Ambient pH Signaling Regulates Nuclear Localization of the Aspergillus nidulans PacC Transcription Factor

Abstract

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...

Regulation of Gene Expression by Ambient pH in Filamentous Fungi and Yeasts

“…Since the C terminus of this mutant protein is close to that of the processed form(s) (within residues 252 to 254), this finding indicates that processing leads to the activation of the otherwise inactive pacC translation product. In view of the facts that PacC , PacC , and PacC polypeptides tagged at their C termini (in all three cases close to the processing site) with green fluorescent protein (GFP) are localized in the nucleus in a pal-independent manner (81,82) and that PacC tagged at its N terminus with GFP is also localized in the nucleus in a pH-and pal-independent manner (81,82), PacC proteolytic activation clearly at least promotes the nuclear localization of a transcriptionally active polypeptide. The model accommodating this finding in references 82 and 95 has been modified to take into account the fact that the proteolytic activation of PacC takes place in two steps (see below and Fig.…”

“…pacC has been thoroughly analyzed genetically, and the molecular characterization of pacC mutations (33,41,43,82,95,122) has been crucial for the understanding of PacC activation by proteolysis (see below). Mutational truncations removing between 92 and 412 residues from the C terminus of PacC result in an alkalinity-mimicking (pacC c ) phenotype (83,95,122;Rainbow et al,unpublished), which revealed that the C-terminal region of PacC contains a negative-acting domain that is inactivated in the presence of alkaline-ambient-pH signaling.…”

“…High-resolution EMSA (33,82) discriminates between the open and closed PacC-DNA complexes. PacC in the open complex is not recognized by anti-PacC[529-678] antiserum or, in the case of C-terminally tagged PacC, by antisera against the cognate tag (33), strongly suggesting that open PacC is not the full-length translation product and actually lacks C-terminal residues.…”

Expression and promoter characterization of BbPacC, a pH response transcription factor gene of the entomopathogenic fungus Beauveria bassiana

“…This results in the removal of the C-terminal negative repression domain (Díez et al, 2002). The remaining N-terminal activation domain goes into the nucleus (Mingot et al, 2001) where it activates transcription of genes preferentially expressed under alkaline conditions and represses those expressed under acidic conditions. In both cases, this is by binding to GCCARG ] 50 300 0 P 3 8 7 P 5 0 2 P 5 7 2 P 7 4 2 P 8 8 8 P 1 0 4 0 P 1 2 4 7 P 1 4 7 9 P t r p C W T Fig.…”

Virulence Regulation of Phytopathogenic Fungi by pH

“…Under acidic ambient conditions, PACC remains cytosolic and inaccessible for proteolysis, but in response to pH signaling, the Pal apparatus activates PACC. In its active form PACC 27 is translocated to the nucleus, where the zinc-finger region recognizes the consensus DNA site 5¢-GCCARG (96,147). PACC regulation has been most extensively studied in the genus Aspergillus, but it was also found in many other fungi (6,117).…”