Mechanistic basis of proteolytic signalling of the SltB-SltA dual system mediating cation homeostasis and alkaline pH response in Aspergillus nidulans

Cation homeostasis and adaptation to alkaline pH in Aspergillus nidulans depend on the Slt pathway, comprising the transcription factor SltA and the signalling protease SltB. However, the molecular mechanisms controlling SltA activation and subcellular regulation remain poorly understood. We have identified the proteolytic and localisation determinants required for SltA function. Basal SltB levels are sufficient to initiate the proteolytic conversion of inactive SltA, supporting a model in which pathway activation starts independently of SltA-dependent transcriptional regulation. Mutational and domain analyses in SltA demonstrate that a conserved RSMA motif is required for cleavage by SltB to generate the transcriptionally active 32 kDa form, independently of the poorly conserved C-terminal region or zinc-finger domain. Proteolytic processing is required for SltA nuclear import and nucleolar accumulation, which relies on a functional nuclear localisation signal adjacent to the zinc-finger domain and is enhanced by residues within the C-terminal region. Zinc fingers differentially contribute to nucleolar targeting and nucleoplasmic retention. Structural predictions aided in demonstrating that correct SltB auto-activation and stability depend on an RSKV motif followed by a K/R-rich region. Together, these findings define the molecular basis of proteolytic mechanisms in Slt system, which is highly conserved in the fungal kingdom, and link SltA proteolytic activation to regulated nuclear/nucleolar localisation.