components of these pathways in several fungal species based on the insight gained from studying pseudohyphal differentiation in Saccharomyces cerevisiae. In C. albicans, two major pathways implicated in dimorphism could be established: the mitogen-activated protein kinase and the cyclic AMP (cAMP)/protein kinase A transduction pathways (for a review, see reference 19).Initial biochemical studies indicate that high cAMP levels promote the yeast-to-hypha transition in C. albicans (23,31). In addition, we have shown that in vivo inhibition of protein kinase A blocks hyphal growth induced by N-acetylglucosamine (GlcNAc) (6). Recent genetic studies allowed the identification of the genes involved in the cAMP/protein kinase A pathway. A transduction cascade similar to that of S. cerevisiae, with regard to location and function of the homologous components, has been established. Thus, CaRa...

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Candida albicans cAMP-dependent protein kinase (PKA) is coded by two catalytic subunits (TPK1 and TPK2 ) and one regulatory subunit (BCY1 ). In this organism the cAMP/PKA signalling pathway mediates basic cellular processes, such as the yeastto-hyphae transition and cell cycle regulation. In the present study, we investigated the role of C. albicans PKA in response to saline, heat and oxidative stresses as well as in glycogen storage. To fine-tune the analysis, we performed the studies on several C. albicans PKA mutants having heterozygous or homozygous deletions of TPK1 and/or TPK2 in a different BCY1 genetic background. We observed that tpk1 /tpk1 strains developed a lower tolerance to saline exposure, heat shock and oxidative stress, while wild-type and tpk2 /tpk2 mutants were resistant to these stresses, indicating that both isoforms play different roles in the stress response pathway. We also found that regardless of the TPK background, heterozygous and homozygous BCY1 mutants were highly sensitive to heat treatment. Surprisingly, we observed that those strains devoid of one or both TPK1 alleles were defective in glycogen storage, while strains lacking Tpk2 accumulated higher levels of the polysaccharide, indicating that Tpk1 and Tpk2 have opposite roles in carbohydrate metabolism.