NapA Mediates a Redox Regulation of the Antioxidant Response, Carbon Utilization and Development in Aspergillus nidulansThe 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 H 2 O 2 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 gpxA, tpxA, and tpxB single, double and triple peroxiredoxin mutants in wild type or napA backgrounds shows that none of these Prxs is required for NapA function in H 2 O 2 and menadione resistance. However, these Prxs participate in a minor NapA-independent H 2 O 2 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, napA 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).
Yap1 homologs mediate more than the redox regulation of the antioxidant response in filamentous fungiThe 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.
Positively charged amino acids at the N terminus of select mitochondrial proteins mediate early recognition by import proteins αβ′-NAC and Sam37A 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 αβ-NAC and αβ'-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 αβ'-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 αβ'-NAC during their sorting to the mitochondria. We used targeting signals of different mitochondrial proteins (αβ'-NAC-dependent Oxa1 and αβ'-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 αβ'-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 αβ'-NAC and Sam37.