Activation of theAspergillusPacC zinc finger transcription factor requires two proteolytic steps

Abstract

The Aspergillus PacC transcription factor undergoes proteolytic activation in response to alkaline ambient pH. In acidic environments, the 674 residue translation product adopts a ‘closed’ conformation, protected from activation through intramolecular interactions involving the ≤150 residue C‐terminal domain. pH signalling converts PacC to an accessible conformation enabling processing cleavage within residues 252–254. We demonstrate that activation of PacC requires two sequential proteolytic steps. First, the ‘closed’ translation product is converted to an accessible, committed intermediate by proteolytic elimination of the C‐terminus. This ambient pH‐regulated cleavage is required for the final, pH‐independent processing reaction and is mediated by a distinct signalling protease (possibly PalB). The signalling protease cleaves PacC between residues 493 and 500, within a conserved 24 residue ‘signalling protease box’. Precise deletion or Leu498Ser substitution prevents formation of the committed and processed forms, demonstrating that signalling cleavage is essential for final processing. In contrast, signalling cleavage is not required for processing of the Leu340Ser protein, which lacks interactions preventing processing. In its two‐step mechanism, PacC processing can be compared with regulated intramembrane proteolysis.

Physiological Involvement in pH Signaling of Vps24-mediated Recruitment of Aspergillus PalB Cysteine Protease to ESCRT-III

“…4D) (10,16,18,22,28). (After ϳ1 h at alkaline pH, newly synthesized PacC 72 becomes detectable again, possibly as a result of a negative feedback loop that regulates its processing).…”

“…PacC 27 is a transcriptional activator of alkaline-expressed genes and a repressor of acid-expressed genes (13,14). The alkaline ambient pH-dependent conversion of PacC 72 into PacC 53 is almost certainly catalyzed by the calpain-like cysteine protease PalB (10,15,16) (see Refs. 7, 8, 17 for reviews).…”

Evidence for the Direct Involvement of the Proteasome in the Proteolytic Processing of the Aspergillus nidulans Zinc Finger Transcription Factor PacC

“…674-residue PacC contains an N-terminal DNA binding domain with three canonical C 2 H 2 zinc fingers (22) and undergoes two-step proteolytic activation removing ϳ425 residues from the C terminus in response to alkaline ambient pH (22)(23)(24). The 72-kDa PacC 72 translation product is converted to 53-kDa PacC 53 by a "signaling" protease.…”

“…(v) The site(s) of processing is dictated by sequence or structure determinants that are remote from the processing site (32). (vi) A processing efficiency determinant has been mapped to a region located C-terminal to the processing site, which would be exposed after signaling protease cleavage (24). (vii) The zinc finger regions of PacC and Ci share distinctive structural features (18,20), and indeed the C-terminal fingers (those that the proteasome would first encounter) share, in addition to the zinc finger fold, high amino acid sequence identity (19).…”

“…This C-terminally truncated version of PacC resembles PacC 53 (whose C terminus lies within residues 493-500) and is therefore processed to PacC 27 in an ambient pH-independent manner (23,24). The pacC c 14 product is processed ex vivo in S. cerevisiae (32), allowing us to use extant viable mutants impairing the yeast proteasome, following the approach reported by the Maniatis group for their analysis of proteolytic processing of NF-B p105 (2,13).…”