Expression levels and subcellular localization of Bcy1p in Candida albicans mutant strains devoid of one BCY1 allele results in a defective morphogenetic behavior

Abstract

We investigated expression, functionality and subcellular localization of C. albicans Bcy1p, the PKA regulatory subunit, in mutant strains having one BCY1 allele fused to a green fluorescent protein (GFP). DE-52 column chromatography of soluble extracts of yeast cells from strains bearing one BCY1 allele (fused or not to GFP) showed co-elution of Bcy1p and Bcy1p-GFP with phosphotransferase activity, suggesting that interaction between regulatory and catalytic subunits was not impaired by the GFP tag. Subcellular localization of Bcy1p-GFP supports our previous hypothesis on the nuclear localization of the regulatory subunit, which can thus tether the PKA catalytic subunit to the nucleus. Protein modeling of CaBcy1p, showed that the fusion of the GFP tag to Bcy1p C-terminus did not significantly disturb its proper folding. Bcy1p levels in mutant strains having one or both BCY1 alleles, led us to establish a direct correlation between the amount of protein and the number of alleles, indicating that deletion of one BCY1 allele is not fully compensated by overexpression of the other. The morphogenetic behavior of several C. albicans mutant strains bearing one or both BCY1 alleles, in a wild-type and in a TPK2 null genetic background, was assessed in N-acetylglucosamine (GlcNAc) liquid medium at 37 degrees C. Strains with one BCY1 allele tagged or not, behaved similarly, displaying pseudohyphae and true hyphae. In contrast, hyphal morphology was almost exclusive in strains having both BCY1 alleles, irrespective of the GFP insertion. It can be inferred that a tight regulation of PKA activity is needed for hyphal growth.

Novel Mechanism Coupling Cyclic AMP-Protein Kinase A Signaling and Golgi Trafficking via Gyp1 Phosphorylation in Polarized Growth

“…4C). This notion is supported by the report that mutants with constitutive activation of PKA are defective in hyphal growth (44,45 To test our hypothesis that Gyp1 PKA phosphorylation controls Golgi polarization, GFP-tagged Gyp1 4A and Gyp1 4E were expressed in gyp1⌬/⌬ cells and their localization in yeast and hyphal cells was examined. The two mutant versions of Gyp1 exhibited greatly distinct localization patterns.…”

Multiple roles and diverse regulation of the Ras/cAMP/protein kinase A pathway in Candida albicans

“…The major components of the Ras/cAMP/PKA pathway of C. albicans include two Ras GTPases (Ras1 and Ras2) (Feng et al, 1999;Zhu et al, 2009), an adenylyl cyclase (Cyr1, also named as Cdc35) (Rocha et al, 2001), a low (Pde1) and a high (Pde2) affinity cyclic nucleotide phosphodiesterase (Hoyer et al, 1994;Jung and Stateva, 2003) and the PKA comprised of regulatory and catalytic subunits Giacometti et al, 2006;Ding et al, 2016;Cao et al, 2017). C. albicans Ras1 shares high similarity to Ras proteins of other fungi in both the amino acid sequence and domain organization (Feng et al, 1999;Zhu et al, 2009).…”

“…PKA consists of two catalytic and two regulatory subunits and is activated when cAMP binds to the regulatory subunits to release the catalytic subunits. In C. albicans, the PKA catalytic subunits have two isoforms encoded by TPK1 and TPK2 , whereas the regulatory subunit is encoded by a single gene BCY1 (Giacometti et al, 2006;Ding et al, 2016). The downstream targets of PKA include transcriptional regulators and other effectors to control cellular responses.…”

The Regulatory Subunit of Protein Kinase A (Bcy1) in Candida albicans Plays Critical Roles in Filamentation and White-Opaque Switching but Is Not Essential for Cell Growth

“…The authors further conclude that the two isoforms of the catalytic subunit are essential for cell viability because they were unable to generate the tpk1/tpk1 tpk2/tpk2 double mutant (Bockmühl et al, 2001; Giacometti et al, 2009). The PKA regulatory subunit Bcy1 plays a negative role in the regulation of the cAMP signaling pathway in fungal species (Cassola et al, 2004; Giacometti et al, 2006, 2012; Schaekel et al, 2013). Cassola et al (2004) demonstrated that it is not possible to generate a BCY1 null mutant in a WT strain of C. albicans , since inactivation of BCY1 leads to constitutive activation of the cAMP/PKA pathway (Cassola et al, 2004).…”