Interconnections between the Cation/Alkaline pH-Responsive Slt and the Ambient pH Response of PacC/Pal Pathways in Aspergillus nidulans

Abstract

In the filamentous ascomycete Aspergillus nidulans, at least three high hierarchy transcription factors are required for growth at extracellular alkaline pH: SltA, PacC and CrzA. Transcriptomic profiles depending on alkaline pH and SltA function showed that pacC expression might be under SltA regulation. Additional transcriptional studies of PacC and the only pH-regulated pal gene, palF, confirmed both the strong dependence on ambient pH and the function of SltA. The regulation of pacC expression is dependent on the activity of the zinc binuclear (C6) cluster transcription factor PacX. However, we found that the ablation of sltA in the pacX^- mutant background specifically prevents the increase in pacC expression levels without affecting PacC protein levels, showing a novel specific function of the PacX factor. The loss of sltA function causes the anomalous proteolytic processing of PacC and a reduction in the post-translational modifications of PalF. At alkaline pH, in a null sltA background, PacC^72kDa accumulates, detection of the intermediate PacC^53kDa form is extremely low and the final processed form of 27 kDa shows altered electrophoretic mobility. Constitutive ubiquitination of PalF or the presence of alkalinity-mimicking mutations in pacC, such as pacC^c14 and pacC^c700, resembling PacC^53kDa and PacC^27kDa, respectively, allowed the normal processing of PacC but did not rescue the alkaline pH-sensitive phenotype caused by the null sltA allele. Overall, data show that Slt and PacC/Pal pathways are interconnected, but the transcription factor SltA is on a higher hierarchical level than PacC on regulating the tolerance to the ambient alkalinity in A. nidulans.

Keywords: abiotic stress; cross regulation; post-translational modifications; signalling; transcriptional factors.

Figure 1

Gene expression profile of genes from slt and ambient pH regulatory pathways in wild-type and sltAΔ strains under alkaline pH condition. (A) Expression levels of sltA and sltB in the wild-type strain MAD3652 (WT). (B) Expression of pacC in the WT and sltAΔ strain (MAD3816). (C) Expression levels of palF in the WT and sltAΔ strain. Mycelia were grown in AMM in independent samples for each experimental time. For alkalinisation of extracellular pH, 100 mM Na₂HPO₄ were added to culture media. Mycelia were collected after 15, 30, 60 and 120 min. Relative expression data were normalised using the control condition expression levels of each strain. The β-tubulin-encoding gene benA/An1182 was used as a reference gene. Error bars represent the standard deviations of three replicates for each sample result. *, p < 0.01–0.05; **, p < 0.001–0.01; ***, p < 0.0001–0.001; ****, p < 0.00001. Not significant p-values (≥0.05) are not indicated in graphs.

Figure 2

Immunodetection of tagged forms of PacC and PalF proteins under alkaline pH in wild-type and sltAΔ strains. (A) PacC900 (MYC₃-PacC protein) was detected using specific anti-myc antibody (α-myc) in total protein extracts from wild-type (MAD7627) and sltAΔ (MAD7630) strains. Red arrow indicates the low mobility band for PacC full proteolysed version (B) Detection of PalF-HA-tagged protein (PalF500) using a specific anti-HA antibody (α-HA). Detection of α-tubulin was used as a loading control. A highly exposed image (+exp) was taken for a better visualisation of PalF post-translational modifications (Ub&PP) at alkaline pH conditions.

Figure 3

Epistatic effect of slt- mutations over pacX20 mutation. (A) Phenotypic analysis of strains combining the absence of PacX function by a pacX20 allele and null alleles of sltA and sltB, or the complete loss of function allele sltA1. Growth of single pacX20 strain (MAD1652) did not show differences compared to wild-type strain (MAD6669) under 1 M NaCl, pH 8 (100 mM Na₂HPO4) and 0.3 M LiCl. Double mutant strains sltAΔ pacX20 (MAD7621), sltBΔ pacX20 (MAD7623) and sltA1 pacX20 (MAD7622) displayed similar phenotype as the single slt- mutants, sltAΔ (MAD3816), sltBΔ (MAD3693) and sltA1 (MAD1132) under stressful conditions. Strains were grown at 37 °C for 48 h and colonial growth was imaged. (B) Transcriptional profile of pacX gene in wild-type (WT) and sltAΔ strains under alkaline pH stress conditions. (C) Relative expression levels of pacX in mycelia of WT and single and double sltAΔ pacX20 mutant strains at alkaline and acid (Control) ambient pH. The benA gene was used as reference gene and relative expression level was normalised according to control condition of wild-type strain. Error bars represent the standard deviations of three replicates for each sample result. *, p < 0.01–0.05; **, p < 0.001–0.01; ***, p < 0.0001–0.001; ****, p < 0.00001. Not significant p-values (≥0.05) are not indicated in graphs.

Figure 4

Absence of PacX function does not rescue pacC or palF transcriptional levels or protein patterns in a null sltA background. Expression levels at pH 8 of pacC (A) and palF (B) in the double mutant pacX20 sltAΔ background (strains used as in Figure 3B). The β-tubulin benA gene was used as reference to measure relative expression. Visualization by immunodetection of changes in the proteolytic patterns of MYC and HA-tagged proteins PacC900 (C) and PalF500 (D) in a single pacX20 (MAD7628) and the double mutant pacX20 sltAΔ (MAD7629) strains. Detection of α-tubulin protein was used as the loading control. Error bars represent the standard deviations of three replicates for each sample result. ****, p < 0.00001. Not significant p-values (≥0.05) are not indicated in graphs.

Figure 5

Recruitment of the Vps23 protein by post-translationally modified PalF protein at alkaline pH. (A) Schematic representation of PalF protein showing arrestin domains N and C (purple), the N-terminal PalH-binding region and the possible Vps23 binding regions (box 1 and box 2 in grey). Locations of the palF15 mutation (pink) and the detected ubiquitination site at alkaline pH (light blue). (B) Fluorescence microscopy visualization of GFP-tagged Vps23 protein in standard (Control) and alkaline conditions (pH8) in WT (MAD7669) and null-sltA (MAD7670) strains. The palF15 mutant (MAD3369) was used as an additional control to show Vps23 mislocalization at pH 8. The three strains were grown for 15 h at 25 °C in WMM. For alkalinisation assays, the WMM was replaced by WMM containing 100 mM Na₂HPO₄. Arrowheads indicate static cortical Vps23 spots visualized in images that are a sum projection of a time-lapse stack (interval 1 s for 46 s) following the methodology previously described [22]. Bar scale represents 5 µm.

Figure 6

Effect of double null palB and sltA mutants in PacC proteolytic pattern and environmental stress phenotype. (A) Phenotype of wild-type strain (MAD7627), single mutants null palB and null sltA (MAD1775 and MAD7630), and of double mutant palBΔ sltAΔ (MAD8108) inoculated in alkaline and high concentration of sodium and molybdate conditions. Strains were grown at 37 °C for 48 h and colonial growth was imaged. (B) Immunodetecion of PacC proteolytic pattern in acid and alkaline-induced conditions in the strains indicated in panel (A). α-tubulin was used as loading control protein. * Indicates the band corresponding to very low mobility form of PacC fully proteolysed version, ** indicates the band corresponding to the standard PacC^27kDa proteolysed form, see the text for additional information.

Figure 7

Effect of constitutive activation of the Pal pathway through PalF ubiquitination in a null sltA background. (A) Comparative patterns of PTMs visualized by the immunodetection of tagged forms of PacC and PalF in wild-type and sltAΔ background (MAD7627 and MAD7630), and in wild-type and sltAΔ strains (MAD4499 and MAD7634) carrying the palF::ub construct that expresses a constitutively ubiquitinated version of PalF. A longer exposure of PalF immunoblot is shown (+exp) to better show the presence or absence of ubiquitinated and phosphorylated forms (Ub&PP). α-tubulin was used as the loading control protein. (B) Phenotypic analysis of strains used in panel (A) tested under alkaline pH conditions and two sodium molybdate concentrations. Strains were grown at 37 °C for 48 h and colonial growth was imaged. Numbers indicate tracks cited in the text.

Figure 8

Analysis of the ambient pH-independent mutant pacC^c14900 in the null sltA. (A) Comparative colonial growth of pacC^c14900 sltAΔ double mutant (MAD4296) with that shown by the wild-type (WT), sltAΔ and single pacC^c14900 mutant (MAD1445) strains on AMM containing different abiotic stresses. (B) Immunodetection of myc-PacC forms in the strains used in (A). (B, right) Coomassie staining blue was used for total protein loading control due to detection problems of α-tubulin in pacC^c14900 mutants. Numbers indicate tracks cited in the text.

Figure 9

Effect of pacC^c700 allele in a null sltA background. (A) Phenotypes of single mutant pacC^c700 (GFP-PacC5-250) (MAD7632) and sltAΔ (MAD3816) strains and the pacC^c700 sltAΔ double mutant (MAD7633). Strains were incubated at 37 °C for 48 h and colony growths were imaged. (B) Immunodetection of PacC700 in a wild-type (sltA⁺) and the null sltA background. As in Figure 7, Coomassie staining was used as the protein loading control. (C) Fluorescence microscopy of WT and null sltA strains expressing GFP-PacC5-250 (PacC700) in WMM showing the accumulation of fluorescence in nuclei. DIC indicates Nomarsky optics for cell visualization. Scale bar represents 5 μm.

Figure 10

A model of the interconnection between Slt and Pal/PacC systems in the transcriptional response to ambient alkaline pH. The scheme integrates data from Bussink and collaborators [29] and the results obtained in this study. SltA is proposed to positively act (green arrows), regulating pacC expression and the correct processing of the PacC protein via the activation of the Pal signalling pathway at alkaline pH. SltA is epistatic to PacC in regulating the tolerance to ambient alkaline pH (blue and orange lines). Green lines indicate positive actions by the regulators and in red the negative activity of PacX. The ? symbol indicates undefined regulatory activities by PacC proteolysed forms.