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.

The Aspergillus nidulans transcription factor PacC, which mediates pH regulation, is proteolytically processed to a functional form in response to ambient alkaline pH. The full-length PacC form is unstable in the presence of an operational pH signal transduction pathway, due to processing to the relatively stable short functional form. We have characterized and used an extensive collection of pacC mutations, including a novel class of "neutrality-mimicking" pacC mutations having aspects of both acidity-and alkalinity-mimicking phenotypes, to investigate a number of important features of PacC processing. Analysis of mutant proteins lacking the major translation initiation residue or truncated at various distances from the C terminus showed that PacC processing does not remove N-terminal residues, indicated that processing yields slightly heterogeneous products, and delimited the most upstream processing site to residues ϳ252 to 254. Faithful processing of three mutant proteins having deletions of a region including the predicted processing site(s) and of a fourth having 55 frameshifted residues following residue 238 indicated that specificity determinants reside at sequences or structural features located upstream of residue 235. Thus, the PacC protease cuts a peptide bond(s) remote from these determinants, possibly thereby resembling type I endonucleases. Downstream of the cleavage site, residues 407 to 678 are not essential for processing, but truncation at or before residue 333 largely prevents it. Ambient pH apparently regulates the accessibility of PacC to proteolytic processing. Alkalinity-mimicking mutations L259R, L266F, and L340S favor the protease-accessible conformation, whereas a protein with residues 465 to 540 deleted retains a protease-inaccessible conformation, leading to acidity mimicry. Finally, not only does processing constitute a crucial form of modulation for PacC, but there is evidence for its conservation during fungal evolution. Transgenic expression of a truncated PacC protein, which was processed in a pH-independent manner, showed that appropriate processing can occur in Saccharomyces cerevisiae.A growing class of transcription factors is activated by the proteolytic removal of protein domains which negatively modulate their activity. These negatively acting domains can be provided in trans (i.e., by another protein in a complex) or in cis (i.e., by a region within the transcription factor's primary translation product). Examples of the former are the p50-p52 NF-B family and their negative regulators, the IB proteins, which regulate human genes involved in immune and inflammatory responses (reviewed in reference 37), and their respective Drosophila homologues dorsal and cactus (reviewed in reference 3), which establish the dorsal-ventral polarity of the fly embryo and mediate the Drosophila immune response (19). Examples of the latter include the p105 precursor of NF-B p50 (whose C-terminal moiety is homologous to a trans-acting member of the IB family); the sterol regulatory element bi...

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The Aspergillus nidulans zinc finger transcription factor PacC is activated by proteolytic processing in response to ambient alkaline pH. The pH-regulated step is the transition of full-length PacC from a closed to an open, protease-accessible conformation. Here we show that in the absence of ambient pH signaling, the C-terminal negative-acting domain prevents the nuclear localization of full-length closed PacC. In contrast, the processed PacC form is almost exclusively nuclear at any ambient pH. In the presence of ambient pH signaling, the fraction of PacC that is in the open conformation but has not yet been processed localizes to the nucleus. Therefore, ambient alkaline pH leads to an increase in nuclear PacC by promoting the proteolytic elimination of the negative-acting domain to yield the processed form and by increasing the proportion of full-length protein that is in the open conformation. These findings explain why mutations resulting in commitment of PacC to processing irrespective of ambient pH lead to permanent PacC activation and alkalinity mimicry. A nuclear import signal that targets Escherichia coli ␤-galactosidase to the nucleus has been located to the PacC zinc finger region. A mutation abolishing DNA binding does not prevent nuclear localization of the processed form, showing that PacC processing does not lead to nuclear localization by passive diffusion of the protein made possible by the reduction in size, followed by retention in the nucleus after DNA binding.Proteolytic processing activation of transcription factors in response to their cognate environmental signals occurs across distant groups of eukaryotic organisms. pH regulation of gene expression in the mold Aspergillus nidulans is one such example. Here, the key regulatory zinc finger protein PacC activates alkaline genes and represses acidic genes according to the needs imposed by ambient pH, thereby providing the organism with one prerequisite for growing in environments as acidic as pH 2.5 or as alkaline as pH 10.5 (7, 59). Other prototypical members of the group of transcription factors activated by proteolytic processing are the immune and inflammatory response regulator NF-B (23, 58), the Drosophila melanogaster cubitus interruptus (Ci) zinc finger factor (the transducer of the hedgehog signal) (29, 53), and the sterol regulatory elementbinding protein (SREBP), which switches on genes for cholesterol biosynthesis and fat metabolism (5, 6).The zinc finger transcription factor PacC is synthesized as a 674-residue precursor. At alkaline ambient pH, a signal transmitted to PacC by the orphan pal gene signal transduction pathway (13,14,37,43,44) results in a conformational change leading to an open conformation in which PacC is accessible to a processing protease (18,41,47). This protease removes ϳ400 residues from the C terminus, which includes a negative-acting domain. The resulting product (248 to 250 residues) (41) is fully competent in transcriptional regulation through 5Ј-GCC ARG-3Ј sites (20) in the promoters of both alkaline...

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...