-
[show abstract]
[hide abstract]
ABSTRACT: We exploit the ease with which highly motile early endosomes are distinguished from static late endosomes in order to study Aspergillus nidulans endosomal traffic. RabS(Rab7) mediates homotypic fusion of late endosomes/vacuoles in a homotypic fusion- and vacuole protein sorting/Vps41-dependent manner. Progression across the endocytic pathway involves endosomal maturation because the end products of the pathway in the absence of RabS(Rab7) are minivacuoles that are competent in multivesicular body sorting and cargo degradation but retain early endosomal features, such as the ability to undergo long-distance movement and propensity to accumulate in the tip region if dynein function is impaired. Without RabS(Rab7), early endosomal Rab5s-RabA and RabB-reach minivacuoles, in agreement with the view that Rab7 homologues facilitate the release of Rab5 homologues from endosomes. RabS(Rab7) is recruited to membranes already at the stage of late endosomes still lacking vacuolar morphology, but the transition between early and late endosomes is sharp, as only in a minor proportion of examples are RabA/RabB and RabS(Rab7) detectable in the same-frequently the less motile-structures. This early-to-late endosome/vacuole transition is coupled to dynein-dependent movement away from the tip, resembling the periphery-to-center traffic of endosomes accompanying mammalian cell endosomal maturation. Genetic studies establish that endosomal maturation is essential, whereas homotypic vacuolar fusion is not.
Molecular biology of the cell 03/2012; 23(10):1889-901. · 5.98 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Journal of Cell Science 02/2012; 125(Pt 7):1784-95. · 6.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The genetically tractable filamentous ascomycete fungus
Aspergillus nidulans has been successfully exploited to gain
major insight into the eukaryotic cell cycle. More recently, its
amenability to in vivo multidimensional microscopy has fueled
a potentially gilded second age of A. nidulans cell biology
studies. This review specifically deals with studies on intracellular
membrane traffic in A. nidulans. The cellular logistics
are subordinated to the needs imposed by the polarized mode
of growth of the multinucleated hyphal tip cells, whereas
membrane traffic is adapted to the large intracellular distances.
Recent work illustrates the usefulness of this fungus for
morphological and biochemical studies on endosome and
Golgi maturation, and on the role of microtubule-dependent
motors in the long-distance movement of endosomes. The
fungus is ideally suited for genetic studies on the secretory
pathway, as mutations impairing secretion reduce apical
extension rates, resulting in phenotypes detectable by visual
inspection of colonies.
Cellular Logistics. 01/2012;
-
[show abstract]
[hide abstract]
ABSTRACT: The genetically tractable filamentous ascomycete fungus Aspergillus nidulans has been successfully exploited to gain major insight into the eukaryotic cell cycle. More recently, its amenability to in vivo multidimensional microscopy has fueled a potentially gilded second age of A. nidulans cell biology studies. This review specifically deals with studies on intracellular membrane traffic in A. nidulans. The cellular logistics are subordinated to the needs imposed by the polarized mode of growth of the multinucleated hyphal tip cells, whereas membrane traffic is adapted to the large intracellular distances. Recent work illustrates the usefulness of this fungus for morphological and biochemical studies on endosome and Golgi maturation, and on the role of microtubule-dependent motors in the long-distance movement of endosomes. The fungus is ideally suited for genetic studies on the secretory pathway, as mutations impairing secretion reduce apical extension rates, resulting in phenotypes detectable by visual inspection of colonies.
Cellular logistics. 01/2012; 2(1):2-14.
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Journal of Cell Science 12/2011; 124(Pt 23):4064-76. · 6.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Aspergillus nidulans early endosomes display characteristic long-distance bidirectional motility. Simultaneous dual-channel acquisition showed that the two Rab5 paralogues RabB and RabA colocalize in these early endosomes and also in larger, immotile mature endosomes. However, RabB-GTP is the sole recruiter to endosomes of Vps34 PI3K (phosphatidylinositol-3-kinase) and the phosphatidylinositol-3-phosphate [PI(3)P] effector AnVps19 and rabB Delta, leading to thermosensitivity prevents multivesicular body sorting of endocytic cargo. Thus, RabB is the sole mediator of degradative endosomal identity. Importantly, rabB Delta, unlike rabA Delta, prevents early endosome movement. As affinity experiments and pulldowns showed that RabB-GTP recruits AnVps45, RabB coordinates PI(3)P-dependent endosome-to-vacuole traffic with incoming traffic from the Golgi and with long-distance endosomal motility. However, the finding that Anvps45 Delta, unlike rabB Delta, severely impairs growth indicates that AnVps45 plays RabB-independent functions. Affinity chromatography showed that the CORVET complex is a RabB and, to a lesser extent, a RabA effector, in agreement with GST pulldown assays of AnVps8. rabB Delta leads to smaller vacuoles, suggesting that it impairs homotypic vacuolar fusion, which would agree with the sequential maturation of endosomal CORVET into HOPS proposed for Saccharomyces cerevisiae. rabB Delta and rabA Delta mutations are synthetically lethal, demonstrating that Rab5-mediated establishment of endosomal identity is essential for A. nidulans.
Molecular biology of the cell 08/2010; 21(15):2756-69. · 5.98 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The seven-transmembrane receptor PalH and its coupled, positive-acting arrestin-like protein PalF are key components of a molecular sensor that in Aspergillus nidulans and other ascomycete fungi mediates activation of an intracellular signaling cascade by alkaline ambient pH. PalF is ubiquitinated in an alkaline pH- and PalH-dependent manner. We show here that PalF assists the plasma membrane localization of PalH and that PalF overexpression slightly hypersensitizes the pathway to alkaline pH but does not bypass the need for the ambient pH signal receptor in signaling. In contrast, covalent attachment of Ub to PalF activates the signaling pathway under acidic pH conditions in which the pathway is normally inactive, demonstrating a positive role for ubiquitination. We further show that PalF acts upstream of, or in concert with, the Bro1 domain-containing pH signaling protein PalC, which is normally recruited to cortical structures likely to represent active pH signaling foci under neutral/alkaline pH conditions. In agreement with its pathway-activating consequences, expression of PalF-Ub also promotes PalC cortical recruitment under acidic conditions. Notably, our data establish that expression of PalF-Ub, at approximately physiological levels, in a null palH background leads to a considerable degree of signaling even in the complete absence of the receptor. Thus PalF ubiquitination is a key, perhaps the sole, molecular trigger required for transmitting the alkaline pH signal to the downstream elements of the pathway.
Journal of Biological Chemistry 04/2010; 285(23):18095-102. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Fungal Genetics and Biology 03/2010; 47(7):636-46. · 3.74 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Journal of Biological Chemistry 01/2009; 284(7):4404-12. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In fungal hyphal cells, intracellular membrane trafficking is constrained by the relatively long intracellular distances and the mode of growth, exclusively by apical extension. Endocytosis plays a key role in hyphal tip growth, which involves the coupling of secretory membrane delivery to the apical region with subapical compensatory endocytosis. However, the identity, dynamics and function of filamentous fungal endosomal compartments remain largely unexplored. Aspergillus nidulans RabA(Rab5) localizes to a population of endosomes that show long range bidirectional movement on microtubule (MT) tracks and are labelled with FM4-64 shortly after dye internalization. RabA(Rab5) membranes do not overlap with largely static mature endosomes/vacuoles. Impaired delivery of dynein to the MT plus ends or downregulation of cytoplasmic dynein using the dynein heavy chain nudA1(ts)mutation results in accumulation of RabA(Rab5) endosomal membranes in an abnormal NudA1 compartment at the tip, strongly supporting the existence in A. nidulans hyphal tips of a dynein loading region. We show that the SynA synaptobrevin endocytic recycling cargo traffics through this region, which strongly supports the contention that polarized hyphal growth involves the association of endocytic recycling with the plus ends of MTs located at the tip, near the endocytic internalization collar.
Traffic 11/2008; 10(1):57-75. · 4.92 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: PalC, distantly related to Saccharomyces cerevisiae peripheral endosomal sorting complexes required for transport III (ESCRT-III) component Bro1p and one of six Aspergillus nidulans pH 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, Arg442Delta 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. cerevisiae orthologue of PalC has been identified, providing the basis for a unifying hypothesis of gene regulation by ambient pH in ascomycetes.
Traffic 11/2007; 8(10):1346-64. · 4.92 Impact Factor
-
Fungal Genetics and Biology 47(7):636-646. · 3.74 Impact Factor
