Data_Sheet_1.pdf

Most of the metal transporters in Aspergillus fumigatus are yet uncharacterized. Their role in fungal metabolism and virulence remains unclear. This paper describes the novel PIB-type cation ATPase PcaA, which links metal homeostasis and heavy metal tolerance in the opportunistic human pathogen A. fumigatus. The protein possesses conserved ATPase motif and shares 51% amino acid sequence identity with the Saccharomyces cerevisiae cadmium exporter Pca1p. A pcaA deletion, an overexpression and a gfp-pcaA complementation strain of A. fumigatus were constructed and their heavy metal susceptibilities were studied. The pcaA knock out strain showed drastically decreased cadmium tolerance, however, its growth was not affected by the exposure to high concentrations of copper, iron, zinc, or silver ions. Although the lack of PcaA had no effect on copper adaption, we demonstrated that not only cadmium but also copper ions are able to induce the transcription of pcaA in A. fumigatus wild type Af293. Similarly, cadmium and copper ions could induce the copper exporting ATPase crpA. These data imply a general response on the transcriptomic level to heavy metals in A. fumigatus through the induction of detoxification systems. Confocal microscopy of the gfp-pcaA complementation strain expressing functional GFP-PcaA supports the predicted membrane localization of PcaA. The GFP-PcaA fusion protein is located in the plasma membrane of A. fumigatus in the presence of cadmium ions. Virulence assays support a function of PcaA for virulence of A. fumigatus in the Galleria mellonella wax moth larvae model, which might be linked to the elimination of reactive oxygen species.</p

Vacuole fragmentation depends on a novel Atg18-containing retromer-complex

The yeast PROPPIN Atg18 folds as a β-propeller with two binding sites for phosphatidylinositol-3-phosphate (PtdIns3P) and PtdIns(3,5)P2 at its circumference. Membrane insertion of an amphipathic loop of Atg18 leads to membrane tubulation and fission. Atg18 has known functions at the PAS during macroautophagy, but the functional relevance of its endosomal and vacuolar pool is not well understood. Here we show in a proximity-dependent labeling approach and by co-immunoprecipitations that Atg18 interacts with Vps35, a central component of the retromer complex. The binding of Atg18 to Vps35 is competitive with the sorting nexin dimer Vps5 and Vps17. This suggests that Atg18 within the retromer can substitute for both the phosphoinositide binding and the membrane bending capabilities of these sorting nexins. Indeed, we found that Atg18-retromer is required for PtdIns(3,5)P2-dependent vacuolar fragmentation during hyperosmotic stress. The Atg18-retromer is further involved in the normal sorting of the integral membrane protein Atg9. However, PtdIns3P-dependent macroautophagy and the selective cytoplasm-to-vacuole targeting (Cvt) pathway are only partially affected by the Atg18-retromer. We expect that this is due to the plasticity of the different sorting pathways within the endovacuolar system. Abbreviations: BAR: bin/amphiphysin/Rvs; FOA: 5-fluoroorotic acid; PAS: phagophore assembly site; PROPPIN: beta-propeller that binds phosphoinositides; PtdIns3P: phosphatidylinositol-3-phosphate; PX: phox homology.</p

NBR1 is involved in selective pexophagy in filamentous ascomycetes and can be functionally replaced by a tagged version of its human homolog

<p>Macroautophagy/autophagy is a conserved degradation process in eukaryotic cells involving the sequestration of proteins and organelles within double-membrane vesicles termed autophagosomes. In filamentous fungi, its main purposes are the regulation of starvation adaptation and developmental processes. In contrast to nonselective bulk autophagy, selective autophagy is characterized by cargo receptors, which bind specific cargos such as superfluous organelles, damaged or harmful proteins, or microbes, and target them for autophagic degradation. Herein, using the core autophagy protein ATG8 as bait, GFP-Trap analysis followed by liquid chromatography mass spectrometry (LC/MS) identified a putative homolog of the human autophagy cargo receptor NBR1 (NBR1, autophagy cargo receptor) in the filamentous ascomycete <i>Sordaria macrospora</i> (Sm). Fluorescence microscopy revealed that SmNBR1 colocalizes with SmATG8 at autophagosome-like structures and in the lumen of vacuoles. Delivery of SmNBR1 to the vacuoles requires SmATG8. Both proteins interact in an LC3 interacting region (LIR)-dependent manner. Deletion of <i>Smnbr1</i> leads to impaired vegetative growth under starvation conditions and reduced sexual spore production under non-starvation conditions. The human <i>NBR1</i> homolog partially rescues the phenotypic defects of the fungal <i>Smnbr1</i> deletion mutant. The <i>Smnbr1</i> mutant can neither use fatty acids as a sole carbon source nor form fruiting bodies under oxidative stress conditions. Fluorescence microscopy revealed that degradation of a peroxisomal reporter protein is impaired in the <i>Smnbr1</i> deletion mutant. Thus, SmNBR1 is a cargo receptor for pexophagy in filamentous ascomycetes.</p

DenA-DipA cytoplasmatic movements <i>in vivo</i>.

<p><b>(A)</b> Fluorescence microscopy of DenA-GFP subpopulations within vegetative hyphae located the protein in the nucleus (N), in the cytoplasm and there with a specific enrichment at septa (S). Control: wild type without GFP. <b>(B)</b> Bimolecular fluorescence studies (BiFC) of DenA (<i>denA</i>::<i>nyfp</i>) and DipA (<i>dipA</i>::<i>cyfp</i>) showed restricted interaction in the cytoplasm, at septa (S) and close to, but not inside nuclei (N). The septal and the nuclear regions are enlarged (white squares; scale bar: 1 μm). Control: strain co-expressing <i>denA</i>::<i>nyfp</i> and <i>cyfp</i>, respectively. <b>(C)</b> Dynamic co-transport of DenA-DipA between nuclei and septa in time lapse of bimolecular fluorescence strain <i>denA</i>::<i>nyfp</i>-<i>dipA</i>::<i>cyfp</i> over 170 seconds. White arrows mark a single interaction complex. <b>(D</b>) Time lapse microscopy over 110 seconds of <i>denA</i>::<i>nyfp</i>-<i>dipA</i>::<i>cyfp</i> with stained mitochondria (red) with a white arrow marking single DenA-DipA. Expressed <i>rfp</i>::<i>h2A</i> decorates nuclei, membranes were stained with FM4-64 and mitochondria with MitoTracker. Scale bar: 5 μm.</p

αSyn posttranslational modifications and nitrative stress in yeast.

Enhanced intracellular nitrative stress increases the protein nitration level and influences yeast growth and aggregation. The nitration of tyrosine residues acts as trigger for αSyn and A30P toxicity. Wild-type αSyn, which is highly nitrated, inhibits growth and shows a high aggregation rate. A30P is weakly nitrated and therefore, does not inhibit yeast growth and has a low aggregation propensity. Yhb1 and its human homolog NGB protect the cells against accumulation of nitrative species and diminish the aggregate formation. Di-tyrosine crosslinked dimers are formed in reverse correlation to cytotoxicity and do not depend on Yhb1. A30P forms twice as many dimers as the toxic αSyn variant, suggesting that the di-tyrosine crosslinked dimers are not toxic species and are presumably part of a cellular detoxification pathway, sequestering the protein off-pathway of αSyn nucleation. The C-terminal tyrosine modifications have dual effect on the toxicity of the protein. Y133, which is nitrated and phosphorylated, is required for the protective phosphorylation at S129 and for the autophagy degradation of αSyn aggregates. Non-modified Y133 promotes the proteasomal degradation of αSyn aggregates. N: nitration; P: phosphorylation.</p

Phenotypical characterization of mutant strains lacking functional DipA.

<p><b>(A)</b> Top and bottom view of point-inoculated wild type (WT), <i>dipA</i>* (codon exchange of catalytic core), <i>dipA</i> deletion (Δ<i>dipA</i>) and complementation (Compl.) strains incubated for three days under asexually development inducing conditions. Zoomed view represents binocular images of respective strains with asexual structures (conidiophores, co) and the sexual fruiting bodies (cleistothecia, cl) after seven days. Scale bar: 100 μm. <b>(B)</b> Colony diameter of point-inoculated asexually grown colonies measured for six days. The mean values with standard deviations derived from three independent experiments are shown. <b>(C)</b> Quantification of conidiospores after four days. The mean values with standard deviations from three independent experiments are shown. <b>(D)</b> Diagram illustrates distances between septa. Data derived from analyzing 70 hyphae of each strain. Shown are the mean values with standard deviations. <b>(E)</b> Fluorescence microscopy of hyphae of WT and <i>dipA</i> deletion strain. Membranes/septa were stained with FM4-64. White arrows are highlighting septa. Scale bar: 5 μm.</p

DenA and DenA interacting proteins (Dip) identified by tandem affinity purification or GFP-trap enrichment.

<p>DenA and DenA interacting proteins (Dip) identified by tandem affinity purification or GFP-trap enrichment.</p

Changes in deneddylase activity and its consequences for the cellular pool of neddylated proteins and fungal development.

<p><b>(A)</b> Western analyses with Nedd8 or Tubulin (reprobed as loading control, lower part) antibodies of vegetative grown mycelia of <i>A</i>. <i>nidulans</i>. Wild type (WT) was compared to mutant strains with altered deneddylase activity either with decreased COP9 signalosome (Δ<i>csnG</i>, Δ<i>csnE</i>) or increased <i>denA</i> (OE <i>denA</i>) and combinations of defective CSN and increased DenA (Δ<i>csnG</i>/OE <i>denA</i>, or Δ<i>csnE</i>/OE <i>denA</i>). Neddylated cullins correspond to ≈ 100 kDa and faster migrating bands are summarized as neddylated non-cullin proteins. <b>(B)</b> Semi-quantitative analyses of Nedd8 signal intensities of three independent experiments of strains shown in (A) normalized to Tubulin signals, including standard deviations. <b>(C)</b> Western hybridization using cullinA (CulA) or <b>(D)</b> cullinC (CulC) antibodies and determination of the ratios (lower panels) of neddylated (CulA-N8 or CulC-N8) in comparison to deneddylated cullins of three independent experiments. <b>(E)</b> Cellular deneddylase activity and fungal development. Equal amount of spores of indicated strains were point-inoculated and grown for four days under illumination which induces asexual development in wild type. Respective strains are shown either on minimal (MM) or stress inducing media (+menadione, +SDS).</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Tyrosine 133 is required for phosphorylation of αSyn at serine 129.

<p>(A) Western blotting of αSyn and A30P expressed in <i>YHB1</i> and Δ<i>yhb1</i> yeast enriched by Ni²⁺ pull-down, using Y133 phosphorylation-specific αSyn antibody (pY133) and S129 phosphorylation-specific αSyn antibody (pS129). The same membrane was stripped and re-probed with αSyn antibody. (B) Quantification of αSyn and A30P Y133- and S129-phosphorylation levels in <i>YHB1</i> and Δ<i>yhb1</i> yeast cells. Densitometric analysis of the immunodetection of pY133, pS129 αSyn and A30P relative to the intensity obtained for αSyn. Significance of differences was calculated with one-way ANOVA test (**, <i>p</i> < 0.01; n = 4). (C) Western blotting of crude extracts from yeast cells, expressing different αSyn variants after 6 h induction of protein expression using S129 phosphorylation-specific αSyn antibody (pS129) and αSyn antibody. Cells expressing S129A mutant served as control. (D) Spotting analysis of αSyn and indicated mutant strains, driven by the inducible <i>GAL1-</i>promoter on non-inducing (´OFF`: glucose) and inducing (´ON`: galactose) SC-Ura medium after 3 days. Cells expressing GFP served as control. (E) Quantification of the percentage of cells displaying αSyn aggregates after 6 h induction in galactose-containing SC-Ura medium. Significance of differences was calculated with one-way ANOVA (***, <i>p</i> < 0.001) or Dunnett’s multiple comparison test (#, <i>p</i> < 0.05, ##, <i>p</i> < 0.01 versus αSyn; n = 6). (F) Cell growth analysis of cells expressing different αSyn variants and GFP (control) after 20 h induction of expression. Significance of differences was calculated with one-way ANOVA (****, <i>p</i> < 0.0001) or Dunnett’s multiple comparison test (#, <i>p</i> < 0.05; ###, <i>p</i> < 0.001, n = 4). (G) Quantification of cells expressing different αSyn variants and GFP (control) displaying Propidium Iodide (PI) fluorescence after 20 h induction of αSyn expression, assessed by flow cytometry. The percentage of PI-positive yeast cells with higher fluorescent intensities (P1) than the background is presented. Significance of differences was calculated with one-way ANOVA (****, <i>p</i> < 0.0001) or Dunnett’s multiple comparison test (#, <i>p</i> < 0.05; ###, <i>p</i> < 0.001 versus αSyn; n = 4).</p