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In an in silico search for correlated gene loss with fungal peroxisomal uric acid oxidase (UOX), we identified PMP22-like proteins, some of which function as promiscuous channels in organellar membranes. To investigate whether PMP22 channels have a role in peroxisomal uric acid transport and catabolism, we functionally analyzed the closest homologue in Aspergillus nidulans, named SspA. We confirmed that SspA is a peroxisomal membrane protein that co-localizes significantly with PTS1-tagged mRFP, UOX or HexA, the latter considered a protein of Woronin bodies (WB), organelles originating from peroxisomes that dynamically plug septal pores in ascomycetes. Our results suggest that in A. nidulans, unlike some other ascomycetes, there is no strict protein segregation of peroxisomal and WB-specific proteins. Importantly, genetic deletion of sspA, but not of hexA, led to lack of peroxisomal localization at septal pores, suggesting that SspA is a key factor for septal pore functioning. Additionally, ΔsspA resulted in increased sensitivity to oxidative stress, apparently as a consequence of not only the inability to plug septal pores, but also a recorded reduction in peroxisome biogenesis. However, deleting sspA had no effect on uric acid or purine utilization, as we hypothesized, a result also in line with the observation that expression of SspA was not affected by regulatory mutants and conditions known to control purine catabolic enzymes. Our results are discussed within the framework of previous studies of SspA homologues in other fungi, as well as, the observed gene losses of PMP22 and peroxisomal uric acid oxidase.

Subcellular localization is emerging as an important mechanism for mTORC1 regulation. We report that the tuberous sclerosis complex (TSC) signalling node, TSC1, TSC2 and Rheb, localizes to peroxisomes, where it regulates mTORC1 in response to reactive oxygen species (ROS). TSC1 and TSC2 were bound by peroxisomal biogenesis factors 19 and 5 (PEX19 and PEX5), respectively, and peroxisome-localized TSC functioned as a Rheb GTPase-activating protein (GAP) to suppress mTORC1 and induce autophagy. Naturally occurring pathogenic mutations in TSC2 decreased PEX5 binding, and abrogated peroxisome localization, Rheb GAP activity and suppression of mTORC1 by ROS. Cells lacking peroxisomes were deficient in mTORC1 repression by ROS, and peroxisome-localization-deficient TSC2 mutants caused polarity defects and formation of multiple axons in neurons. These data identify a role for the TSC in responding to ROS at the peroxisome, and identify the peroxisome as a signalling organelle involved in regulation of mTORC1.

Mammalian gene expression constructs are generally prepared in a plasmid vector, in which a promoter and terminator are located upstream and downstream of a protein-coding sequence, respectively. In this study, we found that front terminator constructs-DNA constructs containing a terminator upstream of a promoter rather than downstream of a coding region-could sufficiently express proteins as a result of end joining of the introduced DNA fragment. By taking advantage of front terminator constructs, FLAG substitutions, and deletions were generated using mutagenesis primers to identify amino acids specifically recognized by commercial FLAG antibodies. A minimal epitope sequence for polyclonal FLAG antibody recognition was also identified. In addition, we analyzed the sequence of a C-terminal Ser-Lys-Leu peroxisome localization signal, and identified the key residues necessary for peroxisome targeting. Moreover, front terminator constructs of hepatitis B surface antigen were used for deletion analysis, leading to the identification of regions required for the particle formation. Collectively, these results indicate that front terminator constructs allow for easy manipulations of C-terminal protein-coding sequences, and suggest that direct gene expression with PCR-amplified DNA is useful for high-throughput protein analysis in mammalian cells.

BMC evolutionary biology 15(1), 64 (2015). doi:10.1186/s12862-015-0337-x, Published by BioMed Central, London

The biological functions of human acyl-CoA thioesterase III (ACTEIII/PTE-1), initially identified as an HIV-1 Nef binding protein, have remained unclear. We report herein that the stable overexpression of ACTEIII/PTE-1 in human and murine T-cell lines resulted in an increase in both peroxisome number and lipid droplet formation in a manner dependent on the amount of the protein. Peroxisome proliferation was evidenced by immunofluorescence staining for catalase, a peroxisome marker protein, as well as by direct peroxisome enumeration on electron micrographs. Consistently, the amount of catalase was elevated as the amount of ACTEIII/PTE-1 was increased. ACTEIII/PTE-1 mutants with reduced enzymatic activity or with the defect in peroxisome localization did not induce peroxisome proliferation, indicating that peroxisome proliferation was mediated by metabolites generated by ACTEIII/PTE-1 within peroxisomes. Finally, thymocytes isolated from a T-cell-specific ACTEIII/PTE-1 transgenic mouse as well as human and murine cell lines of lymphoid and non-lymphoid origins exhibited a similar proliferation of peroxisomes. Thus, ACTEIII/PTE-1 may be involved in the metabolic regulation of peroxisome proliferation.

The filamentous fungus Alternaria alternata includes seven pathogenic variants (pathotypes) which produce different host-selective toxins and cause diseases on different plants. The Japanese pear pathotype produces the host-selective AK-toxin, an epoxy-decatrienoic acid ester, and causes black spot of Japanese pear. Previously, we identified four genes, AKT1 , AKT2 , AKT3 , and AKTR , involved in AK toxin biosynthesis. AKT1 , AKT2 , and AKT3 encode enzyme proteins with peroxisomal targeting signal type 1 (PTS1)-like tripeptides, SKI, SKL, and PKL, respectively, at the C-terminal ends. In this study, we verified the peroxisome localization of Akt1, Akt2, and Akt3 by using strains expressing N-terminal green fluorescent protein (GFP)-tagged versions of the proteins. To assess the role of peroxisome function in AK-toxin production, we isolated Aa PEX6 , which encodes a peroxin protein essential for peroxisome biogenesis, from the Japanese pear pathotype and made Aa PEX6 disruption-containing transformants from a GFP-Akt1-expressing strain. The ΔAa PEX6 mutant strains did not grow on fatty acid media because of a defect in fatty acid β oxidation. The import of GFP-Akt1 into peroxisomes was impaired in the ΔAa PEX6 mutant strains. These strains completely lost AK toxin production and pathogenicity on susceptible pear leaves. These data show that peroxisomes are essential for AK-toxin biosynthesis. The ΔAa PEX6 mutant strains showed a marked reduction in the ability to cause lesions on leaves of a resistant pear cultivar with defense responses compromised by heat shock. This result suggests that peroxisome function is also required for plant invasion and tissue colonization in A. alternata . We also observed that mutation of Aa PEX6 caused a marked reduction of conidiation.

International audience; Peroxisomes are involved in a variety of metabolic pathways and developmental processes. In the filamentous fungus Podospora anserina, absence of different peroxins implicated in peroxisome matrix protein import leads to different developmental defects. Lack of the RING-finger complex peroxin PEX2 blocks sexual development at the dikaryotic stage, while in absence of both receptors, PEX5 and PEX7, karyogamy and meiosis can proceed and sexual spores are formed. This suggests a complex role for PEX2 that prompted us to study the developmental involvement of the RING-finger complex. We show that, like PEX2, the two other proteins of the complex, PEX10 and PEX12, are equally implicated in peroxisome biogenesis and that absence of each or all these proteins lead to the same developmental defect. Moreover, we demonstrate that peroxisome localization of PEX2 is not drastically affected in the absence of PEX10 and PEX12 and that the upregulation of these latter RING-finger peroxins does not compensate for the lack of a second one, suggesting that the three proteins work together in development but independent of their function in peroxisome biogenesis.

One of the proteins targeted to the peridinin plastid of the dinoflagellate Lingulodinium polyedrum is the iron-containing superoxide dismutase (LpSOD). Like dinoflagellate plastid proteins of class II, LpSOD carries a bipartite presequence comprising a signal peptide followed by a transit peptide. Our bioinformatic studies suggest that its signal peptide is atypical, however, and that the entire presequence may function as a mitochondrial targeting signal. It is possible that LpSOD represents a new class of proteins in algae with complex plastids, which are co-targeted to the plastid and mitochondrion. In addition to the ambiguous N-terminal targeting signal, LpSOD contains a potential type-1 peroxisome-targeting signal (PTS1) located at its C-terminus. In accordance with a peroxisome localization of this dismutase, its mRNA has two in-frame AUG codons. Our bioinformatic analyses indicate that the first start codon resides in a much weaker oligonucleotide context than the second one. This suggests that synthesis of the plastid/mitochondrion-targeted and peroxisome-targeted isoforms could proceed through so-called leaky scanning. Moreover, our results show that expression of the two isoforms could be regulated by a 'hairpin' structure located between the first and second start codons.