Amino acid sequence alignment of <i>A. fumigatus</i> MedA putative NLSs 1–4 with other MedA homologues.

<p>(A) Sequence alignment and motif prediction using PSORT II identified NLS1 sequence among orthologues of MedA. (B) Sequence alignment of the MedA minimal nuclear localization domain, MedA^346–557 with other MedA orthologues. The sequences representing the putative NLSs 2, 3, and 4 are boxed. The basic amino acids within the putative NLSs of <i>A. fumigatus</i> MedA and the corresponding amino acids in MedA orthologues are highlighted in gray. The presence of an asterisk or a colon below the basic amino acids indicates a fully or strongly conserved residue, respectively. Numbers indicate the amino acid position within the primary amino acid sequence of the protein. Af_MedA: <i>A. fumigatus</i> MedA (GenBank: EAL93620.1), An_MedA: <i>A. nidulans</i> MedA (GenBank: AAC31205.1), Nc_ACON-3: <i>N. crassa</i> ACON-3 (GenBank: ADL28820.1), Mg_Acr1: <i>M. grisea</i> Acr1 (GenBank: BAC41196.1), and Fo_Ren1: <i>F. oxysporum</i> Ren1 (GenBank: BAC55015.1).</p

Phenotypic analysis of Δ<i>medA</i> strain expressing MedA^346–557 domain.

<p>(A) Conidia hydrophobicity and biofilm formation of the indicated strains. (B) Survival assay of <i>G. mellonella</i> larvae. 28 worms/strain were infected with 10⁵ swollen conidia. Af293 indicates the <i>A. fumigatus</i> wild type strain; MedA^346–557 indicates expression of this construct under the control of the <i>medA(p)</i> in the Δ<i>medA</i> strain. Analysis of survival data was performed using the log rank test. Statistically significant differences are indicated by asterisk (P value ≤0.05).</p

Relative gene expression of cytoplasmic and nuclear <i>medA</i> measured by RT-PCR.

<p>Af293 is the <i>A. fumigatus</i> wild type strain; MedA, MedA^ΔNLS1, MedA^ΔNLS2, MedA^ΔNLS3, and MedA^ΔNLS4 indicate expression of the corresponding construct under the control of the <i>medA(p)</i> in the Δ<i>medA</i> strain, normalized to <i>medA</i> expression in strain Af293. Error bars represent the standard error of three triplicates for every strain.</p

Subcellular localization of the MedA-EGFP fusion truncation constructs in <i>A. fumigatus</i>.

<p>(A) Schematic overview of the various truncated <i>medA-egfp</i> gene fusions. Full-length <i>medA</i> and different <i>medA</i> truncations were fused in-frame to <i>egfp</i> under the control of <i>alcA</i> promoter. (B) The cellular localization of various MedA-EGFP fusion proteins expressed in <i>A. fumigatus</i> Af293. Nuclei were stained by Draq5 and mycelia were analyzed by light microscopy. Left, center, and right columns show the GFP, Draq5, and DIC (Differential Interference Contrast), respectively. The MedA-GFP constructs are indicated on the left side. Vertical lines, from left to right, represent amino acids 1, 346, 557, and 683 respectively.</p

Oligonucleotides used in this study.

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

Plasmids used in this study, combination of oligonucleotides, and the corresponding expression strains of <i>A. fumigatus.</i>

<p>Plasmids used in this study, combination of oligonucleotides, and the corresponding expression strains of <i>A. fumigatus.</i></p

Mycelial growth of <i>A. fumigatus</i> is not affected by MedA nuclear localization.

<p>YPD agar plates were spot inoculated with the indicated strains and the colony diameter measured daily. Af293 is the <i>A. fumigatus</i> wild type strain; MedA, MedA^ΔNLS1, MedA^ΔNLS2, MedA^ΔNLS3, and MedA^ΔNLS4 indicate expression of the corresponding construct under the control of the <i>medA(p)</i> in the Δ<i>medA</i> strain.</p

Effect of MedA nuclear localization on restoring wild type phenotype.

<p>(A) Schematic overview of the <i>medA-egfp</i> fusion constructs under the control of the 1.5 kb <i>medA</i> promoter, <i>medA(p)</i> used for complementation of the Δ<i>medA</i> strain. (B) Vegetative growth of strains on Sabouraud agar for 6 days at 37°C; hydrophobicity of conidia; and biofilm formation before and after washing. (C) Survival assay of <i>G. mellonella</i> larvae. 40 worms/strain were infected with 10⁵ swollen conidia. Af293 indicates the <i>A. fumigatus</i> wild type strain; MedA, MedA^ΔNLS1, MedA^ΔNLS2, MedA^ΔNLS3, and MedA^ΔNLS4 indicate expression of the corresponding construct under the control of the <i>medA(p)</i> in the Δ<i>medA</i> strain. Virulence of strains expressing cytoplasmic and nuclear MedA was compared to Af293 and Δ<i>medA</i> strain, respectively, using the log rank test. For all comparisons, P was ≤0.05.</p

Mechanism of liponecrosis, a distinct mode of programmed cell death

<div><p>An exposure of the yeast <i>Saccharomyces cerevisiae</i> to exogenous palmitoleic acid (POA) elicits “liponecrosis," a mode of programmed cell death (PCD) which differs from the currently known PCD subroutines. Here, we report the following mechanism for liponecrotic PCD. Exogenously added POA is incorporated into POA-containing phospholipids that then amass in the endoplasmic reticulum membrane, mitochondrial membranes and the plasma membrane. The buildup of the POA-containing phospholipids in the plasma membrane reduces the level of phosphatidylethanolamine in its extracellular leaflet, thereby increasing plasma membrane permeability for small molecules and committing yeast to liponecrotic PCD. The excessive accumulation of POA-containing phospholipids in mitochondrial membranes impairs mitochondrial functionality and causes the excessive production of reactive oxygen species in mitochondria. The resulting rise in cellular reactive oxygen species above a critical level contributes to the commitment of yeast to liponecrotic PCD by: (1) oxidatively damaging numerous cellular organelles, thereby triggering their massive macroautophagic degradation; and (2) oxidatively damaging various cellular proteins, thus impairing cellular proteostasis. Several cellular processes in yeast exposed to POA can protect cells from liponecrosis. They include: (1) POA oxidation in peroxisomes, which reduces the flow of POA into phospholipid synthesis pathways; (2) POA incorporation into neutral lipids, which prevents the excessive accumulation of POA-containing phospholipids in cellular membranes; (3) mitophagy, a selective macroautophagic degradation of dysfunctional mitochondria, which sustains a population of functional mitochondria needed for POA incorporation into neutral lipids; and (4) a degradation of damaged, dysfunctional and aggregated cytosolic proteins, which enables the maintenance of cellular proteostasis.</p></div