Characterization of the pH signal transduction pathway gene palA of Aspergillus nidulans and identification of possible homologs.
ABSTRACT: We have cloned the palA gene of Aspergillus nidulans, one of six genes participating in ambient pH signal transduction in a regulatory circuit mediating pH regulation of gene expression. The derived 798-residue PalA protein is 29.4% identical over its entire length to a hypothetical protein from the nematode Caenorhabditis elegans and also has possible yeast homologs.
Project description:The Aspergillus nidulans ambient pH signaling pathway involves two transmembrane domain (TMD)-containing proteins, PalH and PalI. We provide in silico and mutational evidence suggesting that PalI is a three TMD (3-TMD) protein with an N-terminal signal peptide, and we show that PalI localizes to the plasma membrane. PalI is not essential for the proteolytic conversion of the PacC translation product into the processed 27-kDa form, but its absence markedly reduces the accumulation of the 53-kDa intermediate after cells are shifted to an alkaline pH. PalI and its homologues contain a predicted luminal, conserved Gly-Cys-containing motif that distantly resembles a Gly-rich dimerization domain. The Gly44Arg and Gly47Asp substitutions within this motif lead to loss of function. The Gly47Asp substitution prevents plasma membrane localization of PalI-green fluorescent protein (GFP) and leads to its missorting into the multivesicular body pathway. Overexpression of the likely ambient alkaline pH receptor, the 7-TMD protein PalH, partially suppresses the null palI32 mutation. Although some PalH-GFP localizes to the plasma membrane, it predominates in internal membranes. However, the coexpression of PalI to stoichiometrically similar levels results in the strong predominance of PalH-GFP in the plasma membrane. Thus, one role for PalI, but possibly not the only role, is to assist with plasma membrane localization of PalH. These data, considered along with previous reports for both Saccharomyces cerevisiae and A. nidulans, strongly support the prevailing model of pH signaling involving two spatially segregated complexes: a plasma membrane complex containing PalH, PalI, and the arrestin-like protein PalF and an endosomal membrane complex containing PalA and PalB, to which PacC is recruited for its proteolytic activation.
Project description:The alkaline ambient pH signal transduction pathway component PalC has no assigned molecular role. Therefore we attempted a gene-specific mutational analysis and obtained 55 new palC loss-of-function alleles including 24 single residue substitutions. Refined similarity searches reveal conserved PalC regions including one with convincing similarity to the BRO1 domain, denoted PCBROH, where clustering of mutational changes, including PCBROH key residue substitutions, supports its structural and/or functional importance. Since the BRO1 domain occurs in the multivesicular body (MVB) pathway protein Bro1/Vps31 and also the pH signal transduction protein PalA (Rim20), both of which interact with MVB component (ESCRT-III protein) Vps32/Snf7, this might reflect a further link between the pH response and endocytosis.
Project description:The Aspergillus nidulans pH-responsive transcription factor PacC is modulated by limited, two-step proteolysis. The first, pH-regulated cleavage occurs in the 24-residue highly conserved "signaling protease box" in response to the alkaline pH signal. This is transduced by the Pal signaling pathway, containing the predicted calpain-like cysteine protease and likely signaling protease, PalB. In this work, we carried out classical mutational analysis of the putative signaling protease PalB, and we describe 9 missense and 18 truncating loss-of-function (including null) mutations. Mutations in the region of and affecting directly the predicted catalytic cysteine strongly support the deduction that PalB is a cysteine protease. Truncating and missense mutations affecting the C terminus highlight the importance of this region. Analysis of three-hemagglutinin-tagged PalB in Western blots demonstrates that PalB levels are independent of pH and Pal signal transduction. We have followed the processing of MYC(3)-tagged PacC in Western blots. We show unequivocally that PalB is essential for signaling proteolysis and is definitely not the processing protease. In addition, we have replaced 15 residues of the signaling protease box of MYC(3)-tagged PacC (pacC900) with alanine. The majority of these substitutions are silent. Leu481Ala, Tyr493Ala, and Gln499Ala result in delayed PacC processing in response to shifting from acidic to alkaline medium, as determined by Western blot analysis. Leu498Ala reduces function much more markedly, as determined by plate tests and processing recalcitrance. Excepting Leu498, this demonstrates that PacC signaling proteolysis is largely independent of sequence in the cleavage region.
Project description:The Aspergillus nidulans xylanase genes xlnA and xlnB are subject to regulation by ambient pH via the zinc finger transcription factor PacC. In the presence of D-xylose, xlnA is expressed under conditions of alkaline ambient pH while xlnB is expressed at acidic ambient pH. These data have been confirmed for acidity- and alkalinity-mimicking A. nidulans mutants.
Project description:An Aspergillus nidulans mutation, designated nmdA1, has been selected as a partial suppressor of a frameshift mutation and shown to truncate the homologue of the Saccharomyces cerevisiae nonsense-mediated mRNA decay (NMD) surveillance component Nmd2p/Upf2p. nmdA1 elevates steady-state levels of premature termination codon-containing transcripts, as demonstrated using mutations in genes encoding xanthine dehydrogenase (hxA), urate oxidase (uaZ), the transcription factor mediating regulation of gene expression by ambient pH (pacC), and a protease involved in pH signal transduction (palB). nmdA1 can also stabilize pre-mRNA (unspliced) and wild-type transcripts of certain genes. Certain premature termination codon-containing transcripts which escape NMD are relatively stable, a feature more in common with certain nonsense codon-containing mammalian transcripts than with those in S. cerevisiae. As in S. cerevisiae, 5' nonsense codons are more effective at triggering NMD than 3' nonsense codons. Unlike the mammalian situation but in common with S. cerevisiae and other lower eukaryotes, A. nidulans is apparently impervious to the position of premature termination codons with respect to the 3' exon-exon junction.
Project description:Invasive aspergillosis mainly occurs in immunocompromised patients and is commonly caused by Aspergillus fumigatus, while A.nidulans is rarely the causative agent. However, in chronic granulomatous disease (CGD) patients, A. nidulans is a frequent cause of invasive aspergillosis and is associated with higher mortality. Immune recognition of A. nidulans was compared to A. fumigatus to offer an insight into why A. nidulans infections are prevalent in CGD. Live cell imaging with J774A.1 macrophage-like cells and LC3-GFP-mCherry bone marrow-derived macrophages (BMDMs) revealed that phagocytosis of A. nidulans was slower compared to A. fumigatus. This difference could be attributed to slower migration of J774A.1 cells and a lower percentage of migrating BMDMs. In addition, delayed phagosome acidification and LC3-associated phagocytosis was observed with A. nidulans. Cytokine and oxidative burst measurements in human peripheral blood mononuclear cells revealed a lower oxidative burst upon challenge with A. nidulans. In contrast, A. nidulans induced significantly higher concentrations of cytokines. Collectively, our data demonstrate that A. nidulans is phagocytosed and processed at a slower rate compared to A. fumigatus, resulting in reduced fungal killing and increased germination of conidia. This slower rate of A. nidulans clearance may be permissive for overgrowth within certain immune settings.
Project description:The zinc finger transcription factor PacC undergoes two-step proteolytic activation in response to alkaline ambient pH. PalA is a component of the fungal ambient pH signal transduction pathway. Its mammalian homologue AIP1/Alix interacts with the apoptosis-linked protein ALG-2. We show that both PalA and AIP1/Alix recognize a protein-protein binding motif that we denote YPXL/I, where Tyr, Pro, and Leu/Ile are crucial for its interactive properties. Two such motifs flanking the signaling protease cleavage site mediate direct binding of PalA to PacC, required for the first and only pH-regulated cleavage of this transcription factor. PalA can bind the "closed" (i.e., wild-type full-length) conformer of PacC, suggesting that PalA binding constitutes the first stage in the two-step proteolytic cascade, recruiting or facilitating access of the signaling protease, presumably PalB. In addition to recognizing YPXL/I motifs, both PalA and AIP1/Alix interact with the Aspergillus class E Vps protein Vps32 homologue, a member of a protein complex involved in the early steps of the multivesicular body pathway, suggesting that this interaction is an additional feature of proteins of the PalA/AIP1/Alix family.
Project description:Activation of the Aspergillus nidulans transcription factor PacC, which mediates ambient pH regulation of gene expression and is recruited to ESCRT-III by the Vps32-interacting scaffold PalA, involves its ambient pH-dependent C-terminal proteolysis. This reaction is almost certainly catalyzed by the PalB calpain-like protease. Here we show that PalB associates with membranes and interacts specifically and directly with ESCRT-III Vps24. The PalB N-terminal MIT domain and the Vps24 C-terminal MIM motif are necessary and sufficient for this interaction. PalB(DeltaMIT), a mutant PalB lacking the MIT domain is inefficiently recruited to membranes and impaired in PacC proteolytic processing. Notably, membrane recruitment is promoted and PacC processing largely restored by covalent attachment of Vps24 to mutant PalB(DeltaMIT). This is the first reported evidence that calpain-like recruitment to ESCRT-III lattices plays a physiological role. It unambiguously positions the calpain-like protease PalB within the ESCRT-III-associated pH signaling complex, underlines the positive role of ESCRT-III in ambient pH signal transduction, and suggests a possible mechanism for PalB activation.
Project description:The fungal pal/RIM signalling pathway, which regulates gene expression in response to environmental pH involves, in addition to dedicated proteins, several components of ESCRT complexes, which suggested that pH signalling proteins assemble on endosomal platforms. In Aspergillus nidulans, dedicated Pal proteins include the plasma membrane receptor PalH and its coupled arrestin, PalF, which becomes ubiquitylated in alkaline pH conditions, and three potentially endosomal ESCRT-III associates, including Vps32 interactors PalA and PalC and Vps24 interactor calpain-like PalB. We studied the subcellular locations at which signalling takes place after activating the pathway by shifting ambient pH to alkalinity. Rather than localising to endosomes, Vps32 interactors PalA and PalC transiently colocalise at alkaline-pH-induced cortical structures in a PalH-, Vps23- and Vps32-dependent but Vps27-independent manner. These cortical structures are much more stable when Vps4 is deficient, indicating that their half-life depends on ESCRT-III disassembly. Pull-down studies revealed that Vps23 interacts strongly with PalF, but co-immunoprecipitates exclusively with ubiquitylated PalF forms from extracts. We demonstrate that Vps23-GFP, expressed at physiological levels, is also recruited to cortical structures, very conspicuous in vps27? cells in which the prominent signal of Vps23-GFP on endosomes is eliminated, in a PalF- and alkaline pH-dependent manner. Dual-channel epifluorescence microscopy showed that PalC arrives at cortical complexes before PalA. As PalC recruitment is PalA independent and PalA recruitment is PalC dependent but PalB independent, these data complete the participation order of Pal proteins in the pathway and strongly support a model in which pH signalling takes place in ESCRT-containing, plasma-membrane-associated, rather than endosome-associated, complexes.
Project description:Experimental evolution was conducted using Drosophila melanogaster populations that developed as larvae on breeding substrate that was infested with Aspergillus nidulans wild type, A. nidulans toxin-impaired mutant strain delta-laeA, the mycotoxin sterigmatocystin, or on fungi and toxin free substrate. Overall population were reared under these conditions for 11 generations, where after each confrontation generation one relaxation generation (fungi and toxin free breeding substrate) was conducted. Nine generations after the last selection treatment, first instar larvae were confronted with 3 days old A. nidulans wild type colonies or control conditions. 24 hours after confrontation start larvae were collected. For each biological replicate 52 larvae were collected from 4 independent confrontation units, balanced design. Three populations per selection regime were conducted, resulting in: 2 conditions x 4 selection regimes x 3 biological replicates (equal to fly population) = 24 samples. Selection regimes: sCO= control; sWT= A. nidulans wild type; sLA= A. nidulans mutant strain; sST= Sterigmatocystin. confrontation condition: cCO= control; cWT= A. nidulans wild type.