The conserved PacC signal transduction pathway mediates many metabolic events involved in ambient pH sensing in A. nidulans, and it is widely accepted that it governs only the response to neutral-to-alkaline pHs. The pacC gene codes for a Zn-finger transcription factor whose transcription is itself induced under alkaline growth conditions. The pal genes (palA, B, C, F, H, and I) are putative members of a signaling cascade, whose function is presumed to promote the proteolytic activation of PacC. Thus, the current model states that the full-length version of PacC (PacC72) is activated at alkaline pH by two sequential proteolytic steps that remove the C-terminal negatively acting domain. The conversion of PacC72 to PacC53 is PalB- and pH-dependent, whereas the conversion of PacC53 to PacC27 is pH-independent. It is assumed that the PacC27 processed form is perhaps the sole functional form of PacC at alkaline pH. This model implies that inactivation of any of the pal genes should lead to an abundant expression of PacC72 irrespective of the extracellular pH. To test this hypothesis we purified PacC as briefly described here: the truncated PacC protein, which contains the three zinc-fingers of gene pacC, was expressed in E. coli, purified by affinity chromatography, and polyclonal antibodies were raised in albino male rabbits. The anti-PacC antibodies was purified, chemically cross-linked to hydrazide-Sepharose resin, and used for purification of PacC by immunoaffinity chromatography. We present evidence that proteolytic processing of PacC72 occurs earlier in the palB7 mutant grown under acidic conditions. Furthermore, proteolysis of PacC is extensively detected in the palB7 mutant grown under alkaline conditions, where PacC72 should be abundant. Thus, the full-length version of PacC was abundantly detectable only in strains in which palB is functional. Financial support: FAPESP, CNPq, FAEPA and CAPES.
Full conference title:
23rd Fungal Genetics Conference
- Fungal Genetics Conference 23rd (2002)