Yeast Npi3/Bro1 is involved in ubiquitin-dependent control of permease trafficking

AbstractThe membrane traffic and stability of the general amino acid permease Gap1 of Saccharomyces cerevisiae are under nitrogen control. Addition of a preferential nitrogen source such as ammonium to cells growing on a poor nitrogen source induces internalization of the permease and its subsequent degradation in the vacuole. This down-regulation requires ubiquitination of Gap1 through a process involving ubiquitin ligase Npi1/Rsp5, ubiquitin hydrolase Npi2/Doa4, and Bul1/2, two Npi1/Rsp5 interacting proteins. Here we report that yet another protein, Npi3, is involved in the regulation of Gap1 trafficking. We show that Npi3 is required for NH4+-induced down-regulation of Gap1, and particularly for efficient ubiquitination of the permease. Npi3 plays a pleiotropic role in permease down-regulation, since it is also involved in ubiquitination and stress-induced down-regulation of the uracil permease Fur4 and in glucose-induced degradation of hexose transporters Hxt6/7. We further provide evidence that Npi3 is required for direct vacuolar sorting of neosynthesized Gap1 permease as it occurs in npr1 mutant cells. NPI3 is identical to BRO1, a gene encoding a protein of unknown biochemical function and recently proposed to be involved in protein turnover. Npi3/Bro1 homologues include fungal proteins required for proteolytic cleavage of zinc finger proteins and the mouse Aip1 protein involved in apoptosis. We propose that proteins of the Npi3/Bro1 family, including homologues from higher species, may play a conserved role in ubiquitin-dependent control of membrane protein trafficking

Mutational analysis of the Aspergillus ambient pH receptor PalH underscores its potential as a target for antifungal compounds.

The pal/RIM ambient pH signalling pathway is crucial for the ability of pathogenic fungi to infect hosts. The Aspergillus nidulans 7-TMD receptor PalH senses alkaline pH, subsequently facilitating ubiquitination of the arrestin PalF. Ubiquitinated PalF triggers downstream signalling events. The mechanism(s) by which PalH transduces the alkaline pH signal to PalF is poorly understood. We show that PalH is phosphorylated in a signal dependent manner, resembling mammalian GPCRs, although PalH phosphorylation, in contrast to mammalian GPCRs, is arrestin dependent. A genetic screen revealed that an ambient-exposed region comprising the extracellular loop connecting TM4-TM5 and ambient-proximal residues within TM5 is required for signalling. In contrast, substitution by alanines of four aromatic residues within TM6 and TM7 results in a weak 'constitutive' activation of the pathway. Our data support the hypothesis that PalH mechanistically resembles mammalian GPCRs that signal via arrestins, such that the relative positions of individual helices within the heptahelical bundle determines the Pro316-dependent transition between inactive and active PalH conformations, governed by an ambient-exposed region including critical Tyr259 that potentially represents an agonist binding site. These findings open the possibility of screening for agonist compounds stabilizing the inactive conformation of PalH, which might act as antifungal drugs against ascomycetes

PalC, One of Two Bro1 Domain Proteins in the Fungal pH Signalling Pathway, Localizes to Cortical Structures and Binds Vps32

PalC, distantly related to Saccharomyces cerevisiaeperipheral endosomal sorting complexes required for transport III (ESCRT-III) component Bro1p and one of six Aspergillus nidulanspH signalling proteins, contains a Bro1 domain. Green fluorescent protein (GFP)-tagged PalC is recruited to plasma membrane-associated punctate structures upon alkalinization, when pH signalling is active. PalC recruitment to these structures is dependent on the seven transmembrane domain (7-TMD) receptor and likely pH sensor PalH. PalC is a two-hybrid interactor of the ESCRT-III Vps20/Vps32 subcomplex and binds Vps32 directly. This binding is largely impaired by Pro439Phe, Arg442Ala and Arg442His substitutions in a conserved region mediating interaction of Bro1p with Vps32p, but these substitutions do not prevent cortical punctate localization, indicating Vps32 independence. In contrast, Arg442Δ impairs Vps32 binding and prevents PalC-GFP recruitment to cortical structures. pH signalling involves a plasma membrane complex including the 7-TMD receptor PalH and the arrestin-like PalF and an endosomal membrane complex involving the PalB protease, the transcription factor PacC and the Vps32 binding, Bro1-domain-containing protein PalA. PalC, which localizes to cortical structures and can additionally bind a component of ESCRT-III, has the features required to bridge these two entities. A likely S. cerevisiaeorthologue of PalC has been identified, providing the basis for a unifying hypothesis of gene regulation by ambient pH in ascomycetes

Aspects of the PH signal transduction pathway in the filamentous fungus Aspergullus nidulans

SIGLEAvailable from British Library Document Supply Centre-DSC:DXN026482 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Refining the pH response in Aspergillus nidulans: a modulatory triad involving PacX, a novel zinc binuclear cluster protein

International audienceThe Aspergillus nidulans PacC transcription factor mediates gene regulation in response to alkaline ambient pH which, signalled by the Pal pathway, results in the processing of PacC(72) to PacC(27) via PacC(53). Here we investigate two levels at which the pH regulatory system is transcriptionally moderated by pH and identify and characterise a new component of the pH regulatory machinery, PacX. Transcript level analysis and overexpression studies demonstrate that repression of acid-expressed palF, specifying the Pal pathway arrestin, probably by PacC(27) and/or PacC(53), prevents an escalating alkaline pH response. Transcript analyses using a reporter and constitutively expressed pacC trans-alleles show that pacC preferential alkaline-expression results from derepression by depletion of the acid-prevalent PacC(72) form. We additionally show that pacC repression requires PacX. pacX mutations suppress PacC processing recalcitrant mutations, in part, through derepressed PacC levels resulting in traces of PacC(27) formed by pH-independent proteolysis. pacX was cloned by impala transposon mutagenesis. PacX, with homologues within the Leotiomyceta, has an unusual structure with an amino-terminal coiled-coil and a carboxy-terminal zinc binuclear cluster. pacX mutations indicate the importance of these regions. One mutation, an unprecedented finding in A. nidulans genetics, resulted from an insertion of an endogenous Fot1-like transposon

Activation of the Aspergillus PacC zinc finger transcription factor requires two proteolytic steps

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

Virulence Comparisons of Aspergillus nidulans Mutants Are Confounded by the Inflammatory Response of p47(phox)(−/−) Mice

While investigating the requirement for phagosomal alkalinization in the host defense against pulmonary aspergillosis, we observed high morbidity of p47(phox)(−/−) mice infected with pH-insensitive Aspergillus nidulans mutants despite a paucity of fungal growth. Fatal infection also resulted from a normally avirulent p-aminobenzoate auxotroph. This demonstrates that p47(phox)(−/−) murine immunity contributes significantly to A. nidulans lethality. These data have wider implications for microbial virulence studies with p47(phox)(−/−) mice

Nonsense-Mediated mRNA Decay Mutation in Aspergillus nidulans

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