Characterization of Aspergillus nidulans TRAPPs uncovers unprecedented similarities between fungi and metazoans and reveals the modular assembly of TRAPPII.

TRAnsport Protein Particle complexes (TRAPPs) are ubiquitous regulators of membrane traffic mediating nucleotide exchange on the Golgi regulatory GTPases RAB1 and RAB11. In S. cerevisiae and metazoans TRAPPs consist of two large oligomeric complexes: RAB11-activating TRAPPII and RAB1-activating TRAPPIII. These share a common core TRAPPI hetero-heptamer, absent in metazoans but detected in minor proportions in yeast, likely originating from in vitro-destabilized TRAPPII/III. Despite overall TRAPP conservation, the budding yeast genome has undergone extensive loss of genes, and lacks homologues of some metazoan TRAPP subunits. With nearly twice the total number of genes of S. cerevisiae, another ascomycete Aspergillus nidulans has also been used for studies on TRAPPs. We combined size-fractionation chromatography with single-step purification coupled to mass-spectrometry and negative-stain electron microscopy to establish the relative abundance, composition and architecture of Aspergillus TRAPPs, which consist of TRAPPII and TRAPPIII in a 2:1 proportion, plus a minor amount of TRAPPI. We show that Aspergillus TRAPPIII contains homologues of metazoan TRAPPC11, TRAPPC12 and TRAPPC13 subunits, absent in S. cerevisiae, and establish that these subunits are recruited to the complex by Tca17/TRAPPC2L, which itself binds to the 'Trs33 side' of the complex. Thus Aspergillus TRAPPs compositionally resemble mammalian TRAPPs to a greater extent than those in budding yeast. Exploiting the ability of constitutively-active (GEF-independent, due to accelerated GDP release) RAB1* and RAB11* alleles to rescue viability of null mutants lacking essential TRAPP subunits, we establish that the only essential role of TRAPPs is activating RAB1 and RAB11, and genetically classify each essential subunit according to their role(s) in TRAPPII (TRAPPII-specific subunits) or TRAPPII and TRAPPIII (core TRAPP subunits). Constitutively-active RAB mutant combinations allowed examination of TRAPP composition in mutants lacking essential subunits, which led to the discovery of a stable Trs120/Trs130/Trs65/Tca17 TRAPPII-specific subcomplex whose Trs20- and Trs33-dependent assembly onto core TRAPP generates TRAPPII.

Endocytic recycling via the TGN underlies the polarized hyphal mode of life.

Intracellular traffic in Aspergillus nidulans hyphae must cope with the challenges that the high rates of apical extension (1µm/min) and the long intracellular distances (>100 µm) impose. Understanding the ways in which the hyphal tip cell coordinates traffic to meet these challenges is of basic importance, but is also of considerable applied interest, as fungal invasiveness of animals and plants depends critically upon maintaining these high rates of growth. Rapid apical extension requires localization of cell-wall-modifying enzymes to hyphal tips. By combining genetic blocks in different trafficking steps with multidimensional epifluorescence microscopy and quantitative image analyses we demonstrate that polarization of the essential chitin-synthase ChsB occurs by indirect endocytic recycling, involving delivery/exocytosis to apices followed by internalization by the sub-apical endocytic collar of actin patches and subsequent trafficking to TGN cisternae, where it accumulates for ~1 min before being re-delivered to the apex by a RAB11/TRAPPII-dependent pathway. Accordingly, ChsB is stranded at the TGN by Sec7 inactivation but re-polarizes to the apical dome if the block is bypassed by a mutation in geaAgea1 that restores growth in the absence of Sec7. That polarization is independent of RAB5, that ChsB predominates at apex-proximal cisternae, and that upon dynein impairment ChsB is stalled at the tips in an aggregated endosome indicate that endocytosed ChsB traffics to the TGN via sorting endosomes functionally located upstream of the RAB5 domain and that this step requires dynein-mediated basipetal transport. It also requires RAB6 and its effector GARP (Vps51/Vps52/Vps53/Vps54), whose composition we determined by MS/MS following affinity chromatography purification. Ablation of any GARP component diverts ChsB to vacuoles and impairs growth and morphology markedly, emphasizing the important physiological role played by this pathway that, we propose, is central to the hyphal mode of growth.

TRAPPII regulates exocytic Golgi exit by mediating nucleotide exchange on the Ypt31 ortholog RabERAB11.

The oligomeric complex transport protein particle I (TRAPPI) mediates nucleotide exchange on the RAB GTPase RAB1/Ypt1. TRAPPII is composed of TRAPPI plus three additional subunits, Trs120, Trs130, and Trs65. Unclear is whether TRAPPII mediates nucleotide exchange on RAB1/Ypt1, RAB11/Ypt31, or both. In Aspergillus nidulans, RabO(RAB1) resides in the Golgi, RabE(RAB11) localizes to exocytic post-Golgi carriers undergoing transport to the apex, and hypA encodes Trs120. RabE(RAB11), but not RabO(RAB1), immunoprecipitates contain Trs120/Trs130/Trs65, demonstrating specific association of TRAPPII with RabE(RAB11) in vivo. hypA1(ts) rapidly shifts RabE(RAB11), but not RabO(RAB1), to the cytosol, consistent with HypA(Trs120) being specifically required for RabE(RAB11) activation. Missense mutations rescuing hypA1(ts) at 42 °C mapped to rabE, affecting seven residues. Substitutions in six, of which four resulted in 7- to 36-fold accelerated GDP release, rescued lethality associated to TRAPPII deficiency, whereas equivalent substitutions in RabO(RAB1) did not, establishing that the essential role of TRAPPII is facilitating RabE(RAB11) nucleotide exchange. In vitro, TRAPPII purified with HypA(Trs120)-S-tag accelerates nucleotide exchange on RabE(RAB11) and, paradoxically, to a lesser yet substantial extent, on RabO(RAB1). Evidence obtained by exploiting hypA1-mediated destabilization of HypA(Trs120)/HypC(Trs130)/Trs65 assembly onto the TRAPPI core indicates that these subunits sculpt a second RAB binding site on TRAPP apparently independent from that for RabO(RAB1), which would explain TRAPPII in vitro activity on two RABs. Using A. nidulans in vivo microscopy, we show that HypA(Trs120) colocalizes with RabE(RAB11), arriving at late Golgi cisternae as they dissipate into exocytic carriers. Thus, TRAPPII marks, and possibly determines, the Golgi-to-post-Golgi transition.

The Aspergillus nidulans syntaxin PepA(Pep12) is regulated by two Sec1/Munc-18 proteins to mediate fusion events at early endosomes, late endosomes and vacuoles.

Syntaxins are target-SNAREs that crucially contribute to determine membrane compartment identity. Three syntaxins, Tlg2p, Pep12p and Vam3p, organize the yeast endovacuolar system. Remarkably, filamentous fungi lack the equivalent of the yeast vacuolar syntaxin Vam3p, making unclear how these organisms regulate vacuole fusion. We show that the nearly essential Aspergillus nidulans syntaxin PepA(Pep12) , present in all endocytic compartments between early endosomes and vacuoles, shares features of Vam3p and Pep12p, and is capable of forming compositional equivalents of all known yeast endovacuolar SNARE bundles including that formed by yeast Vam3p for vacuolar fusion. Our data further indicate that regulation by two Sec1/Munc-18 proteins, Vps45 in early endosomes and Vps33 in early and late endosomes/vacuoles contributes to the wide domain of PepA(Pep12) action. The syntaxin TlgB(Tlg2) localizing to the TGN appears to mediate retrograde traffic connecting post-Golgi (sorting) endosomes with the TGN. TlgB(Tlg2) is dispensable for growth but becomes essential if the early Golgi syntaxin SedV(Sed5) is compromised, showing that the Golgi can function with a single syntaxin, SedV(Sed5) . Remarkably, its pattern of associations with endosomal SNAREs is consistent with SedV(Sed5) playing roles in retrograde pathway(s) connecting endocytic compartments downstream of the post-Golgi endosome with the Golgi, besides more conventional intra-Golgi roles.

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.

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

The 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.

Cytoplasmic dynein and early endosome transport.

Microtubule-based distribution of organelles/vesicles is crucial for the function of many types of eukaryotic cells and the molecular motor cytoplasmic dynein is required for transporting a variety of cellular cargos toward the microtubule minus ends. Early endosomes represent a major cargo of dynein in filamentous fungi, and dynein regulators such as LIS1 and the dynactin complex are both required for early endosome movement. In fungal hyphae, kinesin-3 and dynein drive bi-directional movements of early endosomes. Dynein accumulates at microtubule plus ends; this accumulation depends on kinesin-1 and dynactin, and it is important for early endosome movements towards the microtubule minus ends. The physical interaction between dynein and early endosome requires the dynactin complex, and in particular, its p25 component. The FTS-Hook-FHIP (FHF) complex links dynein-dynactin to early endosomes, and within the FHF complex, Hook interacts with dynein-dynactin, and Hook-early endosome interaction depends on FHIP and FTS.

Aspergillus nidulans Ambient pH Signaling Does Not Require Endocytosis.

Aspergillus nidulans (Pal) ambient pH signaling takes place in cortical structures containing components of the ESCRT pathway, which are hijacked by the alkaline pH-activated, ubiquitin-modified version of the arrestin-like protein PalF and taken to the plasma membrane. There, ESCRTs scaffold the assembly of dedicated Pal proteins acting downstream. The molecular details of this pathway, which results in the two-step proteolytic processing of the transcription factor PacC, have received considerable attention due to the key role that it plays in fungal pathogenicity. While current evidence strongly indicates that the pH signaling role of ESCRT complexes is limited to plasma membrane-associated structures where PacC proteolysis would take place, the localization of the PalB protease, which almost certainly catalyzes the first and only pH-regulated proteolytic step, had not been investigated. In view of ESCRT participation, this formally leaves open the possibility that PalB activation requires endocytic internalization. As endocytosis is essential for hyphal growth, nonlethal endocytic mutations are predicted to cause an incomplete block. We used a SynA internalization assay to measure the extent to which any given mutation prevents endocytosis. We show that none of the tested mutations impairing endocytosis to different degrees, including slaB1, conditionally causing a complete block, have any effect on the activation of the pathway. We further show that PalB, like PalA and PalC, localizes to cortical structures in an alkaline pH-dependent manner. Therefore, signaling through the Pal pathway does not involve endocytosis.

A second component of the SltA-dependent cation tolerance pathway in Aspergillus nidulans.

The transcriptional response to alkali metal cation stress is mediated by the zinc finger transcription factor SltA in Aspergillus nidulans and probably in other fungi of the pezizomycotina subphylum. A second component of this pathway has been identified and characterized. SltB is a 1272 amino acid protein with at least two putative functional domains, a pseudo-kinase and a serine-endoprotease, involved in signaling to the transcription factor SltA. Absence of SltB activity results in nearly identical phenotypes to those observed for a null sltA mutant. Hypersensitivity to a variety of monovalent and divalent cations, and to medium alkalinization are among the phenotypes exhibited by a null sltB mutant. Calcium homeostasis is an exception and this cation improves growth of sltΔ mutants. Moreover, loss of kinase HalA in conjunction with loss-of-function sltA or sltB mutations leads to pronounced calcium auxotrophy. sltA sltB double null mutants display a cation stress sensitive phenotype indistinguishable from that of single slt mutants showing the close functional relationship between these two proteins. This functional relationship is reinforced by the fact that numerous mutations in both slt loci can be isolated as suppressors of poor colonial growth resulting from certain null vps (vacuolar protein sorting) mutations. In addition to allowing identification of sltB, our sltB missense mutations enabled prediction of functional regions in the SltB protein. Although the relationship between the Slt and Vps pathways remains enigmatic, absence of SltB, like that of SltA, leads to vacuolar hypertrophy. Importantly, the phenotypes of selected sltA and sltB mutations demonstrate that suppression of null vps mutations is not dependent on the inability to tolerate cation stress. Thus a specific role for both SltA and SltB in the VPS pathway seems likely. Finally, it is noteworthy that SltA and SltB have a similar, limited phylogenetic distribution, being restricted to the pezizomycotina subphylum. The relevance of the Slt regulatory pathway to cell structure, intracellular trafficking and cation homeostasis and its restricted phylogenetic distribution makes this pathway of general interest for future investigation and as a source of targets for antifungal drugs.

Acute inactivation of the Aspergillus nidulans Golgi membrane fusion machinery: correlation of apical extension arrest and tip swelling with cisternal disorganization.

The mechanisms governing traffic across the Golgi are incompletely understood. We studied, by live-cell microscopy, the consequences of disorganizing the Aspergillus nidulans Golgi, using an extended set of fluorescent protein markers to resolve early from late cisternae. The early Golgi syntaxin SedV(Sed) (5) and the RabO(Rab) (1) regulatory GTPase play essential roles in secretion, cooperating in the ER-Golgi interface. Following a temperature shift-up 'on-the-stage', hyphae carrying engineered sedV(R258G) and rabO(A136D) ts mutations arrest polarized growth. This arrest correlates with overall Golgi disorganization and characteristic hyphal tip swelling. Using v-SNARE SynA as reporter, we show that the sedV(R258G) phenotypes correlate with arrested secretion. Both the morphogenetic defect and the secretory deficit are reversible. Thus downregulation of secretion, like that of endocytosis, has morphogenetic consequences, implying that mechanisms tuning the secretory pathway might be involved in developmental processes. According to the cisternal maturation model, acute impairment of traffic in the ER-Golgi interface should lead to disorganization of both the early and the late Golgi cisternae. Thus, the relatively rapid late Golgi disorganization observed upon shifting ER-Golgi interface mutants to the restrictive temperature seems incompatible with an A. nidulans Golgi network organized on the basis of stable early and late compartments, supporting instead cisternal maturation.

An ordered pathway for the assembly of fungal ESCRT-containing ambient pH signalling complexes at the plasma membrane.

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 vps27Δ 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.

Aspergillus nidulans CkiA is an essential casein kinase I required for delivery of amino acid transporters to the plasma membrane.

Type I casein kinases are highly conserved among Eukaryotes. Of the two Aspergillus nidulans casein kinases I, CkiA is related to the δ/ε mammalian kinases and to Saccharomyces cerevisiae Hrr25p. CkiA is essential. Three recessive ckiA mutations leading to single residue substitutions, and downregulation using a repressible promoter, result in partial loss-of-function, which leads to a pleiotropic defect in amino acid utilization and resistance to toxic amino acid analogues. These phenotypes correlate with miss-routing of the YAT plasma membrane transporters AgtA (glutamate) and PrnB (proline) to the vacuole under conditions that, in the wild type, result in their delivery to the plasma membrane. Miss-routing to the vacuole and subsequent transporter degradation results in a major deficiency in the uptake of the corresponding amino acids that underlies the inability of the mutant strains to catabolize them. Our findings may have important implications for understanding how CkiA, Hrr25p and other fungal orthologues regulate the directionality of transport at the ER-Golgi interface.

Rescue of Aspergillus nidulans severely debilitating null mutations in ESCRT-0, I, II and III genes by inactivation of a salt-tolerance pathway allows examination of ESCRT gene roles in pH signalling.

The Aspergillus pal pathway hijacks ESCRT proteins into ambient pH signalling complexes. We show that components of ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III are nearly essential for growth, precluding assessment of null mutants for pH signalling or trafficking. This severely debilitating effect is rescued by loss-of-function mutations in two cation tolerance genes, one of which, sltA, encodes a transcription factor whose inactivation promotes hypervacuolation. Exploiting a conditional expression sltA allele, we demonstrate that deletion of vps27 (ESCRT-0), vps23 (ESCRT-I), vps36 (ESCRT-II), or vps20 or vps32 (both ESCRT-III) leads to numerous small vacuoles, a phenotype also suppressed by SltA downregulation. This situation contrasts with normal vacuoles and vacuole-associated class E compartments seen in Saccharomyces cerevisiae ESCRT null mutants. Exploiting the suppressor phenotype of sltA(-) mutations, we establish that Vps23, Vps36, Vps20 and Vps32 are essential for pH signalling. Phosphatidylinositol 3-phosphate-recognising protein Vps27 (ESCRT-0) is not, consistent with normal pH signalling in rabB null mutants unable to recruit Vps34 kinase to early endosomes. In contrast to the lack of pH signalling in the absence of Vps20 or Vps32, detectable signalling occurs in the absence of ESCRT-III subunit Vps24. Our data support a model in which certain ESCRT proteins are recruited to the plasma membrane to mediate pH signalling.

The Uso1 globular head interacts with SNAREs to maintain viability even in the absence of the coiled-coil domain.

Uso1/p115 and RAB1 tether ER-derived vesicles to the Golgi. Uso1/p115 contains a globular-head-domain (GHD), a coiled-coil (CC) mediating dimerization/tethering, and a C-terminal region (CTR) interacting with golgins. Uso1/p115 is recruited to vesicles by RAB1. Genetic studies placed Uso1 paradoxically acting upstream of, or in conjunction with RAB1 (Sapperstein et al., 1996). We selected two missense mutations in uso1 resulting in E6K and G540S in the GHD that rescued lethality of rab1-deficient Aspergillus nidulans. The mutations are phenotypically additive, their combination suppressing the complete absence of RAB1, which emphasizes the key physiological role of the GHD. In living hyphae Uso1 recurs on puncta (60 s half-life) colocalizing partially with the Golgi markers RAB1, Sed5, and GeaA/Gea1/Gea2, and totally with the retrograde cargo receptor Rer1, consistent with Uso1 dwelling in a very early Golgi compartment from which ER residents reaching the Golgi recycle back to the ER. Localization of Uso1, but not of Uso1E6K/G540S, to puncta is abolished by compromising RAB1 function, indicating that E6K/G540S creates interactions bypassing RAB1. That Uso1 delocalization correlates with a decrease in the number of Gea1 cisternae supports that Uso1-and-Rer1-containing puncta are where the protein exerts its physiological role. In S-tag-coprecipitation experiments, Uso1 is an associate of the Sed5/Bos1/Bet1/Sec22 SNARE complex zippering vesicles with the Golgi, with Uso1E6K/G540S showing a stronger association. Using purified proteins, we show that Bos1 and Bet1 bind the Uso1 GHD directly. However, Bet1 is a strong E6K/G540S-independent binder, whereas Bos1 is weaker but becomes as strong as Bet1 when the GHD carries E6K/G540S. G540S alone markedly increases GHD binding to Bos1, whereas E6K causes a weaker effect, correlating with their phenotypic contributions. AlphaFold2 predicts that G540S increases the binding of the GHD to the Bos1 Habc domain. In contrast, E6K lies in an N-terminal, potentially alpha-helical, region that sensitive genetic tests indicate as required for full Uso1 function. Remarkably, this region is at the end of the GHD basket opposite to the end predicted to interact with Bos1. We show that, unlike dimeric full-length and CTR∆ Uso1 proteins, the GHD lacking the CC/CTR dimerization domain, whether originating from bacteria or Aspergillus extracts and irrespective of whether it carries or not E6K/G540S, would appear to be monomeric. With the finding that overexpression of E6K/G540S and wild-type GHD complement uso1∆, our data indicate that the GHD monomer is capable of providing, at least partially, the essential Uso1 functions, and that long-range tethering activity is dispensable. Rather, these findings strongly suggest that the essential role of Uso1 involves the regulation of SNAREs.

Siderophore biosynthesis but not reductive iron assimilation is essential for Aspergillus fumigatus virulence.

The ability to acquire iron in vivo is essential for most microbial pathogens. Here we show that Aspergillus fumigatus does not have specific mechanisms for the utilization of host iron sources. However, it does have functional siderophore-assisted iron mobilization and reductive iron assimilation systems, both of which are induced upon iron deprivation. Abrogation of reductive iron assimilation, by inactivation of the high affinity iron permease (FtrA), has no effect on virulence in a murine model of invasive aspergillosis. In striking contrast, A. fumigatus L-ornithine-N5-monooxygenase (SidA), which catalyses the first committed step of hydroxamate-type siderophore biosynthesis, is absolutely essential for virulence. Thus, A. fumigatus SidA is an essential virulence attribute. Combined with the absence of a sidA ortholog-and the fungal siderophore system in general-in mammals, these data demonstrate that the siderophore biosynthetic pathway represents a promising new target for the development of antifungal therapies.

Characterization of Aspergillus nidulans DidB Did2, a non-essential component of the multivesicular body pathway.

ESCRT-III heteropolymers mediate membrane protein cargo sorting into multivesicular endosomes for subsequent vacuolar degradation. We studied the localization of largely uncharacterized Aspergillus nidulans ESCRT-III using its key structural component Vps32 and the 'associated' component DidB(Did2). Vps32-GFP localizes to motile early endosomes as reported, but predominates in aggregates often associated with vacuoles due to inability to dissociate from endosomes. DidB(Did)(2) regulating Vps4 (the ATPase disassembling ESCRT-III) is not essential. Consistent with this accessory role, didB Delta is unable to block the MVB sorting of the glutamate transporter AgtA, but increases its steady-state level and mislocalizes a fraction of the permease to the plasma membrane under conditions promoting its vacuolar targeting. didB Delta exacerbates the dominant-negative growth defect resulting from Vps32-GFP over-expression. A proportion of DidB-GFP is detectable in early endosomes colocalizing with RabA(Rab5) and accumulating in nudA1 tips, suggesting that ESCRT-III assembles on endosomes from the early steps of the endocytic pathway.

Analysis of a novel calcium auxotrophy in Aspergillus nidulans.

In Aspergillus nidulans a combination of null mutations in halA, encoding a protein kinase, and sltA, encoding a zinc-finger transcription factor having no yeast homologues, results in an elevated calcium requirement ('calcium auxotrophy') without impairing net calcium uptake. sltA(-) (+/-halA(-)) mutations result in hypertrophy of the vacuolar system. In halA(-)sltA(-) (and sltA(-)) strains, transcript levels for pmcA and pmcB, encoding vacuolar Ca(2+)-ATPase homologues, are highly elevated, suggesting a regulatory relationship between vacuolar membrane area and certain vacuolar membrane ATPase levels. Deletion of both pmcA and pmcB strongly suppresses the 'calcium auxotrophy'. Therefore the 'calcium auxotrophy' possibly results from excessive vacuolar calcium sequestration, causing cytosolic calcium deprivation. Null mutations in nhaA, homologous to Saccharomyces cerevisiae NHA1, encoding a plasma membrane Na(+)/H(+) antiporter effluxing Na(+) and K(+), and a non-null mutation in trkB, homologous to S. cerevisiae TRK1, encoding a plasma membrane high affinity K(+) transporter, also suppress the calcium auxotrophy.

Ambient pH gene regulation in fungi: making connections.

Many fungi grow over a wide pH range and their gene expression is tailored to the environmental pH. In Aspergillus nidulans, the transcription factor PacC, an activator of genes expressed in alkaline conditions and a repressor of those expressed in acidic conditions, undergoes two processing proteolyses, the first being pH-signal dependent and the second proteasomal. Signal transduction involves a 'go-between' connecting two complexes, one of which comprises two plasma membrane proteins and an arrestin and the other comprises PacC, a cysteine protease, a scaffold and endosomal components. The Saccharomyces cerevisiae PacC orthologue, Rim101p, differs in that it does not undergo the second round of proteolysis and it functions directly as a repressor only. PacC/Rim101-mediated pH regulation is crucial to fungal pathogenicity.

Distinct roles for intra- and extracellular siderophores during Aspergillus fumigatus infection.

Siderophore biosynthesis by the highly lethal mould Aspergillus fumigatus is essential for virulence, but non-existent in humans, presenting a rare opportunity to strategize therapeutically against this pathogen. We have previously demonstrated that A. fumigatus excretes fusarinine C and triacetylfusarinine C to capture extracellular iron, and uses ferricrocin for hyphal iron storage. Here, we delineate pathways of intra- and extracellular siderophore biosynthesis and show that A. fumigatus synthesizes a developmentally regulated fourth siderophore, termed hydroxyferricrocin, employed for conidial iron storage. By inactivation of the nonribosomal peptide synthetase SidC, we demonstrate that the intracellular siderophores are required for germ tube formation, asexual sporulation, resistance to oxidative stress, catalase A activity, and virulence. Restoration of the conidial hydroxyferricrocin content partially rescues the virulence of the apathogenic siderophore null mutant Delta sidA, demonstrating an important role for the conidial siderophore during initiation of infection. Abrogation of extracellular siderophore biosynthesis following inactivation of the acyl transferase SidF or the nonribosomal peptide synthetase SidD leads to complete dependence upon reductive iron assimilation for growth under iron-limiting conditions, partial sensitivity to oxidative stress, and significantly reduced virulence, despite normal germ tube formation. Our findings reveal distinct cellular and disease-related roles for intra- and extracellular siderophores during mammalian Aspergillus infection.

Two zinc finger transcription factors, CrzA and SltA, are involved in cation homoeostasis and detoxification in Aspergillus nidulans.

To investigate cation adaptation and homoeostasis in Aspergillus nidulans, two transcription-factor-encoding genes have been characterized. The A. nidulans orthologue crzA of the Saccharomyces cerevisiae CRZ1 gene, encoding a transcription factor mediating gene regulation by Ca(2+), has been identified and deleted. The crzA deletion phenotype includes extreme sensitivity to alkaline pH, Ca(2+) toxicity and aberrant morphology connected with alterations of cell-wall-related phenotypes such as reduced expression of a chitin synthase gene, chsB. A fully functional C-terminally GFP (green fluorescent protein)-tagged form of the CrzA protein is apparently excluded from nuclei in the absence of added Ca(2+), but rapidly accumulates in nuclei upon exposure to Ca(2+). In addition, the previously identified sltA gene, which has no identifiable homologues in yeasts, was deleted, and the resulting phenotype includes considerably enhanced toxicity by a number of cations other than Ca(2+) and also by alkaline pH. Reduced expression of a homologue of the S. cerevisiae P-type ATPase Na(+) pump gene ENA1 might partly explain the cation sensitivity of sltA-null strains. Up-regulation of the homologue of the S. cerevisiae vacuolar Ca(2+)/H(+) exchanger gene VCX1 might explain the lack of Ca(2+) toxicity to null-sltA mutants, whereas down-regulation of this gene might be responsible for Ca(2+) toxicity to crzA-null mutants. Both crzA and sltA encode DNA-binding proteins, and the latter exerts both positive and negative gene regulation.