Meiotic development initiation in the fungus Podospora anserina requires the peroxisome receptor export machinery

Abstract

Peroxisomes are versatile organelles essential for diverse developmental processes. One such process is the meiotic development of Podospora anserina. In this fungus, absence of the docking peroxin PEX13, the RING-finger complex peroxins, or the PTS2 co-receptor PEX20 blocks sexual development before meiocyte formation. However, this defect is not seen in the absence of the receptors PEX5 and PEX7, or of the docking peroxins PEX14 and PEX14/17. Here we describe the function of the remaining uncharacterized P. anserina peroxins predictably involved in peroxisome matrix protein import. We show that PEX8, as well as the peroxins potentially mediating receptor monoubiquitination (PEX4 and PEX22) and membrane dislocation (PEX1, PEX6 and PEX26) are indeed implicated in peroxisome matrix protein import in this fungus. However, we observed that elimination of PEX4 and PEX22 affects to different extent the import of distinct PEX5 cargoes, suggesting differential ubiquitination-complex requirements for the import of distinct proteins. In addition, we found that elimination of PEX1, PEX6 or PEX26 results in loss of peroxisomes, suggesting that these peroxins restrain peroxisome removal in specific physiological conditions. Finally, we demonstrate that all analyzed peroxins are required for meiocyte formation, and that PEX20 function in this process depends on its potential monoubiquitination target cysteine. Our results suggest that meiotic induction relies on a peroxisome import pathway, which is not dependent on PEX5 or PEX7 but that is driven by an additional cycling receptor. These findings uncover a collection of peroxins implicated in modulating peroxisome activity to facilitate a critical developmental cell fate decision.

Distinct Contributions of the Peroxisome-Mitochondria Fission Machinery During Sexual Development of the Fungus Podospora anserina

“…FOX2 is a peroxisomal protein that lacks canonical peroxisometargeting signals (PTS), but whose import into peroxisomes is dependent on the import receptor PEX5. We have shown that FOX2 peroxisome targeting is not affected by the C-terminal tagging (Suaste-Olmos et al, 2018). In addition, for fis1 we corroborated our observations using an ectopically-encoded GFP possessing the consensus peroxisome-targeting signal 1 (GFP-PTS1).…”

“…Streptomyces noursei nat1 gene was obtained from plasmid pAPI509, a derivative from pAPI508 (El-Khoury et al, 2008). Escherichia coli hph gene was obtained from pBC-Hygro (Silar, 1995), P. anserina GPD (glyceraldehyde-3-phosphate dehydrogenase) gene promoter sequence from pPable (Coppin and Debuchy, 2000), and the mCherry-HygR and GFP-HygR cassettes from plasmids pUC-Cherry and pUC-GFP, respectively (Suaste-Olmos et al, 2018). Oligonucleotide primers used in this research are shown on Supplementary Table 2. Gene Sequences FIS1 (Pa_3_3970), DNM1 (Pa_1_12670), ATP9-7 (Pa_7_20), and IDH1 (Pa_1_5850) sequences were obtained from the P. anserina genome sequence (Espagne et al, 2008).…”

“…Nuclei and mtDNA were stained with DAPI (Molecular Probes) (0.5 µg mL −1 ). For livecell microscopy, hyphae from entire P. anserina colonies grown for 24 h at 27 • C on M2 agarose beds were imaged as described before (Suaste-Olmos et al, 2018). To avoid differences in the physiology of the analyzed hyphae due to storage or aging of the strains, the mycelial explants used to inoculate the analyzed colonies were always issued from young growing cultures (at incubation distances ≤3 cm from the point of inoculation of the ascospore yielding the strain).…”

“…Next, we studied the effect of FIS1 and DNM1 elimination in sexual development. We have shown that peroxisomes are required for karyogamy and meiotic development initiation in P. anserina (Berteaux-Lecellier et al, 1995;Peraza-Reyes et al, 2008Suaste-Olmos et al, 2018); therefore, we tested whether FIS1 and DNM1 elimination affected these processes. As shown before, fis1 and dnm1 are able to produce ascospores in homozygous crosses, implying that FIS1 and DNM1 are not strictly required for karyogamy of meiosis initiation.…”

“…On the other hand, this process involves precise modulation of peroxisome dynamics and biogenesis (Peraza-Reyes et al, 2011;Takano-Rojas et al, 2016). In addition, different stages of sexual development-including karyogamy, meiotic induction and progression-require specific peroxisome biogenesis factors (peroxins) (Berteaux-Lecellier et al, 1995;Bonnet et al, 2006;Peraza-Reyes et al, 2008Suaste-Olmos et al, 2018). Moreover, sexual development also relies on a regulatory system that prevents meiosis progression in absence of mitochondrial citrate synthase, which was identified by its genetic interactions with peroxisomal assembly PEX2 gene (Ruprich-Robert et al, 2002b), and specific peroxisome assembly mutants exhibit defects in mitochondrial morphology, which are consistent with altered mitochondrial fission (Bonnet et al, 2006).…”