Specificity Determinants of Proteolytic Processing of Aspergillus PacC Transcription Factor Are Remote from the Processing Site, and Processing Occurs in Yeast If pH Signalling Is Bypassed

Abstract

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...

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

“…Acidity-, alkalinity-and neutrality-mimicking mutations have been obtained in pacC (17,33,39,83,95,122). This phenotypic heterogeneity reflects the more direct involvement of the pacC product (the key transcription factor, see below) in regulation of gene expression by ambient pH.…”

“…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. Single-residue changes involving residues 259, 266, 340, 573, and 579 also lead to an alkalinity-mimicking pacC c phenotype (41,83). The relatively large size of the region in which alkalinity-mimicking mutations can occur accounts for the ease with which they can be selected and for the erroneous proposal (17), based on their high frequency, that they constitute the loss-of-function class.…”

“…An M5I mutation (pacC504) (122) has been used to show that most, possibly all, translation proceeds from methionine codon 5, which would result in a 674-residue protein (83). (Residue numbering, however, continues to be on the basis of 678 residues to avoid confusion between earlier and later publications [83].)…”

“…For example, they prevent or reduce growth at alkaline pH, reduce alkaline phosphatase levels, increase GABA transport and acid phosphatase levels, and confer neomycin resistance and molybdate hypersensitivity (Table 1). In addition, a small number of mutations have a neutrality-mimicking phenotype irrespective of ambient pH in which the pattern of gene expression approximates that of the wild type grown at pH 6.5 or has a heterogeneous mixture of alkalinity-mimicking and aciditymimicking characteristics (83).…”

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

“…7, 8, 17 for reviews). The pH-independent proteolytic processing of PacC 53 into PacC 27 is in all likelihood mediated by the proteasome (11,18). The pathway is conserved in Saccharomyces cerevisiae, where the PacC orthologue Rim101p is activated by a single proteolytic step (19), presumably catalyzed by the budding yeast PalB orthologue Rim13p (20,21).…”