Prohibitins, Phb1 and Phb2, function as Atg8 receptors to support yeast mitophagy and also play a negative regulatory role in Atg32 processing.

The prohibitins Phb1 and Phb2 assemble at the mitochondrial inner membrane to form a multi-dimeric complex. These scaffold proteins are highly conserved in eukaryotic cells, from yeast to mammals, and have been implicated in a variety of mitochondrial functions including aging, proliferation, and degenerative and metabolic diseases. In mammals, PHB2 regulates PINK1-PRKN mediated mitophagy by interacting with lipidated MAP1LC3B/LC3B. Despite their high conservation, prohibitins have not been linked to mitophagy in budding yeasts. In this study, we demonstrate that both Phb1 and Phb2 are required to sustain mitophagy in Saccharomyces cerevisiae. Prohibitin-dependent mitophagy requires formation of the Phb1-Phb2 complex and a conserved AIM/LIR-like motif identified in both yeast prohibitins. Furthermore, both Phb1 and Phb2 interact and exhibit mitochondrial colocalization with Atg8. Interestingly, we detected a basal C terminus processing of the mitophagy receptor Atg32 that depends on the presence of the i-AAA Yme1. In the absence of prohibitins this processing is highly enhanced but reverted by the inactivation of the rhomboid protease Pcp1. Together our results revealed a novel role of yeast prohibitins in mitophagy through its interaction with Atg8 and regulating an Atg32 proteolytic event. Abbreviation: AIM/LIR: Atg8-family interacting motif/LC3-interacting region; ANOVA: analysis of variance; ATG/Atg: autophagy related; C terminus/C-terminal: carboxyl terminus/carboxyl-terminal; GFP: green fluorescent protein; HA: human influenza hemagglutinin; Idh1: isocitrate dehydrogenase 1; MAP1C3B/LC3B: microtubule associated protein 1 light chain 3 beta; mCh: mCherry; MIM: mitochondrial inner membrane; MOM: mitochondrial outer membrane; N starvation: nitrogen starvation; N terminus: amino terminus; PARL: presenilin associated rhomboid like; Pcp1: processing of cytochrome c peroxidase 1; PCR: polymerase chain reaction; PGAM5: PGAM family member 5 mitochondrial serine/threonine protein phosphatase; PHBs/Phb: prohibitins; PINK1: PTEN induced kinase 1; PMSF: phenylmethylsulfonyl fluoride; PRKN: parkin RBR E3 ubiquitin protein ligase; SD: synthetic defined medium; SDS: sodium dodecyl sulfate; SMD-N: synthetic defined medium lacking nitrogen; WB: western blot; WT: wild type; Yme1: yeast mitochondrial escape 1; YPD: yeast extract-peptone-dextrose medium; YPLac: yeast extract-peptone-lactate medium.

The role of peroxiredoxins PrxA and PrxB in the antioxidant response, carbon utilization and development in Aspergillus nidulans.

In addition to their role in the breakdown of H2O2, some peroxiredoxins (Prxs) have chaperone and H2O2 sensing functions. Acting as an H2O2 sensor, Prx Gpx3 transfers the oxidant signal to the transcription factor Yap1, involved in the antioxidant response in Saccharomyces cerevisiae. We have shown that Aspergillus nidulans Yap1 ortholog NapA is necessary for the antioxidant response, the utilization of arabinose, fructose and ethanol, and for proper development. To address the Prx roles in these processes, we generated and characterized mutants lacking peroxiredoxins PrxA, PrxB, PrxC, or TpxC. Our results show that the elimination of peroxiredoxins PrxC or TpxC does not produce any distinguishable phenotype. In contrast, the elimination of atypical 2-cysteine peroxiredoxins PrxA and PrxB produce different mutant phenotypes. ΔprxA, ΔnapA and ΔprxA ΔnapA mutants are equally sensitive to H2O2 and menadione, while PrxB is dispensable for this. However, the sensitivity of ΔprxA and ΔprxA ΔnapA mutants is increased by the lack of PrxB. Moreover, PrxB is required for arabinose and ethanol utilization and fruiting body cell wall pigmentation. PrxA expression is partially independent of NapA, and the replacement of peroxidatic cysteine 61 by serine (C61S) is enough to cause oxidative stress sensitivity and prevent NapA nuclear accumulation in response to H2O2, indicating its critical role in H2O2 sensing. Our results show that despite their high similarity, PrxA and PrxB play differential roles in Aspergillus nidulans antioxidant response, carbon utilization and development.

Positively charged amino acids at the N terminus of select mitochondrial proteins mediate early recognition by import proteins αß'-NAC and Sam37.

A major challenge in eukaryotic cells is the proper distribution of nuclear-encoded proteins to the correct organelles. For a subset of mitochondrial proteins, a signal sequence at the N terminus (matrix-targeting sequence [MTS]) is recognized by protein complexes to ensure their proper translocation into the organelle. However, the early steps of mitochondrial protein targeting remain undeciphered. The cytosolic chaperone nascent polypeptide-associated complex (NAC), which in yeast is represented as the two different heterodimers αß-NAC and αß'-NAC, has been proposed to be involved during the early steps of mitochondrial protein targeting. We have previously described that the mitochondrial outer membrane protein Sam37 interacts with αß'-NAC and together promote the import of specific mitochondrial precursor proteins. In this work, we aimed to detect the region in the MTS of mitochondrial precursors relevant for their recognition by αß'-NAC during their sorting to the mitochondria. We used targeting signals of different mitochondrial proteins (αß'-NAC-dependent Oxa1 and αß'-NAC-independent Mdm38) and fused them to GFP to study their intracellular localization by biochemical and microscopy methods, and in addition followed their import kinetics in vivo. Our results reveal the presence of a positively charged amino acid cluster in the MTS of select mitochondrial precursors, such as Oxa1 and Fum1, which are crucial for their recognition by αß'-NAC. Furthermore, we explored the presence of this cluster at the N terminus of the mitochondrial proteome and propose a set of precursors whose proper localization depends on both αß'-NAC and Sam37.

Yap1 homologs mediate more than the redox regulation of the antioxidant response in filamentous fungi.

The regulation of gene expression in response to increased levels of reactive oxygen species (ROS) is a ubiquitous response in aerobic organisms. However, different organisms use different strategies to perceive and respond to high ROS levels. Yeast Yap1 is a paradigmatic example of a specific mechanism used by eukaryotic cells to link ROS sensing and gene regulation. The activation of this transcription factor by H2O2 is mediated by peroxiredoxins, which are widespread enzymes that use cysteine thiols to sense ROS, as well as to catalyze the reduction of peroxides to water. In filamentous fungi, Yap1 homologs and peroxiredoxins also are major regulators of the antioxidant response. However, Yap1 homologs are involved in a wider array of processes by regulating genes involved in nutrient assimilation, secondary metabolism, virulence and development. Such novel functions illustrate the divergent roles of ROS and other oxidizing compounds as important regulatory signaling molecules.

NapA Mediates a Redox Regulation of the Antioxidant Response, Carbon Utilization and Development in Aspergillus nidulans.

The redox-regulated transcription factors (TFs) of the bZIP AP1 family, such as yeast Yap1 and fission yeast Pap1, are activated by peroxiredoxin proteins (Prxs) to regulate the antioxidant response. Previously, Aspergillus nidulans mutants lacking the Yap1 ortholog NapA have been characterized as sensitive to H2O2 and menadione. Here we study NapA roles in relation to TFs SrrA and AtfA, also involved in oxidant detoxification, showing that these TFs play different roles in oxidative stress resistance, catalase gene regulation and development, during A. nidulans life cycle. We also uncover novel NapA roles in repression of sexual development, normal conidiation, conidial mRNA accumulation, and carbon utilization. The phenotypic characterization of ΔgpxA, ΔtpxA, and ΔtpxB single, double and triple peroxiredoxin mutants in wild type or ΔnapA backgrounds shows that none of these Prxs is required for NapA function in H2O2 and menadione resistance. However, these Prxs participate in a minor NapA-independent H2O2 resistance pathway and NapA and TpxA appear to regulate conidiation along the same route. Using transcriptomic analysis we show that during conidial development NapA-dependent gene expression pattern is different from canonical oxidative stress patterns. In the course of conidiation, NapA is required for regulation of at least 214 genes, including ethanol utilization genes alcR, alcA and aldA, and large sets of genes encoding proteins involved in transcriptional regulation, drug detoxification, carbohydrate utilization and secondary metabolism, comprising multiple oxidoreductases, membrane transporters and hydrolases. In agreement with this, ΔnapA mutants fail to grow or grow very poorly in ethanol, arabinose or fructose as sole carbon sources. Moreover, we show that NapA nuclear localization is induced not only by oxidative stress but also by growth in ethanol and by carbon starvation. Together with our previous work, these results show that SakA-AtfA, SrrA and NapA oxidative stress-sensing pathways regulate essential aspects of spore physiology (i.e., cell cycle arrest, dormancy, drug production and detoxification, and carbohydrate utilization).