Ste18p is a positive control element in the mating process of Candida albicans

Abstract

Heterotrimeric G proteins are an important class of eukaryotic signaling molecules that have been identified as central elements in the pheromone response pathways of many fungi. In the fungal pathogen Candida albicans, the STE18 gene (ORF19.6551.1) encodes a potential γ subunit of a heterotrimeric G protein; this protein contains the C-terminal CAAX box characteristic of γ subunits and has sequence similarity to γ subunits implicated in the mating pathways of a variety of fungi. Disruption of this gene was shown to cause sterility of MTLa mating cells and to block pheromone-induced gene expression and shmoo formation; deletion of just the CAAX box residues is sufficient to inactivate Ste18 function in the mating process. Intriguingly, ectopic expression behind the strong ACT1 promoter of either the Gα or the Gβ subunit of the heterotrimeric G protein is able to suppress the mating defect caused by deletion of the Gγ subunit and restore both pheromone-induced gene expression and morphology changes.

FIG 1

Alignment of candidate G protein γ subunit sequences encoded by 14 species of ascomycete yeasts. The deduced amino acid sequences of Gγ subunits encoded by the C. albicans STE18 gene and its homologs in 13 related ascomycete yeasts are shown, and the conserved C-terminal tail is highlighted.

FIG 2

Ste18 null mutant strains are sterile. Mating was assayed by auxotrophic marker complementation between strains of opposite mating types as described in Materials and Methods. The mating assay for MTLa ste18Δ strains, strains with STE18 reintegrated (ste18Δ + STE18), and strains with STE18ΔC reintegrated (ste18Δ + STE18ΔC) is shown. No colonies were formed by complementation of the ste18Δ strains (LH002 and LH006) and the strain with STE18ΔC reintegrated (LH005), while the strain with STE18 reintegrated (LH004) reverted the sterile phenotype. WT, wild type. The original mating cross is shown at the bottom corner.

FIG 3

Transcriptional response to pheromone treatment. The values shown represent the averages from 3 independent biological samples for 17 pheromone-inducible genes from C. albicans. The stronger signals have the deeper red color. The final column presents a graphical summary of the data. Deletion of the STE18 gene eliminates the pheromone induction of the genes, while ectopic expression of either STE4 or CAG1 enhances the responsiveness to induction by pheromone treatment. The complete data files are accessible at GEO through accession no. GSE54031.

FIG 4

Strains with C termini of CaGγ subunits (CCTIV) deleted are sterile. The mating assay was done as described in Materials and Methods. No prototrophic colonies from the ste18ΔC strain LH012 were detected after 5 days of incubation at room temperature. WT, wild type.

FIG 5

Ectopic expression the STE4 gene in a ste18Δ strain reverts the sterile phenotype. The mating assay was done as described in Materials and Methods. Prototrophic colonies from the ste18Δ strain with the STE4 gene overexpressed were detected after 5 days of incubation at room temperature. WT, wild type.

FIG 6

Ectopic expression of the CAG1 gene in a ste18Δ strain reverts the sterile phenotype. The mating assay was done as described in Materials and Methods. Prototrophic colonies from the ste18Δ strain with the CAG1 gene overexpressed (LH022) were detected after 5 days of incubation at room temperature. WT, wild type.

FIG 7

Roles of G protein subunits in the mating process in C. albicans. All three G protein subunits are required for the mating (A). Loss of any one of them leads to full sterility (B, C, and D). The CAAX box of the Ste18p is critical for mating of C. albicans (E). Ectopic expression behind the strong ACT1 promoter of the STE4 gene did not rescue the sterility caused by absence of the Gα subunit, while the ectopic expression of the CAG1 gene was similarly unable to suppress the sterility caused by the absence of the Gβ subunit (H and I). Intriguingly, the ectopic expression of either the CAG1 gene or the STE4 gene is able to suppress the mating defect caused by deletion of the Gγ subunit (F and G).