6 research outputs found

    Yeast-two hybrid interaction of SmATG12 with SmATG7, SmATG8 and SmATG3.

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    <p>Full-length cDNAs of <i>Smatg12</i>, <i>Smatg7</i> and <i>Smatg3</i> were used to generate GAL4-DNA binding domain (BD) and activation domain (AD) plasmids. <i>Smatg8</i> two-hybrid vectors were previously described in Voigt and Pöggeler [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157960#pone.0157960.ref034" target="_blank">34</a>]. To select for the presence of both plasmids 20 μl of cells were spotted in serial delutions on SD medium lacking tryptophan and leucine (SD -Trp, -Leu) or to verify the interactions of the proteins on medium lacking additionally histidine or adenine (SD -Trp, -Leu, -His/-Ade). (A) SmATG12 and SmATG7 interacted only when SmATG7 was expressed as GAL4-BD fusion protein. (B) SmATG12 and SmATG3 interacted with each other as bait and prey proteins, respectively. (C) SmATG8 did not interact with SmATG12. Transformants carrying a bait plasmid and pAD-ranBPM [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157960#pone.0157960.ref069" target="_blank">69</a>] were used to confirm the appropriate expression of the bait proteins. Strains carrying empty plasmids pGADT7 and pGBKT7 served as negative control (A). Plates were incubated for 3–5 days at 30°C.</p

    Multiple sequence alignment of ATG12 orthologs from fungi, plants and animals.

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    <p>ClustalX alignment was created using the following sequences: Smac [<i>S</i>. <i>macrospora</i>, Accession No. XP_003349162.1, excluding 56 C-terminal amino acids)], Ncra [<i>Neurospora crassa</i>, Q7S083.1], Pans [<i>Podospora anserina</i>, XP_001906089.1], Cglo [<i>Chaetomium globosum</i>, Q2GSG9.2], Mory [<i>Magnaporthe oryzae</i>, XP_368646.1], Anid [<i>Aspergillus nidulans</i>, Q5BCH0.2], Pchr [<i>Penicillium chrysogenum</i>, XP_002557636.1], Celg [<i>Caenorhabditis elegans</i>, CCD61524.1], Hsap [<i>Homo sapiens</i>, NP_004698.3], Dmel [<i>Drosophila melanogaster</i>, NP_648551.3], AthaB [<i>Arabidopsis thaliana</i>, Q9LVK3.1], AthaA [<i>A</i>. <i>thaliana</i>, Q8S924.1], Scer [<i>S</i>. <i>cerevisiae</i>, P38316]. Identical amino acids, which are conserved in all proteins, are shaded in black; residues conserved in at least 10 of 13 sequences are shaded in dark grey and residues conserved in at least eight sequences are shaded in light grey. The conserved C-terminal glycine residue for the covalent linkage to ATG5 and the conserved phenylalanine residue corresponding to Phe154 in the <i>S</i>. <i>cerevisiae</i> Atg12 is labelled in red [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157960#pone.0157960.ref067" target="_blank">67</a>], amino acids important for non-covalent interactions between ATG12 and ATG5 in <i>S</i>. <i>cerevisiae</i> according to Noda et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157960#pone.0157960.ref065" target="_blank">65</a>] are marked by asterisks. Non-covalent contacts between ATG12 and ATG5 identified in the human homologs according to Otomo et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157960#pone.0157960.ref066" target="_blank">66</a>] are marked by black squares. The red bar represents the turn—loop—alpha helix 2 segment (Asn105 –Phe123 of the human ATG12) which is associated with the interaction surface of ATG5 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157960#pone.0157960.ref066" target="_blank">66</a>]. White squares mark residues of the human ATG12, which are important for binding of ATG3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157960#pone.0157960.ref064" target="_blank">64</a>], #, indicates residues of the non-canonical AIM of ATG12 involved in interaction with ATG8 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157960#pone.0157960.ref024" target="_blank">24</a>]. The region of the BH3 domain identified in the human ATG12 homolog is indicated and the conserved aspartic acid residue is indicated in green. Amino-acid identity in % is given at the right margin.</p

    EGFP-SmATG8 protein degradation in the ΔSmatg12 strain compared to the corresponding complemented ΔSmatg8::egfp-Smatg8<sup>ect</sup> strain.

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    <p>Protein crude extracts of <i>S</i>. <i>macrospora</i> wt, ΔSmatg12::egfp-Smatg8<sup>ect</sup> and ΔSmatg8::egfp-Smatg8<sup>ect</sup> strains expressing EGFP or EGFP-SmATG8 were separated on a 12% SDS-PAGE gel. The Western blot hybridization using an anti-EGFP antibody verified the degradation of the EGFP-SmATG8 fusion protein in the complemented ΔSmatg8 strain by accumulation of free EGFP, whereas in the ΔSmatg12 mutant the EGFP-SmATG8 fusion protein accumulated.</p

    Fluorescence microscopic localization of EGFP-SmATG12 and EGFP-SmATG8.

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    <p>(A) Mutant ΔSmatg12 was transformed with plasmid pegfp-Smatg12. Expression of the EGFP-SmATG12 fusion construct complemented the sterile phenotype of ΔSmatg12. EGFP-SmATG12 localizes to the cytoplasm and at phagophore assembly sites indicated by small arrows and cup-shaped phagophores indicated by long arrows. Vacuolar membranes were stained using FM4-64. The merged picture shows a close up. (B) Localization of EGFP-SmATG12 in the ΔSmatg8 mutant [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157960#pone.0157960.ref034" target="_blank">34</a>]. The fluorescence signals are larger and more distinct than signals in the complemented mutant ΔSmatg8::egfp-Smatg8<sup>ect</sup> (compare to E). (C) The fluorescence signal of free EGFP in the wt strain transformed with plasmid p1783-1 served as control. (D) When the mutant strain ΔSmatg12 expressed EGFP-SmATG8 (plasmid pRS-egfp-Smatg8 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157960#pone.0157960.ref034" target="_blank">34</a>]), the fluorescence protein displays an equal diffused signal in the cytoplasm with large accumulating spots (arrow head) which are excluded from the vacuole. Vacuolar membranes were co-stained with FM4-64 and pictures were merged. (E) The previously constructed strain ΔSmatg8::egfp-Smatg8<sup>ect</sup> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157960#pone.0157960.ref034" target="_blank">34</a>] was used to compare the localization of EGFP-SmATG8 in the complemented ΔSmatg8 mutant. Autophagosomes are indicated by arrows. DIC, differential interference contrast; EGFP, enhanced green fluorescence protein. Autophagy was induced by the addition of 2.5 mM 3-AT to the SWG medium or by nitrogen starvation conditions (SWG-N, without KNO<sub>3</sub> and arginine). Scale bars as depicted.</p

    Phenotypic characterization of <i>S</i>. <i>macrospora</i> wt, <i>Smatg12</i> deletion and complementation strain.

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    <p>(A) Phenotype of wt, ΔSmatg12 and rescued strain ΔSmatg12::egfp-Smatg12<sup>ect</sup> grown on SWG or SWG medium supplemented with 2.5 mM 3-AT in petri dishes. Insets show a detailed view of perithecia or sterile mycelium. The images were taken seven days post inoculation. (B) Foraging capacity of indicated strains. Agar plugs of 0.5-cm diameter were transferred into empty cell-culture plates (6 well, 17.2 ml) and incubated for 5 d at 27°C in a damp chamber before photographed. Scale bars as depicted. (C) The growth velocity of wt, ΔSmatg12 and the complemented strain was analyzed by measuring the growth velocity in cm/day in 30-cm race tubes. Growth rates on SWG medium shown are averages from 7 independent measurements of three independent experiments (n = 21), standard deviations are indicated by error bars. Asterisks indicate significant differences according to Student´s t-test (p<0.0000001). (D) Microscopic investigation of sexual development of ΔSmatg12 compared to wt and the complemented strain. Strains were grown on SWG medium. Expression of the EGFP-SmATG12 fusion construct complemented the sterile phenotype of ΔSmatg12. The wt and the complemented strain ΔSmatg12::egfp-Smatg12<sup>ect</sup> form ascogonia at day 3, and protoperithecia at day 4 post inoculation. These develop to pigmented protoperithecia at day 5 and to mature perithecia at day 7. Sexual development of the mutant ΔSmatg12 is blocked at the stage of protoperithecia formation. ΔSmatg12 neither forms pigmented protoperithecia nor perithecia and ascospores. Scale bars as depicted.</p

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

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    <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