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.

Cell wall dynamics stabilize tip growth in a filamentous fungus.

Hyphal tip growth allows filamentous fungi to colonize space, reproduce, or infect. It features remarkable morphogenetic plasticity including unusually fast elongation rates, tip turning, branching, or bulging. These shape changes are all driven from the expansion of a protective cell wall (CW) secreted from apical pools of exocytic vesicles. How CW secretion, remodeling, and deformation are modulated in concert to support rapid tip growth and morphogenesis while ensuring surface integrity remains poorly understood. We implemented subresolution imaging to map the dynamics of CW thickness and secretory vesicles in Aspergillus nidulans. We found that tip growth is associated with balanced rates of CW secretion and expansion, which limit temporal fluctuations in CW thickness, elongation speed, and vesicle amount, to less than 10% to 20%. Affecting this balance through modulations of growth or trafficking yield to near-immediate changes in CW thickness, mechanics, and shape. We developed a model with mechanical feedback that accounts for steady states of hyphal growth as well as rapid adaptation of CW mechanics and vesicle recruitment to different perturbations. These data provide unprecedented details on how CW dynamics emerges from material secretion and expansion, to stabilize fungal tip growth as well as promote its morphogenetic plasticity.

Live-Cell Imaging of Dynein-Mediated Cargo Transport in Aspergillus nidulans.

Filamentous fungi have been used for studying long-distance transport of cargoes driven by cytoplasmic dynein. Aspergillus nidulans is a well-established genetic model organism used for studying dynein function and regulation in vivo. Here, we describe how we grow A. nidulans strains for live-cell imaging and how we observe the dynein-mediated distribution of early endosomes and secretory vesicles. Using an on-stage incubator and culture chambers for inverted microscopes, we can image fungal hyphae that naturally attach to the bottom of the chambers, using wide-field epifluorescence microscopes or the new Zeiss LSM 980 (with Airyscan 2) microscope. In addition to methods for preparing cells for imaging, a procedure for A. nidulans transformation is also described.

The type V myosin-containing complex HUM is a RAB11 effector powering movement of secretory vesicles.

In the apex-directed RAB11 exocytic pathway of Aspergillus nidulans, kinesin-1/KinA conveys secretory vesicles (SVs) to the hyphal tip, where they are transferred to the type V myosin MyoE. MyoE concentrates SVs at an apical store located underneath the PM resembling the presynaptic active zone. A rod-shaped RAB11 effector, UDS1, and the intrinsically disordered and coiled-coil HMSV associate with MyoE in a stable HUM (HMSV-UDS1-MyoE) complex recruited by RAB11 to SVs through an interaction network involving RAB11 and HUM components, with the MyoE globular tail domain (GTD) binding both HMSV and RAB11-GTP and RAB11-GTP binding both the MyoE-GTD and UDS1. UDS1 bridges RAB11-GTP to HMSV, an avid interactor of the MyoE-GTD. The interaction between the UDS1-HMSV sub-complex and RAB11-GTP can be reconstituted in vitro. Ablating UDS1 or HMSV impairs actomyosin-mediated transport of SVs to the apex, resulting in spreading of RAB11 SVs across the apical dome as KinA/microtubule-dependent transport gains prominence.

Comment on Dimou et al. Profile of Membrane Cargo Trafficking Proteins and Transporters Expressed under N Source Derepressing Conditions in Aspergillus nidulans. J. Fungi 2021, 7, 560.

Contrary to the opinion recently offered by Dimou et al., our previously published biochemical, subcellular and genetic data supported our contention that AN11127 corresponds to the A. nidulans gene encoding Sec12, which is the guanine nucleotide exchange factor (GEF) specific for SAR1. We add here additional bioinformatics evidence that fully disprove the otherwise negative evidence reported by Dimou et al., highlighting the dangers associated with the lax interpretation of genomic data. On the positive side, we establish guidelines for the identification of this key secretory gene in other species of Ascomycota and Basidiomycota, including species of medical and applied interest.

The fungal RABOME: RAB GTPases acting in the endocytic and exocytic pathways of Aspergillus nidulans (with excursions to other filamentous fungi).

RAB GTPases are major determinants of membrane identity that have been exploited as highly specific reporters to study intracellular traffic in vivo. A score of fungal papers have considered individual RABs, but systematic, integrated studies on the localization and physiological role of these regulators and their effectors have been performed only with Aspergillus nidulans. These studies have influenced the intracellular trafficking field beyond fungal specialists, leading to findings such as the maturation of trans-Golgi (TGN) cisternae into post-Golgi RAB11 secretory vesicles, the concept that these RAB11 secretory carriers are loaded with three molecular nanomotors, the understanding of the role of endocytic recycling mediated by RAB6 and RAB11 in determining the hyphal mode of life, the discovery that early endosome maturation and the ESCRT pathway are essential, the identification of specific adaptors of dynein-dynactin to RAB5 endosomes, the exquisite dependence that autophagy displays on RAB1 activity, the role of TRAPPII as a GEF for RAB11, or the conclusion that the RAB1-to-RAB11 transition is not mediated by TRAPP maturation. A remarkable finding was that the A. nidulans Spitzenkörper contains four RABs: RAB11, Sec4, RAB6, and RAB1. How these RABs cooperate during exocytosis represents an as yet outstanding question.

Tracking exocytosis of a GPI-anchored protein in Aspergillus nidulans.

Secretion of the glycosylphosphatidylinositol-anchored protein (GPI-AP) EglC was investigated in the filamentous fungus Aspergillus nidulans, exploiting a sucrose-inducible promoter to conditionally express the protein in cells blocked at different steps of exocytosis. EglC is delivered to the cell surface in a polarized fashion, but appears to redistribute rapidly toward apico-distal regions. Inactivation of SarASar1 mediating COPII vesicle biogenesis resulted in the accumulation of EglC in the endoplasmic reticulum (ER) but, rather than concentrating in ER-exit-sites, the reporter labeled the ER uniformly. Abnormal posttranslational modifications of EglC were detected in sarAts and sed5ts mutants, suggesting that blocking COPII biogenesis or traffic in the ER/Golgi interface might affect GPI remodeling. EglC delivery to the plasma membrane requires, besides Golgi function, the TRAPPII complex mediating the biogenesis of RAB11 secretory vesicles at the TGN, but is unaffected by the absence of RAB5, the key regulator of early endosome biogenesis/maturation. Thus, unlike the soluble extracellular enzyme inulinase, EglC is directly delivered from the TGN to the plasma membrane without involvement of endosomes. We conclude that in A. nidulans, GPI-APs follow a direct secretory pathway from the ER to the plasma membrane.

En bloc TGN recruitment of Aspergillus TRAPPII reveals TRAPP maturation as unlikely to drive RAB1-to-RAB11 transition.

Transport protein particle (TRAPP) complexes regulate membrane traffic. TRAPPII and TRAPPIII share a core hetero-heptamer, also denoted TRAPPI. In fungi TRAPPIII and TRAPPII mediate GDP exchange on RAB1 and RAB11, respectively, regulating traffic across the Golgi, with TRAPPIII also activating RAB1 in autophagosomes. Our finding that Aspergillus nidulans TRAPPII can be assembled by addition of a TRAPPII-specific subcomplex onto core TRAPP prompted us to investigate the possibility that TRAPPI and/or TRAPPIII already residing in the Golgi matures into TRAPPII to determine a RAB1-to-RAB11 conversion as Golgi cisternae progress from early Golgi to TGN identity. By time-resolved microscopy, we determine that the TRAPPII reporter Trs120 (the homolog of metazoan TRAPPC9) is recruited to existing trans-Golgi network (TGN) cisternae slightly before RAB11 arrives, and resides for ∼45 s on them before cisternae tear off into RAB11 secretory carriers. Notably, the core TRAPP reporter Bet3 (the homolog of metazoan TRAPPC3) was not detectable in early Golgi cisternae, being instead recruited to TGN cisternae simultaneously with Trs120, indicating en bloc recruitment of TRAPPII to the Golgi and arguing strongly against the TRAPP maturation model.

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.

Identification of the guanine nucleotide exchange factor for SAR1 in the filamentous fungal model Aspergillus nidulans.

In spite of its basic and applied interest, the regulation of ER exit by filamentous fungi is insufficiently understood. In previous work we isolated a panel of conditional mutations in sarA encoding the master GTPase SarASAR1 in A. nidulans and demonstrated its key role in exocytosis and hyphal morphogenesis. However, the SAR1 guanine nucleotide exchange factor (GEF), Sec12, has not been characterized in any filamentous fungus, largely due to the fact that SEC12 homologues share little amino acid sequence identity beyond a GGGGxxxxGϕxN motif involved in guanine nucleotide exchange. Here we demonstrate that AN11127 encodes A. nidulans Sec12, which is an essential protein that localizes to the ER and that, when overexpressed, rescues the growth defect resulting from a hypomorphic sarA6ts mutation at 37 °C. Using purified, bacterially expressed proteins we demonstrate that the product of AN11127 accelerates nucleotide exchange on SarASAR1, but not on its closely related GTPase ArfAARF1, as expected for a bona fide GEF. The unequivocal characterization of A. nidulans Sec12 paves the way for the tailored modification of ER exit in a model organism that is closely related to industrial species of filamentous fungi.

COPI localizes to the early Golgi in Aspergillus nidulans.

Coatomer-I (COPI) is a heteromeric protein coat that facilitates the budding of membranous carriers mediating Golgi-to-ER and intra-Golgi transport. While the structural features of COPI have been thoroughly investigated, its physiological role is insufficiently understood. Here we exploit the amenability of A. nidulans for studying intracellular traffic, taking up previous studies by Breakspear et al. (2007) with the α-COP/CopA subunit of COPI. Endogenously tagged α-COP/CopA largely localizes to SedVSed5 syntaxin-containing early Golgi cisterna, and acute inactivation of ER-to-Golgi traffic delocalizes COPI to a haze, consistent with the cisternal maturation model. In contrast, the Golgi localization of COPI is independent of the TGN regulators HypBSec7 and HypATrs120, implying that COPI budding predominates at the SedVSed5 early Golgi, with lesser contribution of the TGN. This finding agrees with the proposed role of COPI-mediated intra-Golgi retrograde traffic in driving cisternal maturation, which predicts that the capacity of the TGN to generate COPI carriers is low. The COPI early Golgi compartments intimately associates with Sec13-containing ER exit sites. Characterization of the heat-sensitive copA1ts (sodVIC1) mutation showed that it results in a single residue substitution in the ε-COP-binding Carboxyl-Terminal-Domain of α-COP that likely destabilizes its folding. However, we show that Golgi disorganization by copA1ts necessitates >150 min-long incubation at 42 °C. This weak subcellular phenotype makes it unsuitable for inactivating COPI traffic acutely for microscopy studies, and explains the aneuploidy-stabilizing role of the mutation at subrestrictive temperatures.

Genetic dissection of the secretory route followed by a fungal extracellular glycosyl hydrolase.

Hyphal tip cells of Aspergillus nidulans are > 100 µm-long, which challenges intracellular traffic. In spite of the basic and applied interest of the secretory pathway of filamentous fungi, only recently has it been investigated in detail. We used InuA, an inducible and highly glycosylated inulinase, and mutations affecting different intracellular membranous compartments, to investigate the route by which the enzyme traffics to the extracellular medium. InuA is core-N-glycosylated in the ER and hyperglycosylated during transit across the Golgi. Hyperglycosylation was prevented by ts mutations in sarASAR1 impeding ER exit, and in sedVSED5 and rabORAB1 dissipating the early Golgi, but not by mutations in the TGN regulators hypATRS120 and hypBSEC7 , implicating the early Golgi in cargo glycosylation. podB1ts (cog2ts ) affecting the COG complex also prevents glycosylation, without disassembling early Golgi cisternae. That InuA exocytosis is prevented by inactivation of any of the above genes shows that it follows a conventional secretory pathway. However, ablation of RabBRAB5 regulating early endosomes (EEs), but not of RabSRAB7 , its equivalent in late endosomes, also prevents InuA accumulation in the medium, indicating that EEs are specifically required for InuA exocytosis. This work provides a framework to understand the secretion of enzyme cargoes by industrial filamentous fungi.

Molecular basis of resistance to the microtubule-depolymerizing antitumor compound plocabulin.

Plocabulin (PM060184) is a microtubule depolymerizing agent with potent antiproliferative activity undergoing phase II clinical trials for the treatment of solid tumors. Plocabulin shows antifungal activity virtually abolishing growth of the filamentous fungus Aspergillus nidulans. A. nidulans hyphae depend both on mitotic and interphase microtubules, as human cells. Here, we exploited the A. nidulans genetic amenability to gain insight into the mechanism of action of plocabulin. By combining mutations in the two A. nidulans ß-tubulin isotypes we obtained a plocabulin-insensitive strain, showing that ß-tubulin is the only molecular target of plocabulin in fungal cells. From a genetic screen, we recovered five mutants that show plocabulin resistance but do not carry mutations in ß-tubulin. Resistance mutations resulted in amino acid substitutions in (1) two subunits of the eukaryotic translation initiation factor eIF2B activating the General Amino Acid Control, (2) TIM44, an essential component of the inner mitochondrial membrane translocase, (3) two transcription factors of the binuclear zinc cluster family potentially interfering with the uptake or efflux of plocabulin. Given the conservation of some of the identified proteins and their respective cellular functions in the tumor environment, our results pinpoint candidates to be tested as potential biomarkers for determination of drug efficiency.

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.

Probing the effect of tip pressure on fungal growth: Application to Aspergillus nidulans.

The study of fungal cells is of great interest due to their importance as pathogens and as fermenting fungi and for their appropriateness as model organisms. The differential pressure between the hyphal cytoplasm and the bordering medium is essential for the growth process, because the pressure is correlated with the growth rate. Notably, during the invasion of tissues, the external pressure at the tip of the hypha may be different from the pressure in the surrounding medium. We report the use of a method, based on the micropipette-aspiration technique, to study the influence of this external pressure at the hyphal tip. Moreover, this technique makes it possible to study hyphal growth mechanics in the case of very thin hyphae, not accessible to turgor pressure probes. We found a correlation between the local pressure at the tip and the growth rate for the species Arpergillus nidulans. Importantly, the proposed method allows one to measure the pressure at the tip required to arrest the hyphal growth. Determining that pressure could be useful to develop new medical treatments for fungal infections. Finally, we provide a mechanical model for these experiments, taking into account the cytoplasm flow and the wall deformation.

Aspergillus nidulans BapH is a RAB11 effector that connects membranes in the Spitzenkörper with basal autophagy.

Using affinity chromatography we identified the Aspergillus nidulans F-BAR-and-PH domain-containing protein BapH as a RabERAB11 effector. BapH localizes to the Spitzenkörper (SPK) in an F-actin- and Sec7-dependent manner, becoming cytosolic after inactivation of Trs120 in TRAPPII, the oligomeric GEF for RabERAB11 . Therefore, RabERAB11 contributes to the recruitment of BapH to secretory vesicles in vivo. BapH has a close homologue, SlmA, which is related to yeast Slm1p/Slm2p, localizes to eisosomes and does not bind RabERAB11 . bapHΔ, slmAΔ and double bapHΔ slmAΔ mutations do not affect growth, although slmAΔ results in myriocin hypersensitivity. Both the PH and the F-BAR domain in BapH are necessary to recruit the protein to membranes, whereas its C-terminal moiety negatively regulates localization to the SPK. Strong overexpression of full-length BapH or of BapH lacking the C-terminal moiety impairs growth. The tandemly duplicated PHBapH domain is recruited to the plasma membrane in a manner dependent on critical Lys residues in its 'noncanonical' lipid binding pocket, suggesting that it binds to biological membranes containing PtdIns(4,5)P2 . Ablation of BapH, or deletion of the PH or BAR domains critical for the SPK localization increases autophagy under nitrogen-replete conditions. Therefore, BapH localizing to SPK vesicles influences basal levels of autophagy.

Cell Biology of Hyphal Growth.

Filamentous fungi are a large and ancient clade of microorganisms that occupy a broad range of ecological niches. The success of filamentous fungi is largely due to their elongate hypha, a chain of cells, separated from each other by septa. Hyphae grow by polarized exocytosis at the apex, which allows the fungus to overcome long distances and invade many substrates, including soils and host tissues. Hyphal tip growth is initiated by establishment of a growth site and the subsequent maintenance of the growth axis, with transport of growth supplies, including membranes and proteins, delivered by motors along the cytoskeleton to the hyphal apex. Among the enzymes delivered are cell wall synthases that are exocytosed for local synthesis of the extracellular cell wall. Exocytosis is opposed by endocytic uptake of soluble and membrane-bound material into the cell. The first intracellular compartment in the endocytic pathway is the early endosomes, which emerge to perform essential additional functions as spatial organizers of the hyphal cell. Individual compartments within septated hyphae can communicate with each other via septal pores, which allow passage of cytoplasm or organelles to help differentiation within the mycelium. This article introduces the reader to more detailed aspects of hyphal growth in fungi.

Genetic studies on the physiological role of CORVET in Aspergillus nidulans.

CORVET and HOPS are protein complexes mediating the maturation of early endosomes (EEs) into late endosomes (LEs)/vacuoles. These hetero-hexamers share four 'core' components, Vps11, Vps16, Vps18 and Vps33, and differ in two specific subunits, CORVET Vps8 and Vps3 and HOPS Vps39 and Vps41. Whereas ablating HOPS-specific components has minor growth effects, ablating any CORVET constituent severely debilitates Aspergillus nidulans growth, buttressing previous work indicating that maturation of EEs into LEs is physiologically crucial. A genetic screen revealed that impairing the slt cation homeostasis pathway rescues the growth defect resulting from inactivation of the 'core' protein Vps33. Subsequent genetic analyses showed that the defect resulting from lack of any one of the five other CORVET components could similarly be rescued by sltAΔ eliminating the slt regulator SltA. Whereas double deletants lacking functionally non-equivalent components of the CORVET and HOPS complexes are rescued by sltAΔ, those lacking functionally equivalent components are not, suggesting that intermediate 'hybrid' complexes previously detected in yeast are physiologically relevant. vps3Δ, vps8Δ, vps39Δ and vps41Δ result in small vacuoles. This phenotype is remediable by sltAΔ in the case of CORVET-specific, but not in the case of HOPS-specific deletants, indicating that the slt- effect on vacuolar size necessitates HOPS.

Transport of fungal RAB11 secretory vesicles involves myosin-5, dynein/dynactin/p25, and kinesin-1 and is independent of kinesin-3.

Hyphal tip cells of the fungus Aspergillus nidulans are useful for studying long-range intracellular traffic. Post-Golgi secretory vesicles (SVs) containing the RAB11 orthologue RabE engage myosin-5 as well as plus end- and minus end-directed microtubule motors, providing an experimental system with which to investigate the interplay between microtubule and actin motors acting on the same cargo. By exploiting the fact that depolymerization of F-actin unleashes SVs focused at the apex by myosin-5 to microtubule-dependent motors, we establish that the minus end-directed transport of SVs requires the dynein/dynactin supercomplex. This minus end-directed transport is largely unaffected by genetic ablation of the Hook complex adapting early endosomes (EEs) to dynein but absolutely requires p25 in dynactin. Thus dynein recruitment to two different membranous cargoes, namely EEs and SVs, requires p25, highlighting the importance of the dynactin pointed-end complex to scaffold cargoes. Finally, by studying the behavior of SVs and EEs in null and rigor mutants of kinesin-3 and kinesin-1 (UncA and KinA, respectively), we demonstrate that KinA is the major kinesin mediating the anterograde transport of SVs. Therefore SVs arrive at the apex of A. nidulans by anterograde transport involving cooperation of kinesin-1 with myosin-5 and can move away from the apex powered by dynein.

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.