Comparison of AOX1 and choline oxidase active sites.

<p>AOX1 monomer is colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149846#pone.0149846.g002" target="_blank">Fig 2C</a>, the CO is colored grey. The residues delimiting the substrate binding cavity are labeled and represented as sticks. Modified a-FAD is depicted in ball-and-stick representation. (a) The active site of AOX1 with cavities depicted in atom colored surface representation. The substrate binding cavity is marked with blurred red rim. The water molecule, which is bound close to the isoalloxazine ring, is depicted as sphere. Polar interactions are marked as dashed lines. (b) The active site of Choline oxidase with cavities depicted in surface representation. The substrate binding cavity is marked with blurred red rim. (c) Cartoon representation of superimposed AOX1 and Choline Oxidase monomers.</p

Interaction network of MAK-2 pathway components.

<p>(<b>A</b>) Proteins associated with the MAK-2 cascade were identified in affinity purification experiments coupled to mass spectrometry (AP-MS), and identified proteins were filtered against control purifications using GFP as bait (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004762#pgen.1004762.s007" target="_blank">Table S1</a>). Only proteins identified in two biological replicates and absent from the control data set are shown. Protein coverage by peptides and the number of identified total and unique peptides identified are given for the better of the two purifications. Bold numbers indicate the GFP-fusion protein used as bait. (<b>B</b>) Physical interactions between MAK-2 pathway components were mapped in yeast two-hybrid (Y2H) tests. The indicated constructs were co-expressed in strain AH109 and yeast growth was analyzed on the indicated selective media. The reciprocal Y2H assays are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004762#pgen.1004762.s002" target="_blank">Figure S2</a>. (<b>C</b>) Summary schema of Y2H-based interactions of the indicated proteins and their domains. Color-coded lines below the protein schemas and dashed connectors indicate the used constructs and detected interactions, respectively.</p

STE-50 functions as regulatory subunit of NRC-1.

<p>(<b>A</b>) GFP-STE-50 localizes in a dynamic manner to opposing tips of two communicating germlings and the site of cell-cell contact. The oscillation period is approximately three to five minutes. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004762#pgen.1004762.s010" target="_blank">Video S1</a> for time course. (<b>B</b>) GFP-STE-50 localizes in a diffuse cytosolic manner in non-communicating hyphae and is excluded from nuclei (left image). Moreover, GFP-STE-50 accumulates at septa (arrowheads) and the contact point (arrow) of communicating hyphal tips (right image). (<b>C</b>) Δ<i>ste-50</i> fully phenocopies Δ<i>mak-2</i> defects (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004762#pgen.1004762.s003" target="_blank">Figure S3</a> for comparative characterization of mutant defects), and the mutant characteristics are complemented by expression of a constitutive active NRC-1(P488S) allele in Δ<i>ste-50</i>. The slant images represent macroscopic appearance and conidiation pattern, while the Table summarizes rates of mycelial extension, protoperithecia (PP) formation, germling communication and MAK-2 activities of the indicated strains.</p

The MAK-2 pathway elements STE-20 and RAS-2 are important for cell-cell communication.

<p>(<b>A</b>) STE-20-GFP localizes in a stable manner to the apices of two communicating germlings and marks the site of contact. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004762#pgen.1004762.s015" target="_blank">Video S6</a> for time course. (<b>B</b>) GFP-RAS-2 associates with the entire plasma membrane of germinating spores and localizes at the contact point of two communicating cells. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004762#pgen.1004762.s016" target="_blank">Video S7</a> for time course. (<b>C</b>) Time course experiments to determine rates of germination and of chemotropic interactions of the indicated mutants. Note that due to delayed germination Δ<i>cr-1</i> and Δ<i>cap-1</i> strains are assayed at later time points than the other mutants. (<b>D</b>) Quantification of basal and stress-induced MAK-2 phosphorylation levels (detected with p42/44 antibodies) in cell extracts of exponentially growing liquid cultures of the indicated strains (n = 3). A representative Western blot is depicted below; tubulin was used as loading control.</p

Active site comparison between AOX1 and choline oxidase complexed with glycine betaine.

<p>AOX1 monomer is colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149846#pone.0149846.g002" target="_blank">Fig 2C</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149846#pone.0149846.g004" target="_blank">Fig 4</a>, the CO is colored grey. Residues delimiting the substrate binding cavity are labeled and represented as sticks. Modified a-FAD is depicted in ball-and-stick representation. Superposition with the closely related CO in a complex with product glycine betaine (PDB id: 4MJW) indicates that the water molecule in the AOX1 active site, depicted as magenta sphere, occupies similar position as one oxygen of the carboxylic group of the bound product molecule.</p

Mutant characteristics of MAK-2 pathway components.

1<p>assayed at 6 h post inoculation except for strains marked with *, which were analyzed 8 h post inoculation due to reduced germination rate and abnormal growth.</p>2<p>in %±SEM.</p>3<p>not determined.</p>4<p>heterokaryotic deletion strain.</p><p>Mutant characteristics of MAK-2 pathway components.</p

Model depicting network organization of the MAK-2 pathway and putative regulatory mechanisms.

<p>Ligand-induced activation of an unknown receptor may be transmitted through plasma membrane-associated STE-20, RAS-2 and CAP-1, which signal toward the NRC-1/STE-50 complex and recruit the MAPK cascade through activation and clustering of the scaffold HAM-5 at intracellular puncta. MAK-2 activation triggers nuclear gene expression through interaction with the transcription factor PP-1 and the RCO-1/RCM-1 complex and the cytosolic activation of the secretory pathway and cell polarity machineries to coordinate pulsed signal release and chemotrophic growth towards the partner cell, respectively. MAK-2 activity is also required for termination of the receiver phase, potentially through negative feedback phosphorylation of the MAPK and disassembly of the MAK-2/HAM-5 module. MAK-2 pathway function may also be regulated through the STRIPAK complex, the CK2 heterodimer, membrane lipid composition, the septum-associated septation initiation network SIN and motor protein-dependent vesicle trafficking.</p

The structure of one <i>P</i>. <i>pastoris</i> AOX1 subunit.

<p>The two views are related by rotating the molecule by 180 degrees. (a) Rainbow colored cartoon representation of the AOX1 monomer. FAD is depicted in ball-and-stick representation; the position of the PP loop (residues 13–17) is highlighted as blue balls and sticks. N- and C-termini are labeled. (b) Superposition with Choline oxidase (PDB: 3LJP) and domain organization of the AOX1 subunit, both proteins represented as cartoon. The FAD-binding domain (AOX1, residues 1–155, 192–306 and 568–663) is colored wheat and a substrate-binding domain (AOX1, residues 156–191 and 307–567) is colored dirty violet. Residues forming insertions are colored purple and red for substrate- and FAD-binding domains, respectively. Choline oxidase is colored grey. (c) Monomer of AOX1 with residues involved in octamer formation depicted as surface representation, colored as in b (d) Monomer of AOX1 with residues involved in dimer formation (inter tetramer interactions) highlighted with yellow dots. Colors are chosen as in (b) and (c).</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

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p