| Summary: | In the filamentous ascomycete <i>Aspergillus nidulans</i>, 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 <i>pacC</i> expression might be under SltA regulation. Additional transcriptional studies of PacC and the only pH-regulated <i>pal</i> gene, <i>palF</i>, confirmed both the strong dependence on ambient pH and the function of SltA. The regulation of <i>pacC</i> expression is dependent on the activity of the zinc binuclear (C6) cluster transcription factor PacX. However, we found that the ablation of <i>sltA</i> in the <i>pacX<sup>−</sup></i> mutant background specifically prevents the increase in <i>pacC</i> expression levels without affecting PacC protein levels, showing a novel specific function of the PacX factor. The loss of <i>sltA</i> function causes the anomalous proteolytic processing of PacC and a reduction in the post-translational modifications of PalF. At alkaline pH, in a null <i>sltA</i> background, PacC<sup>72kDa</sup> accumulates, detection of the intermediate PacC<sup>53kDa</sup> 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 <i>pacC</i>, such as <i>pacC<sup>c</sup>14</i> and <i>pacC<sup>c</sup>700</i>, resembling PacC<sup>53kDa</sup> and PacC<sup>27kDa</sup>, respectively, allowed the normal processing of PacC but did not rescue the alkaline pH-sensitive phenotype caused by the null <i>sltA</i> 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 <i>A. nidulans</i>.
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