Translocation of nutrient transporters to cell membrane via Golgi bypass in Aspergillus nidulans.
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ABSTRACT:
SUBMITTER: Dimou S
PROVIDER: S-EPMC7332984 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Nutrient transporters, being polytopic membrane proteins, are believed, but not formally shown, to traffic from their site of synthesis, the ER, to the plasma membrane through Golgi-dependent vesicular trafficking. Here, we develop a novel genetic system to investigate the trafficking of a neosynthesized model transporter, the well-studied UapA purine transporter of Aspergillus nidulans. We show that sorting of neosynthesized UapA to the plasma membrane (PM) bypasses the Golgi and does not necessitate key Rab GTPases, AP adaptors, microtubules or endosomes. UapA PM localization is found to be dependent on functional COPII vesicles, actin polymerization, clathrin heavy chain and the PM t-SNARE SsoA. Actin polymerization proved to primarily affect COPII vesicle formation, whereas the essential role of ClaH seems indirect and less clear. We provide evidence that other evolutionary and functionally distinct transporters of A. nidulans also follow the herein identified Golgi-independent trafficking route of UapA. Importantly, our findings suggest that specific membrane cargoes drive the formation of distinct COPII subpopulations that bypass the Golgi to be sorted non-polarly to the PM, serving house-keeping cell functions.
| S-SCDT-EMBOR-2019-49929-T | biostudies-other
Project description:To characterize the mechanisms involved in glucose transport, in the filamentous fungus Aspergillus nidulans, we have identified four glucose transporter encoding genes hxtB-E. We evaluated the ability of hxtB-E to functionally complement the Saccharomyces cerevisiae EBY.VW4000 strain that is unable to grow on glucose, fructose, mannose or galactose as single carbon source. In S. cerevisiae HxtB-E were targeted to the plasma membrane. The expression of HxtB, HxtC and HxtE was able to restore growth on glucose, fructose, mannose or galactose, indicating that these transporters accept multiple sugars as a substrate through an energy dependent process. A tenfold excess of unlabeled maltose, galactose, fructose, and mannose were able to inhibit glucose uptake to different levels (50 to 80 %) in these s. cerevisiae complemented strains. Moreover, experiments with cyanide-m-chlorophenylhydrazone (CCCP), strongly suggest that hxtB, -C, and -E mediate glucose transport via active proton symport. The A. nidulans ΔhxtB, ΔhxtC or ΔhxtE null mutants showed ~2.5-fold reduction in the affinity for glucose, while ΔhxtB and -C also showed a 2-fold reduction in the capacity for glucose uptake. The ΔhxtD mutant had a 7.8-fold reduction in affinity, but a 3-fold increase in the capacity for glucose uptake. However, only the ΔhxtB mutant strain showed a detectable decreased rate of glucose consumption at low concentrations and an increased resistance to 2-deoxyglucose.
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Project description: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-?m 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.
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Project description:After dephosphorylation by the phosphatase calcineurin, the fungal transcription factor CrzA enters the nucleus and activates the transcription of genes responsible for calcium homeostasis and many other calcium-regulated activities. A lack of CrzA confers calcium-sensitivity to the filamentous fungus Aspergillus nidulans. To further understand calcium signaling in filamentous fungi and to identify genes that interact genetically with CrzA, we selected for mutations that were able to suppress crzA? calcium intolerance and identified three genes. Through genetic mapping, gene sequencing, and mutant rescue, we were able to identify these as cnaB (encoding the calcineurin regulatory subunit), folA (encoding an enzyme involved in folic acid biosynthesis, dihydroneopterin aldolase), and scrC (suppression of crzA(-), encoding a hypothetical protein). By using a calcium indicator, Fluo-3, we were able to determine that the wild-type and the suppressor strains were either able to regulate intracellular calcium levels or were able to take up and or store calcium correctly. The increased expression of calcium transporters, pmcA and/or pmcB, in suppressor mutants possibly enabled tolerance to high levels of calcium. Our results suggest that a cnaB suppressor mutation confers calcium tolerance to crzA? strains through restoration of calcium homeostasis. These results stress that in A. nidulans there are calcineurin-dependent and CrzA-independent pathways. In addition, it is possible that CrzA is able to contribute to the modulation of folic acid biosynthesis.
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Project description:Solute and ion transporters are proteins essential for cell nutrition, detoxification, signaling, homeostasis and drug resistance. Being polytopic transmembrane proteins, they are co-translationally inserted and folded into the endoplasmic reticulum (ER) of eukaryotic cells and subsequently sorted to their final membrane destination via vesicular secretion. During their trafficking and in response to physiological/stress signals or prolonged activity, transporters undergo multiple quality control processes and regulated turnover. Consequently, transporters interact dynamically and transiently with multiple proteins. To further dissect the trafficking and turnover mechanisms underlying transporter subcellular biology, we herein describe a novel mass spectrometry-based proteomic protocol adapted to conditions allowing for maximal identification of proteins related to N source uptake in A. nidulans. Our analysis led to identification of 5690 proteins, which to our knowledge constitutes the largest protein dataset identified by omics-based approaches in Aspergilli. Importantly, we detected possibly all major proteins involved in basic cellular functions, giving particular emphasis to factors essential for membrane cargo trafficking and turnover. Our protocol is easily reproducible and highly efficient for unearthing the full A. nidulans proteome. The protein list delivered herein will form the basis for downstream systematic approaches and identification of protein-protein interactions in living fungal cells.
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Project description:Aspergillus nidulans hyphae grow exclusively by apical extension. Golgi equivalents (GEs) labeled with mRFP-tagged PH(OSBP) domain form a markedly polarized, dynamic network of ring-shaped and fenestrated cisternae that remains intact during "closed" mitosis. mRFP-PH(OSBP) GEs advance associated with the growing apex where secretion predominates but do not undergo long-distance movement toward the tip that could account for their polarization. mRFP-PH(OSBP) GEs overlap with the trans-Golgi resident Sec7 but do not colocalize with also polarized accretions of the early Golgi marker GrhA(Grh1)-GFP, indicating that early and late Golgi membranes segregate spatially. AnSec23-GFP ER exit sites (ERES) are numerous, relatively static foci localizing across the entire cell. However, their density is greatest near the tip, correlating with predominance of early and trans-Golgi elements in this region. Whereas GrhA-GFP structures and ERES reach the apical dome, mRFP-PH(OSBP) GEs are excluded from this region, which contains the endosome dynein loading zone. After latrunculin-mediated F-actin disruption, mRFP-PH(OSBP) GEs fragment and, like AnSec23-GFP ERES, depolarize. Brefeldin A transiently collapses late and early GEs into distinct aggregates containing Sec7/mRFP-PH(OSBP) and GrhA-GFP, respectively, temporarily arresting apical extension. Rapid growth reinitiates after washout, correlating with reacquisition of the normal Golgi organization that, we conclude, is required for apical extension.
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Project description:Type I casein kinases are highly conserved among Eukaryotes. Of the two Aspergillus nidulans casein kinases I, CkiA is related to the ?/? 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.
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Project description:The asexual spore is one of the most crucial factors contributing to the fecundity and fitness of filamentous fungi. Although the developmental activator FluG was shown to be necessary for activation of asexual sporulation (conidiation) and production of the carcinogenic mycotoxin sterigmatocystin (ST) in the model filamentous fungus Aspergillus nidulans, the molecular mechanisms underlying the developmental switch have remained elusive. In this study, we report that the FluG-mediated conidiation in A. nidulans occurs via derepression. Suppressor analyses of fluG led to the identification of the sfgA gene encoding a novel protein with the Gal4-type Zn(II)2Cys6 binuclear cluster DNA-binding motif at the N terminus. Deletion (delta) and 31 other loss-of-function sfgA mutations bypassed the need for fluG in conidiation and production of ST. Moreover, both delta sfgA and delta sfgA delta fluG mutations resulted in identical phenotypes in growth, conidiation, and ST production, indicating that the primary role of FluG is to remove repressive effects imposed by SfgA. In accordance with the proposed regulatory role of SfgA, overexpression of sfgA inhibited conidiation and delayed/reduced expression of conidiation- and ST-specific genes. Genetic analyses demonstrated that SfgA functions downstream of FluG but upstream of transcriptional activators (FlbD, FlbC, FlbB, and BrlA) necessary for normal conidiation.
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