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.

pH by the products of the six pal genes (Denison et al., Universidad del País Vasco, Apdo. 1072, San Sebastián 20080, Spain, 1995, 1998 Maccheroni et al., 1997 form containing the~248-250 N-terminal residues Mingot et al., 1999), which activates expres- 5 Corresponding author e-mail: penalva@cib.csic.es sion of genes expressed preferentially under alkaline growth conditions (Espeso et al., 1993;Tilburn et al., In response to alkaline ambient pH, the Aspergillus 1995; Espeso and Peñalva, 1996) and represses genes nidulans PacC transcription factor mediating pH reguexpressed preferentially at acidic pH (Tilburn et al., lation of gene expression is activated by proteolytic 1995; Hutchings et al., 1999; E.A.Espeso and H.N.Arst, removal of a negative-acting C-terminal domain. We submitted). The PacC DNA binding domain (DBD), demonstrate interactions involving the~150 C-terminal containing three Cys 2 His 2 zinc fingers and binding to PacC residues and two regions located immediately GCCARG promoter sites ; Espeso downstream of the DNA binding domain. Our data et al., 1997), is located centrally within the processed form. indicate two full-length PacC conformations whoseThe alkaline pH-sensitive step in the regulatory cascade relative amounts depend upon ambient pH: one 'open' appears to be the accessibility of the PacC primary and accessible for processing, the other 'closed' and translation product to the processing protease, suggesting inaccessible. The location of essential determinants for that PacC alternates between protease-resistant and proteolytic processing within the two more upstream protease-sensitive conformations in response to ambient interacting regions probably explains why the interpH (Mingot et al., 1999). Loss-of-function pal -mutations actions prevent processing, whereas the direct preventing ambient pH signal transduction and PacC involvement of the C-terminal region in processingproteolytic processing (Caddick et al., 1986; Denison preventing interactions explains why C-terminal trun Tilburn et al., 1995;Negrete-Urtasun et al., cating mutations result in alkalinity mimicry and pH-1999) lead to an acidity-mimicking phenotype, as do null independent processing. A mutant PacC deficient in (pacC -) or partial loss-of-function (pacC ϩ/-) mutations in pH signal response and consequent processing behaves pacC. Gain-of-function pacC c mutations have an alkalinas though locked in the 'closed' form. Single-residue ity-mimicking phenotype, obviating the need for ambient substitutions, obtained as mutations bypassing the need pH signalling and resulting in constitutive (i.e. pH-indefor pH signal transduction, identify crucial residues in pendent) PacC processing (Caddick et al., 1986; Orejas each of the three interactive regions and overcome Tilburn et al., 1995;Mingot et al., 1999). processing deficiency in the 'permanently closed ' Most extant pacC c mutations result in truncation of 100-mutant.412 residues from the C-terminus of PacC, suggesting a Keywords: Aspergillus nidulans/PacC/pH regulation...

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