Peroxisomal Import Pathway Research Articles

MrPEX33 is involved in infection-related morphogenesis and pathogenicity of Metarhizium robertsii

Peroxisomes, being indispensable organelles, play an important role in different biological processes in eukaryotes. PEX33, a filamentous fungus-specific peroxin of the docking machinery of peroxisomes, is involved in the virulence and development of other fungal pathogens. However, it is not clear whether PEX33 is necessary for the pathogenicity and development of an insect pathogenic fungus. In the present study, we report the presence of homologs of PEX33, namely MrPEX33 (MAA_05331), in the entomopathogenic fungus, Metarhizium robertsii. An M. robertsii transgenic strain expressing the fusion protein with MrPEX33-GFP and mCherry-PTS1 showed that MrPEX33 localizes to peroxisomes. The results also demonstrated that MrPEX33 is involved in the peroxisomal import pathway by peroxisomal targeting signals. Targeted gene deletion of MrPEX33 led to a significant decline in the asexual sporulation capacity, which was accompanied by downregulation of several conidiation-associated genes, such as wetA, abaA, and brlA. More importantly, our bioassay results showed that the virulence of ∆MrPEX33 mutants, against Galleria mellonella through cuticle infection, was greatly reduced. This was further accompanied by a significant drop in appressorium formation and cuticle penetration. Additionally, ∆MrPEX33 mutants showed a significant decrease in tolerance to cell wall integrity and oxidative stress. Taken together, our results suggest that MrPEX33 is involved in the cuticle infection-related morphogenesis and pathogenicity. KEY POINTS: • MrPEX33 is a specific peroxin of the docking machinery of peroxisomes. • MrPEX33 localizes to peroxisomes and is involved in the import of matrix proteins. • MrPEX33 is involved in the pathogenicity associated with cuticle infections.

Identification and Localization of Peroxisomal Biogenesis Proteins Indicates the Presence of Peroxisomes in the Cryptophyte Guillardia theta and Other "Chromalveolates".

Peroxisomes are single-membrane-bound organelles with a huge metabolic versatility, including the degradation of fatty acids (β-oxidation) and the detoxification of reactive oxygen species as most conserved functions. Although peroxisomes seem to be present in the majority of investigated eukaryotes, where they are responsible for many eclectic and important spatially separated metabolic reactions, knowledge about their existence in the plethora of protists (eukaryotic microorganisms) is scarce. Here, we investigated genomic data of organisms containing complex plastids with red algal ancestry (so-called “chromalveolates”) for the presence of genes encoding peroxins—factors specific for the biogenesis, maintenance, and division of peroxisomes in eukaryotic cells. Our focus was on the cryptophyte Guillardia theta, a marine microalga, which possesses two phylogenetically different nuclei of host and endosymbiont origin, respectively, thus being of enormous evolutionary significance. Besides the identification of a complete set of peroxins in G. theta, we heterologously localized selected factors as GFP fusion proteins via confocal and electron microscopy in the model diatom Phaeodactylum tricornutum. Furthermore, we show that peroxins, and thus most likely peroxisomes, are present in haptophytes as well as eustigmatophytes, brown algae, and alveolates including dinoflagellates, chromerids, and noncoccidian apicomplexans. Our results indicate that diatoms are not the only “chromalveolate” group devoid of the PTS2 receptor Pex7, and thus a PTS2-dependent peroxisomal import pathway, which seems to be absent in haptophytes (Emiliania huxleyi) as well. Moreover, important aspects of peroxisomal biosynthesis and protein import in “chromalveolates”are highlighted.

Towards designer organelles by subverting the peroxisomal import pathway

The development of ‘designer’ organelles could be a key strategy to enable foreign pathways to be efficiently controlled within eukaryotic biotechnology. A fundamental component of any such system will be the implementation of a bespoke protein import pathway that can selectively deliver constituent proteins to the new compartment in the presence of existing endogenous trafficking systems. Here we show that the protein–protein interactions that control the peroxisomal protein import pathway can be manipulated to create a pair of interacting partners that still support protein import in moss cells, but are orthogonal to the naturally occurring pathways. In addition to providing a valuable experimental tool to give new insights into peroxisomal protein import, the variant receptor-signal sequence pair forms the basis of a system in which normal peroxisomal function is downregulated and replaced with an alternative pathway, an essential first step in the creation of a designer organelle.

PTS1 Peroxisomal Import Pathway Plays Shared and Distinct Roles to PTS2 Pathway in Development and Pathogenicity of Magnaporthe oryzae

Peroxisomes participate in various important metabolisms and are required in pathogenicity of fungal plant pathogens. Peroxisomal matrix proteins are imported from cytoplasm into peroxisomes through peroxisomal targeting signal 1 (PTS1) or peroxisomal targeting signal 2 (PTS2) import pathway. PEX5 and PEX7 genes participate in the two pathways respectively. The involvement of PEX7 mediated PTS2 import pathway in fungal pathogenicity has been documented, while that of PTS1 remains unclear. Through null mutant analysis of MoPEX5, the PEX5 homolog in Magnaporthe oryzae, we report the crucial roles of PTS1 pathway in the development and host infection in the rice blast fungus, and compared with those of PTS2. We found that MoPEX5 disruption specifically blocked the PTS1 pathway. Δmopex5 was unable to use lipids as sole carbon source and lost pathogenicity completely. Similar as Δmopex7, Δmopex5 exhibited significant reduction in lipid utilization and mobilization, appressorial turgor genesis and H2O2 resistance. Additionally, Δmopex5 presented some distinct defects which were undetected in Δmopex7 in vegetative growth, conidial morphogenesis, appressorial morphogenesis and melanization. The results indicated that the PTS1 peroxisomal import pathway, in addition to PTS2, is required for fungal development and pathogenicity of the rice blast fungus, and also, as a main peroxisomal import pathway, played a more predominant role than PTS2.

Ubiquitination of the peroxisomal import receptor Pex5p is required for its recycling

Pex5p, which is the import receptor for peroxisomal matrix proteins harboring a type I signal sequence (PTS1), is mono- and polyubiquitinated in Saccharomyces cerevisiae. We identified Pex5p as a molecular target for Pex4p-dependent monoubiquitination and demonstrated that either poly- or monoubiquitination of the receptor is required for the ATP-dependent release of the protein from the peroxisomal membrane to the cytosol as part of the receptor cycle. Therefore, the energy requirement of the peroxisomal import pathway has to be extended by a second ATP-dependent step, namely receptor monoubiquitination.

The absence of Pmp47, a putative yeast peroxisomal transporter, causes a defect in transport and folding of a specific matrix enzyme.

Candida boidinii Pmp47, an integral peroxisomal membrane protein, belongs to a family of mitochondrial solute transporters (e.g., ATP/ADP exchanger), and is the only known peroxisomal member of this family. However, its physiological and biochemical functions have been unrevealed because of the difficulties in the molecular genetics of C. boidinii. In this study, we first isolated the PMP47 gene, which was the single gene encoding for Pmp47 in a gene-engineerable strain S2 of C. boidinii. Sequence analysis revealed that it was very similar to PMP47A and PMP47B genes from a polyploidal C. Boidinii strain (ATCC32195). Next, the PMP47 gene was disrupted and the disruption strain (pmp47delta) was analyzed. Depletion of PMP47 from strain S2 resulted in a retarded growth on oleate and a complete loss of growth on methanol. Both growth substrates require peroxisomal metabolism. EM observations revealed the presence of peroxisomes in methanol- and oleate-induced cells of pmp47delta, but in reduced numbers, and the presence of material of high electron density in the cytoplasm in both cases. Methanol-induced cells of pmp47delta were investigated in detail. The activity of one of the methanol-induced peroxisome matrix enzymes, dihydroxyacetone synthase (DHAS), was not detected in pmp47delta. Further biochemical and immunocytochemical experiments revealed that the DHAS protein aggregated in the cytoplasm as an inclusion body, while two other peroxisome matrix enzymes, alcohol oxidase (AOD) and catalase, were active and found in peroxisomes. Two peroxisome-deficient mutants, strains M6 and M13 (described in previous studies), retained DHAS activity although it was mislocalized to the cytoplasm and the nucleus. We disrupted PMP47 in these peroxisome-deficient mutants. In both strains, M6-pmp47delta and M13-pmp47delta, DHAS was enzymatically active and was located in the cytoplasm and the nucleus. We suggest that an unknown small molecule, which PMP47 transports, is necessary for the folding or the translocation machinery of DHAS within peroxisomes. Pmp47 does not catalyze folding directly because active DHAS is observed in the M6-pmp47delta and M13-pmp47delta strains. Since both AOD and DHAS have the PTS1 motif sequences at their carboxyl terminal, our results first show that depletion of Pmp47 could dissect the peroxisomal import pathway (PTS1 pathway) of these proteins.

PAS7 encodes a novel yeast member of the WD-40 protein family essential for import of 3-oxoacyl-CoA thiolase, a PTS2-containing protein, into peroxisomes.

To identify components of the peroxisomal import pathway in yeast, we have isolated pas mutants affected in peroxisome biogenesis. Two mutants assigned to complementation group 7 define a new gene, PAS7, whose product is necessary for import of thiolase, a PTS2-containing protein, but not for that of SKL (PTS1)-containing proteins, into peroxisomes. We have cloned PAS7 by complementation of the oleic acid non-utilizing phenotype of the pas7-1 strain. The DNA sequence predicts a 42.3 kDa polypeptide of 375 amino acids encoding a novel member of the beta-transducin related (WD-40) protein family. A Myc epitope-tagged Pas7p, expressed under the control of the CUP1 promotor, was functionally active. Subcellular localization studies revealed that in the presence of thiolase this epitope-tagged Pas7p in part associates with peroxisomes. However, in a thiolase-deficient mutant, Pas7p was entirely found in the cytoplasm. We suggest that Pas7p mediates the binding of thiolase to these organelles.

Primary hyperoxaluria type 1 and peroxisome-to-mitochondrion mistargeting of alanine: glyoxylate aminotransferase

Under the influence of dietary selection pressure, the intracellular compartmentalization of alanine:glyoxylate amino-transferase (AGT) has changed on many occasions during the evolution of mammals. In some mammals, AGT is peroxisomal in others it is mainly mitochondrial while in yet others it is more-or-less equally divided between both organelles. Although in normal human liver AGT is usually found exclusively within the peroxisomes, in some individuals a small proportion (≈5%) is found also in the mitochondria. This apparently trivial intracellular redistribution of AGT is caused by the presence of a Pro 11Leu polymorphism which allows the N-terminus of AGT to fold into a conformation ( ie a positively-charged amphiphilic α-helix) which functions as a mitochondrial targeting sequence. In one third of patients with the autosomal recessive disease primary hyperoxaluria type 1, there is a further redistribution of AGT so that the great majority (≈90%) is located in the mitochondria and only a small minority (10%) in the peroxisomes. AGT cannot fulfil its proper metabolic role in human liver ( ie glyoxylate detoxification) when located in the mitochondria. This erroneous compartmentalization is due to the presence of a Gly 170Arg mutation superimposed upon the Pro 11Leu polymorphism. The Gly 170Arg mutation appears to have no direct effect on mitochondrial targeting and is predicted tlo enhance mitochondrial important of AGT by interfering with its peroxisomal targeting and/or import. The mitochondrial targeting sequence generated by the Pro 11Leu polymorphism is not homologous to that found in the AGT of other mammals which localise AGT within the mitochondria normally. The identity of the peroxisomal targeting sequence in AGT is unkown, but the Gly 170Arg mutation is found in a highly conserved region of the protein which might be involved in some aspect of the peroxisomal import pathway for AGT.