Clathrin adaptors mediate two sequential pathways of intra-Golgi recycling.

The pathways of membrane traffic within the Golgi apparatus are not fully known. This question was addressed using the yeast Saccharomyces cerevisiae, in which the maturation of individual Golgi cisternae can be visualized. We recently proposed that the AP-1 clathrin adaptor mediates intra-Golgi recycling late in the process of cisternal maturation. Here, we demonstrate that AP-1 cooperates with the Ent5 clathrin adaptor to recycle a set of Golgi transmembrane proteins, including some that were previously thought to pass through endosomes. This recycling can be detected by removing AP-1 and Ent5, thereby diverting the AP-1/Ent5-dependent Golgi proteins into an alternative recycling loop that involves traffic to the plasma membrane followed by endocytosis. Unexpectedly, various AP-1/Ent5-dependent Golgi proteins show either intermediate or late kinetics of residence in maturing cisternae. We infer that the AP-1/Ent5 pair mediates two sequential intra-Golgi recycling pathways that define two classes of Golgi proteins. This insight can explain the polarized distribution of transmembrane proteins in the Golgi.

A Kinetic View of Membrane Traffic Pathways Can Transcend the Classical View of Golgi Compartments.

A long-standing assumption is that the cisternae of the Golgi apparatus can be grouped into functionally distinct compartments, yet the molecular identities of those compartments have not been clearly described. The concept of a compartmentalized Golgi is challenged by the cisternal maturation model, which postulates that cisternae form de novo and then undergo progressive biochemical changes. Cisternal maturation can potentially be reconciled with Golgi compartmentation by defining compartments as discrete kinetic stages in the maturation process. These kinetic stages are distinguished by the traffic pathways that are operating. For example, a major transition occurs when a cisterna stops producing COPI vesicles and begins producing clathrin-coated vesicles. This transition separates one kinetic stage, the "early Golgi," from a subsequent kinetic stage, the "late Golgi" or "trans-Golgi network (TGN)." But multiple traffic pathways drive Golgi maturation, and the periods of operation for different traffic pathways can partially overlap, so there is no simple way to define a full set of Golgi compartments in terms of kinetic stages. Instead, we propose that the focus should be on the series of transitions experienced by a Golgi cisterna as various traffic pathways are switched on and off. These traffic pathways drive changes in resident transmembrane protein composition. Transitions in traffic pathways seem to be the fundamental, conserved determinants of Golgi organization. According to this view, the initial goal is to identify the relevant traffic pathways and place them on the kinetic map of Golgi maturation, and the ultimate goal is to elucidate the logic circuit that switches individual traffic pathways on and off as a cisterna matures.

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.

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.

The Golgi apparatus: insights from filamentous fungi.

Cargo passage through the Golgi, albeit an undoubtedly essential cellular function, is a mechanistically unresolved and much debated process. Although the main molecular players are conserved, diversification of the Golgi among different eukaryotic lineages is providing us with tools to resolve standing controversies. During the past decade the Golgi apparatus of model filamentous fungi, mainly Aspergillus nidulans, has been intensively studied. Here an overview of the most important findings in the field is provided. Golgi architecture and dynamics, as well as the novel cell biology tools that were developed in filamentous fungi in these studies, are addressed. An emphasis is placed on the central role the Golgi has as a crossroads in the endocytic and secretory-traffic pathways in hyphae. Finally the major advances that the A. nidulans Golgi biology has yielded so far regarding our understanding of key Golgi regulators, such as the Rab GTPases RabC(Rab6) and RabE(Rab11), the oligomeric transport protein particle, TRAPPII, and the Golgi guanine nucleotide exchange factors of Arf1, GeaA(GBF1/Gea1) and HypB(BIG/Sec7), are highlighted.

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.

Conditional inactivation of Aspergillus nidulans†sarA(SAR1) uncovers the morphogenetic potential of regulating endoplasmic reticulum (ER) exit.

In the genetic model Aspergillus nidulans, hyphal growth is exquisitely dependent on exocytic traffic. Following mutagenic PCR and gene replacement, we characterized thermosensitive mutations in sarA(SAR1) encoding a key regulator of endoplasmic reticulum (ER) exit. Six sarA(ts) alleles permitting relatively normal growth at 30°C prevented it at 42°C. This growth phenotype correlated with markedly reduced SarA levels at high temperature, suggesting that these alleles cause temperature-dependent SarA misfolding. sarA8 results in Ser substitution for conserved P-loop Gly27. sarA5 (Trp185Cys) and sarA6 (Ser186Pro) substitutions underscore the importance of the C-terminal α-helix on SarA(Sar1) function/stability. sarA6 markedly diminishing growth at 37°C was useful for microscopy experiments in which ER exit was impaired by shifting the incubation temperature. Early and late Golgi cisternae, labeled with the integral membrane syntaxins SedV(Sed5) and TlgB(Tlg2) , respectively, were rapidly dissipated by sarA6. However, whereas SedV(Sed5) was shifted toward the ER, TlgB(Tlg2) relocalized to a haze, underscoring the asymmetry of Golgi organization. This rapid Golgi dissipation that takes place after blocking anterograde COPII traffic is consistent with the cisternal maturation model. Incubation of sarA6 cells at 37°C led to the formation of apical balloons resembling specialized fungal structures. The formation of these balloons highlights the morphogenetic consequences of impairing ER exit.

GBF/Gea mutant with a single substitution sustains fungal growth in the absence of BIG/Sec7.

Golgi Arf1-guanine nucleotide exchange factors (GEFs) belong to two subfamilies: GBF/Gea and BIG/Sec7. Both are conserved across eukaryotes, but the physiological role of each is not well understood. Aspergillus nidulans has a single member of the early Golgi GBF/Gea-subfamily, geaA, and the late Golgi BIG/Sec7-subfamily, hypB. Both geaA and hypB are essential. hypB5 conditionally blocks secretion. We sought extragenic hypB5 suppressors and obtained geaA1. geaA1 results in Tyr1022Cys within a conserved GBF/Gea-specific S(Y/W/F)(L/I) motif in GeaA. This mutation alters GeaA localization. Remarkably, geaA1 suppresses hypBΔ, indicating that a single mutant Golgi Arf1-GEF suffices for growth.

Maturation of late Golgi cisternae into RabE(RAB11) exocytic post-Golgi carriers visualized in vivo.

The mechanism(s) by which proteins traverse and exit the Golgi are incompletely understood. Using Aspergillus nidulans hyphae, we show that late Golgi cisternae undergo changes in composition to gradually lose Golgi identity while acquiring post-Golgi RabE(RAB11) identity. This behavior of late Golgi cisternae is consistent with the cisternal maturation model. Post-Golgi RabE(RAB11) carriers travel to, and accumulate at, the apex, indicating that fusion is rate limiting for exocytosis. These carriers, which are loaded with kinesin, dynein, and MyoE(MYO5), move on a microtubule-based bidirectional conveyor belt relaying them to actin, which ultimately focuses exocytosis at the apex. Dynein drags RabE(RAB11) carriers away if engagement of MyoE(MYO5) to actin cables fails. Microtubules seemingly cooperating with F-actin capture can sustain secretion if MyoE(MYO5) is absent. Thus, filamentous fungal secretion involving post-Golgi carriers is remarkably similar, mechanistically, to the transport of melanosomes in melanocyte dendrites, even though melanosome biogenesis involves lysosomes rather than Golgi.

The Aspergillus nidulans peripheral ER: disorganization by ER stress and persistence during mitosis.

The genetically amenable fungus Aspergillus nidulans is well suited for cell biology studies involving the secretory pathway and its relationship with hyphal tip growth by apical extension. We exploited live-cell epifluorescence microscopy of the ER labeled with the translocon component Sec63, endogenously tagged with GFP, to study the organization of 'secretory' ER domains. The Sec63 A. nidulans ER network includes brightly fluorescent peripheral strands and more faintly labeled nuclear envelopes. In hyphae, the most abundant peripheral ER structures correspond to plasma membrane-associated strands that are polarized, but do not invade the hyphal tip dome, at least in part because the subapical collar of endocytic actin patches constrict the cortical strands in this region. Thus the subapical endocytic ring might provide an attachment for ER strands, thereby ensuring that the growing tip remains 'loaded' with secretory ER. Acute disruption of secretory ER function by reductive stress-mediated induction of the unfolded protein response results in the reversible aggregation of ER strands, cessation of exocytosis and swelling of the hyphal tips. The secretory ER is insensitive to brefeldin A treatment and does not undergo changes during mitosis, in agreement with the reports that apical extension continues at normal rates during this period.

New interfacial microtubule inhibitors of marine origin, PM050489/PM060184, with potent antitumor activity and a distinct mechanism.

We have investigated the target and mechanism of action of a new family of cytotoxic small molecules of marine origin. PM050489 and its dechlorinated analogue PM060184 inhibit the growth of relevant cancer cell lines at subnanomolar concentrations. We found that they are highly potent microtubule inhibitors that impair mitosis with a distinct molecular mechanism. They bind with nanomolar affinity to unassembled αß-tubulin dimers, and PM050489 binding is inhibited by known Vinca domain ligands. NMR TR-NOESY data indicated that a hydroxyl-containing analogue, PM060327, binds in an extended conformation, and STD results define its binding epitopes. Distinctly from vinblastine, these ligands only weakly induce tubulin self-association, in a manner more reminiscent of isohomohalichondrin B than of eribulin. PM050489, possibly acting like a hinge at the association interface between tubulin heterodimers, reshapes Mg(2+)-induced 42 S tubulin double rings into smaller 19 S single rings made of 7 ± 1 αß-tubulin dimers. PM060184-resistant mutants of Aspergillus nidulans map to ß-tubulin Asn100, suggesting a new binding site different from that of vinblastine at the associating ß-tubulin end. Inhibition of assembly dynamics by a few ligand molecules at the microtubule plus end would explain the antitumor activity of these compounds, of which PM060184 is undergoing clinical trials.

Live-cell imaging of Aspergillus nidulans autophagy: RAB1 dependence, Golgi independence and ER involvement.

We exploited the amenability of the fungus Aspergillus nidulans to genetics and live-cell microscopy to investigate autophagy. Upon nitrogen starvation, GFP-Atg8-containing pre-autophagosomal puncta give rise to cup-shaped phagophores and circular (0.9-µm diameter) autophagosomes that disappear in the vicinity of the vacuoles after their shape becomes irregular and their GFP-Atg8 fluorescence decays. This 'autophagosome cycle' gives rise to characteristic cone-shaped traces in kymographs. Autophagy does not require endosome maturation or ESCRTs, as autophagosomes fuse with vacuoles directly in a RabS (homolog of Saccharomyces cerevisiae Ypt7 and mammalian RAB7; written hereafter as RabS(RAB7))-HOPS-(homotypic fusion and vacuole protein sorting complex)-dependent manner. However, by removing RabS(RAB7) or Vps41 (a component of the HOPS complex), we show that autophagosomes may still fuse, albeit inefficiently, with the endovacuolar system in a process almost certainly mediated by RabA(RAB5)/RabB(RAB5) (yeast Vps21 homologs)-CORVET (class C core vacuole/endosome tethering complex), because acute inactivation of HbrA/Vps33, a key component of HOPS and CORVET, completely precludes access of GFP-Atg8 to vacuoles without affecting autophagosome biogenesis. Using a FYVE 2-GFP probe and endosomal PtdIns3P-depleted cells, we imaged PtdIns3P on autophagic membranes. PtdIns3P present on autophagosomes decays at late stages of the cycle, preceding fusion with the vacuole. Autophagy does not require Golgi traffic, but it is crucially dependent on RabO(RAB1). TRAPPIII-specific factor AN7311 (yeast Trs85) localizes to the phagophore assembly site (PAS) and RabO(RAB1) localizes to phagophores and autophagosomes. The Golgi and autophagy roles of RabO(RAB1) are dissociable by mutation: rabO(A136D) hyphae show relatively normal secretion at 28°C but are completely blocked in autophagy. This finding and the lack of Golgi traffic involvement pointed to the ER as one potential source of membranes for autophagy. In agreement, autophagosomes form in close association with ring-shaped omegasome-like ER structures resembling those described in mammalian cells.

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.

Searching for gold beyond mitosis: Mining intracellular membrane traffic in Aspergillus nidulans.

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.

Endosomal maturation by Rab conversion in Aspergillus nidulans is coupled to dynein-mediated basipetal movement.

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.

Mutational analysis and modeling reveal functionally critical residues in transmembrane segments 1 and 3 of the UapA transporter.

Earlier, we identified mutations in the first transmembrane segment (TMS1) of UapA, a uric acid-xanthine transporter in Aspergillus nidulans, that affect its turnover and subcellular localization. Here, we use one of these mutations (H86D) and a novel mutation (I74D) as well as genetic suppressors of them, to show that TMS1 is a key domain for proper folding, trafficking and turnover. Kinetic analysis of mutants further revealed that partial misfolding and deficient trafficking of UapA does not affect its affinity for xanthine transport, but reduces that of uric acid and confers a degree of promiscuity towards the binding of other purines. This result strengthens the idea that subtle interactions among domains not directly involved in substrate binding refine the selectivity of UapA. Characterization of second-site suppressors of H86D revealed a genetic interaction of TMS1 with TMS3, the latter segment shown for the first time to be important for UapA function. Systematic mutational analysis of polar and conserved residues in TMS3 showed that Ser154 is crucial for UapA transport activity. Our results are in agreement with a topological model of UapA built on the recently published structure of UraA, a bacterial homolog of UapA.

Characterization of Aspergillus nidulans RabC/Rab6.

The Aspergillus nidulans Golgi is not stacked. Early and late Golgi equivalents (GEs) are intermingled but can be resolved by epifluorescence microscopy. RabC, the Aspergillus ortholog of mammalian Rab6, is present across the Golgi, preferentially associated with early GEs near the tip and with late GEs in tip-distal regions. rabCΔ mutants, showing markedly impaired apical extension, have conspicuously fragmented, brefeldin A-insensitive early and late GEs, indicating that the Golgi network organization requires RabC. rabCΔ Golgi fragmentation is paralleled by an increase in early endosome abundance. rabCΔ reduces extracellular levels of the major secretable protease, suggesting that it impairs secretion. Notably, the Spitzenkörper, an apical intracellular structure in which secretory carriers accumulate awaiting fusion with the adjacent plasma membrane (PM), contains RabC. rabCΔ leads to abnormally increased accumulation of carriers, detectable with secretory v-SNARE GFP-SynA and FM4-64, in this structure. VpsT(Vps10) , present across the Golgi, recycles between endosomes and Golgi and is mislocalized to a cytosolic haze by rabCΔ that, in contrast, does not affect SynA recycling between endosomes and the PM, indicating that SynA follows a RabC-independent pathway. tlg2Δ mutants grow normally but are synthetically lethal with rabCΔ, indicating that RabC plays Tlg2-independent roles.

Aspergillus RabB Rab5 integrates acquisition of degradative identity with the long distance movement of early endosomes.

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.