The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Yukio Fujiki - One of the best experts on this subject based on the ideXlab platform.
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peroxisomes control mitochondrial dynamics and the mitochondrion dependent apoptosis pathway
Journal of Cell Science, 2019Co-Authors: Hideaki Tanaka, Yukio Fujiki, Tomohiko Okazaki, Saeko Aoyama, Mutsumi Yokota, Masato Koike, Yasushi Okada, Yukiko GotohAbstract:Peroxisomes cooperate with mitochondria in the performance of cellular metabolic functions, such as fatty acid oxidation and the maintenance of redox homeostasis. However, whether peroxisomes also regulate mitochondrial fission-fusion dynamics or mitochondrion-dependent apoptosis remained unclear. We now show that genetic ablation of the peroxins Pex3 or PEX5, which are essential for peroxisome biogenesis, results in mitochondrial fragmentation in mouse embryonic fibroblasts (MEFs) in a manner dependent on Drp1 (also known as DNM1L). Conversely, treatment with 4-PBA, which results in peroxisome proliferation, resulted in mitochondrial elongation in wild-type MEFs, but not in Pex3-knockout MEFs. We further found that peroxisome deficiency increased the levels of cytosolic cytochrome c and caspase activity under basal conditions without inducing apoptosis. It also greatly enhanced etoposide-induced caspase activation and apoptosis, which is indicative of an enhanced cellular sensitivity to death signals. Taken together, our data unveil a previously unrecognized role for peroxisomes in the regulation of mitochondrial dynamics and mitochondrion-dependent apoptosis. Effects of peroxin gene mutations on mitochondrion-dependent apoptosis may contribute to pathogenesis of peroxisome biogenesis disorders.This article has an associated First Person interview with the first author of the paper.
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identification of peroxisomal protein complexes with pts receptors PEX5 and pex7 in mammalian cells
Sub-cellular biochemistry, 2018Co-Authors: Kanji Okumoto, Non Miyata, Yukio FujikiAbstract:PEX5 and Pex7 are cytosolic receptors for peroxisome targeting signal type-1 (PTS1) and type-2 (PTS2), respectively, and play a pivotal role in import of peroxisomal matrix proteins. Recent advance in mass spectrometry analysis has facilitated comprehensive analysis of protein-protein interaction network by a combination with immunoprecipitation or biochemical purification. In this chapter, we introduce several findings obtained by these methods applied to mammalian cells. Exploring PEX5-binding partners in mammalian cells revealed core components comprising the import machinery complex of matrix proteins and a number of PTS1-type cargo proteins. Biochemical purification of the PEX5-export stimulating factor from rat liver cytosol fraction identified Awp1, providing further insight into molecular mechanisms of the export step of mono-ubiquitinated PEX5. Identification of DDB1 (damage-specific DNA-binding protein 1), a component of CRL4 (Cullin4A-RING ubiquitin ligase) E3 complex, as a Pex7-interacting protein revealed that quality control of Pex7 by CRL4A is important for PTS2 protein import by preventing the accumulation of dysfunctional Pex7. Furthermore, analysis of binding partners of an intraperoxisomal processing enzyme, trypsin-domain containing 1 (Tysnd1), showed a protein network regulating peroxisomal fatty acid β-oxidation.
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localization of protein kinase ndr2 to peroxisomes and its role in ciliogenesis
Journal of Biological Chemistry, 2017Co-Authors: Tomoaki Nagai, Yukio Fujiki, Kanji Okumoto, Moe Masukawa, Yuta Homma, Kensaku MizunoAbstract:Abstract Nuclear Dbf2-related (NDR) kinases, comprising NDR1 and NDR2, are serine/threonine kinases that play crucial roles in the control of cell proliferation, apoptosis, and morphogenesis. We recently showed that NDR2, but not NDR1, is involved in primary cilium formation; however, the mechanism underlying their functional difference in ciliogenesis is unknown. To address this issue, we examined their subcellular localization. Despite their close sequence similarity, NDR2 exhibited punctate localization in the cytoplasm, whereas NDR1 was diffusely distributed within the cell. Notably, NDR2 puncta mostly co-localized with the peroxisome marker proteins, catalase and CFP-SKL (cyan fluorescent protein carrying the C-terminal typical peroxisome-targeting signal type-1 (PTS1) sequence, Ser-Lys-Leu). NDR2 contains the PTS1-like sequence, Gly-Lys-Leu, at the C-terminal end, whereas the C-terminal end of NDR1 is Ala-Lys. An NDR2 mutant lacking the C-terminal Leu, NDR2(ΔL), exhibited almost diffuse distribution in cells. Additionally, NDR2, but neither NDR1 nor NDR2(ΔL), bound to the PTS1 receptor PEX5p. Together, these findings indicate that NDR2 localizes to the peroxisome by using the C-terminal GKL sequence. Intriguingly, topology analysis of NDR2 suggests that NDR2 is exposed to the cytosolic surface of the peroxisome. The expression of wild-type NDR2, but not NDR2(ΔL), recovered the suppressive effect of NDR2 knockdown on ciliogenesis. Furthermore, knockdown of peroxisome biogenesis factor genes (PEX1 or PEX3 ) partially suppressed ciliogenesis. These results suggest that the peroxisomal localization of NDR2 is implicated in its function to promote primary cilium formation.
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PEX5p stabilizes pex14p a study using a newly isolated PEX5 cho cell mutant zpeg101
Biochemical Journal, 2013Co-Authors: Ryuichi Natsuyama, Kanji Okumoto, Yukio FujikiAbstract:PEX5p [PTS (peroxisome-targeting signal) type 1 receptor] plays an essential role in peroxisomal matrix protein import. In the present study, we isolated a novel PEX5 -deficient CHO (Chinese-hamster ovary) cell mutant, termed ZPEG101, showing typical peroxisomal import defects of both PTS1 and PTS2 proteins. ZPEG101 is distinct from other known PEX5 CHO mutants in its PEX5p expression. An undetectable level of PEX5p in ZPEG101 results in unstable Pex14p, which is due to inefficient translocation to the peroxisomal membrane. All of the mutant phenotypes of ZPEG101 are restored by expression of wild-type PEX5pL, a longer form of PEX5p, suggesting a role for PEX5p in sustaining the levels of Pex14p in addition to peroxisomal matrix protein import. Complementation analysis using various PEX5p mutants revealed that in the seven pentapeptide WXXXF/Y motifs in PEX5pL, known as the multiple binding sites for Pex14p, the fifth motif is an auxiliary binding site for Pex14p and is required for Pex14p stability. Furthermore, we found that PEX5p–Pex13p interaction is essential for the import of PTS1 proteins as well as catalase, but not for that of PTS2 proteins. Therefore ZPEG101 with no PEX5p would be a useful tool for investigating PEX5p function and delineating the mechanisms underlying peroxisomal matrix protein import.
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awp1 zfand6 functions in PEX5 export by interacting with cys monoubiquitinated PEX5 and pex6 aaa atpase
Traffic, 2012Co-Authors: Non Miyata, Kanji Okumoto, Satoru Mukai, Masafumi Noguchi, Yukio FujikiAbstract:During biogenesis of the peroxisome, a subcellular organelle, the peroxisomal-targeting signal 1 (PTS1) receptor PEX5 functions as a shuttling receptor for PTS1-containing peroxisomal matrix proteins. However, the precise mechanism of receptor shuttling between peroxisomes and cytosol remains elusive despite the identification of numerous peroxins involved in this process. Herein, a new factor was isolated by a combination of biochemical fractionation and an in vitro PEX5 export assay, and was identified as AWP1/ZFAND6, a ubiquitin-binding NF-κB modulator. In the in vitro PEX5 export assay, recombinant AWP1 stimulated PEX5 export and an anti-AWP1 antibody interfered with PEX5 export. AWP1 interacted with Pex6 AAA ATPase, but not with Pex1-Pex6 complexes. Preferential binding of AWP1 to the cysteine-ubiquitinated form of PEX5 rather than to unmodified PEX5 was mediated by the AWP1 A20 zinc-finger domain. Inhibition of AWP1 by RNA interference had a significant effect on PTS1-protein import into peroxisomes. Furthermore, in AWP1 knock-down cells, PEX5 stability was decreased, similar to fibroblasts from patients defective in Pex1, Pex6 and Pex26, all of which are required for PEX5 export. Taken together, these results identify AWP1 as a novel cofactor of Pex6 involved in the regulation of PEX5 export during peroxisome biogenesis.
Bonnie Bartel - One of the best experts on this subject based on the ideXlab platform.
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a PEX5 missense allele preferentially disrupts pts1 cargo import into arabidopsis peroxisomes
Plant direct, 2019Co-Authors: Khushali J Patel, Yunting Kao, Roxanna J Llinas, Bonnie BartelAbstract:The sorting of eukaryotic proteins to various organellar destinations requires receptors that recognize cargo protein targeting signals and facilitate transport into the organelle. One such receptor is the peroxin PEX5, which recruits cytosolic cargo carrying a peroxisome-targeting signal (PTS) type 1 (PTS1) for delivery into the peroxisomal lumen (matrix). In plants and mammals, PEX5 is also indirectly required for peroxisomal import of proteins carrying a PTS2 signal because PEX5 binds the PTS2 receptor, bringing the associated PTS2 cargo to the peroxisome along with PTS1 cargo. Despite PEX5 being the PTS1 cargo receptor, previously identified Arabidopsis PEX5 mutants display either impairment of both PTS1 and PTS2 import or defects only in PTS2 import. Here we report the first Arabidopsis PEX5 mutant with an exclusive PTS1 import defect. In addition to markedly diminished GFP-PTS1 import and decreased PEX5-2 protein accumulation, this PEX5-2 mutant shows typical peroxisome-related defects, including inefficient β-oxidation and reduced growth. Growth at reduced or elevated temperatures ameliorated or exacerbated PEX5-2 peroxisome-related defects, respectively, without markedly changing PEX5-2 protein levels. In contrast to the diminished PTS1 import, PTS2 processing was only slightly impaired and PTS2-GFP import appeared normal in PEX5-2. This finding suggests that even minor peroxisomal localization of the PTS1 protein DEG15, the PTS2-processing protease, is sufficient to maintain robust PTS2 processing.
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A pex1 missense mutation improves peroxisome function in a subset of Arabidopsis pex6 mutants without restoring PEX5 recycling.
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Kim L. Gonzalez, Sarah E. Ratzel, Kendall H. Burks, Charles H. Danan, Jeanne M. Wages, Bethany K. Zolman, Bonnie BartelAbstract:Peroxisomes are eukaryotic organelles critical for plant and human development because they house essential metabolic functions, such as fatty acid β-oxidation. The interacting ATPases PEX1 and PEX6 contribute to peroxisome function by recycling PEX5, a cytosolic receptor needed to import proteins targeted to the peroxisomal matrix. Arabidopsis pex6 mutants exhibit low PEX5 levels and defects in peroxisomal matrix protein import, oil body utilization, peroxisomal metabolism, and seedling growth. These defects are hypothesized to stem from impaired PEX5 retrotranslocation leading to PEX5 polyubiquitination and consequent degradation of PEX5 via the proteasome or of the entire organelle via autophagy. We recovered a pex1 missense mutation in a screen for second-site suppressors that restore growth to the pex6-1 mutant. Surprisingly, this pex1-1 mutation ameliorated the metabolic and physiological defects of pex6-1 without restoring PEX5 levels. Similarly, preventing autophagy by introducing an atg7-null allele partially rescued pex6-1 physiological defects without restoring PEX5 levels. atg7 synergistically improved matrix protein import in pex1-1 pex6-1, implying that pex1-1 improves peroxisome function in pex6-1 without impeding autophagy of peroxisomes (i.e., pexophagy). pex1-1 differentially improved peroxisome function in various pex6 alleles but worsened the physiological and molecular defects of a pex26 mutant, which is defective in the tether anchoring the PEX1-PEX6 hexamer to the peroxisome. Our results support the hypothesis that, beyond PEX5 recycling, PEX1 and PEX6 have additional functions in peroxisome homeostasis and perhaps in oil body utilization.
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disparate peroxisome related defects in arabidopsis pex6 and pex26 mutants link peroxisomal retrotranslocation and oil body utilization
Plant Journal, 2017Co-Authors: Kim L. Gonzalez, Wendell A Fleming, Yunting Kao, Zachary J Wright, Savina V Venkova, Meredith J Ventura, Bonnie BartelAbstract:Catabolism of fatty acids stored in oil bodies is essential for seed germination and seedling development in Arabidopsis. This fatty acid breakdown occurs in peroxisomes, organelles that sequester oxidative reactions. Import of peroxisomal enzymes is facilitated by peroxins including PEX5, a receptor that delivers cargo proteins from the cytosol to the peroxisomal matrix. After cargo delivery, a complex of the PEX1 and PEX6 ATPases and the PEX26 tail-anchored membrane protein removes ubiquitinated PEX5 from the peroxisomal membrane. We identified Arabidopsis pex6 and pex26 mutants by screening for inefficient seedling β-oxidation phenotypes. The mutants displayed distinct defects in growth, response to a peroxisomally metabolized auxin precursor, and peroxisomal protein import. The low PEX5 levels in these mutants were increased by treatment with a proteasome inhibitor or by combining pex26 with peroxisome-associated ubiquitination machinery mutants, suggesting that ubiquitinated PEX5 is degraded by the proteasome when the function of PEX6 or PEX26 is reduced. Combining pex26 with mutations that increase PEX5 levels either worsened or improved pex26 physiological and molecular defects, depending on the introduced lesion. Moreover, elevating PEX5 levels via a 35S:PEX5 transgene exacerbated pex26 defects and ameliorated the defects of only a subset of pex6 alleles, implying that decreased PEX5 is not the sole molecular deficiency in these mutants. We found peroxisomes clustered around persisting oil bodies in pex6 and pex26 seedlings, suggesting a role for peroxisomal retrotranslocation machinery in oil body utilization. The disparate phenotypes of these pex alleles may reflect unanticipated functions of the peroxisomal ATPase complex.
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elevated growth temperature decreases levels of the PEX5 peroxisome targeting signal receptor and ameliorates defects of arabidopsis mutants with an impaired pex4 ubiquitin conjugating enzyme
BMC Plant Biology, 2015Co-Authors: Yunting Kao, Bonnie BartelAbstract:Peroxisomes house critical metabolic reactions. For example, fatty acid β-oxidation enzymes, which are essential during early seedling development, are peroxisomal. Peroxins (PEX proteins) are needed to bring proteins into peroxisomes. Most matrix proteins are delivered to peroxisomes by PEX5, a receptor that forms transient pores to escort proteins across the peroxisomal membrane. After cargo delivery, a peroxisome-tethered ubiquitin-conjugating enzyme (PEX4) and peroxisomal ubiquitin-protein ligases mono- or polyubiquitinate PEX5 for recycling back to the cytosol or for degradation, respectively. Arabidopsis pex mutants β-oxidize fatty acids inefficiently and therefore fail to germinate or grow less vigorously. These defects can be partially alleviated by providing a fixed carbon source, such as sucrose, in the growth medium. Despite extensive characterization of peroxisome biogenesis in Arabidopsis grown in non-challenged conditions, the effects of environmental stressors on peroxisome function and pex mutant dysfunction are largely unexplored. We surveyed the impact of growth temperature on a panel of pex mutants and found that elevated temperature ameliorated dependence on external sucrose and reduced PEX5 levels in the pex4-1 mutant. Conversely, growth at low temperature exacerbated pex4-1 physiological defects and increased PEX5 levels. Overexpressing PEX5 also worsened pex4-1 defects, implying that PEX5 lingering on the peroxisomal membrane when recycling is impaired impedes peroxisome function. Growth at elevated temperature did not reduce the fraction of membrane-associated PEX5 in pex4-1, suggesting that elevated temperature did not restore PEX4 enzymatic function in the mutant. Moreover, preventing autophagy in pex4-1 did not restore PEX5 levels at high temperature. In contrast, MG132 treatment increased PEX5 levels, implicating the proteasome in degrading PEX5, especially at high temperature. We conclude that growth at elevated temperature increases proteasomal degradation of PEX5 to reduce overall PEX5 levels and ameliorate pex4-1 physiological defects. Our results support the hypothesis that efficient retrotranslocation of PEX5 after cargo delivery is needed not only to make PEX5 available for further rounds of cargo delivery, but also to prevent the peroxisome dysfunction that results from PEX5 lingering in the peroxisomal membrane.
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a viable arabidopsis pex13 missense allele confers severe peroxisomal defects and decreases PEX5 association with peroxisomes
Plant Molecular Biology, 2014Co-Authors: Sarah E. Ratzel, Wendell A Fleming, Andrew Woodward, Sarah E Burkhart, Marta Bjornson, Bonnie BartelAbstract:Peroxisomes are organelles that catabolize fatty acids and compartmentalize other oxidative metabolic processes in eukaryotes. Using a forward-genetic screen designed to recover severe peroxisome-defective mutants, we isolated a viable allele of the peroxisome biogenesis gene PEX13 with striking peroxisomal defects. The pex13-4 mutant requires an exogenous source of fixed carbon for pre-photosynthetic development and is resistant to the protoauxin indole-3-butyric acid. Delivery of peroxisome-targeted matrix proteins depends on the PEX5 receptor docking with PEX13 at the peroxisomal membrane, and we found severely reduced import of matrix proteins and less organelle-associated PEX5 in pex13-4 seedlings. Moreover, pex13-4 physiological and molecular defects were partially ameliorated when PEX5 was overexpressed, suggesting that PEX5 docking is partially compromised in this mutant and can be improved by increasing PEX5 levels. Because previously described Arabidopsis pex13 alleles either are lethal or confer only subtle defects, the pex13-4 mutant provides valuable insight into plant peroxisome receptor docking and matrix protein import.
Paul A M Michels - One of the best experts on this subject based on the ideXlab platform.
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ubiquitination of the glycosomal matrix protein receptor PEX5 in trypanosoma brucei by pex4 displays novel features
Biochimica et Biophysica Acta, 2013Co-Authors: Melisa Gualdronlopez, Pierre J Courtoy, Nathalie Chevalier, Patrick Van Der Smissen, Daniel J Rigden, Paul A M MichelsAbstract:Trypanosomatids contain peroxisome-like organelles called glycosomes. Peroxisomal biogenesis involves a cytosolic receptor, PEX5, which, after its insertion into the organellar membrane, delivers proteins to the matrix. In yeasts and mammalian cells, transient PEX5 monoubiquitination at the membrane serves as the signal for its retrieval from the organelle for re-use. When its recycling is impaired, PEX5 is polyubiquitinated for proteasomal degradation. Stably monoubiquitinated TbPEX5 was detected in cytosolic fractions of Trypanosoma brucei, indicative for its role as physiological intermediate in receptor recycling. This modification's resistance to dithiothreitol suggests ubiquitin conjugation of a lysine residue. T. brucei PEX4, the functional homologue of the ubiquitin-conjugating (UBC) enzyme responsible for PEX5 monoubiquitination in yeast, was identified. It is associated with the cytosolic face of the glycosomal membrane, probably anchored by an identified putative TbPEX22. The involvement of TbPEX4 in TbPEX5 ubiquitination was demonstrated using procyclic ∆PEX4 trypanosomes. Surprisingly, glycosomal matrix protein import was only mildly affected in this mutant. Since other UBC homologues were upregulated, it might be possible that these have partially rescued PEX4's function in PEX5 ubiquitination. In addition, the altered expression of UBCs, notably of candidates involved in cell-cycle control, could be responsible for observed morphological and motility defects of the ∆PEX4 mutant.
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processing of the glycosomal matrix protein import receptor PEX5 of trypanosoma brucei
Biochemical and Biophysical Research Communications, 2013Co-Authors: Melisa Gualdronlopez, Paul A M MichelsAbstract:Glycolysis in kinetoplastid protists such as Trypanosoma brucei is compartmentalized in peroxisome-like organelles called glycosomes. Glycosomal matrix-protein import involves a cytosolic receptor, PEX5, which recognizes the peroxisomal-targeting signal type 1 (PTS1) present at the C-terminus of the majority of matrix proteins. PEX5 appears generally susceptible to in vitro proteolytic processing. On western blots of T. brucei, two PEX5 forms are detected with apparent M(r) of 100kDa and 72kDa. 5'-RACE-PCR showed that TbPEX5 is encoded by a unique transcript that can be translated into a protein of maximally 72kDa. However, recombinant PEX5 migrates aberrantly in SDS-PAGE with an apparent M(r) of 100kDa, similarly as observed for the native peroxin. In vitro protease susceptibility analysis of native and (35)S-labelled PEX5 showed truncation of the 100kDa form at the N-terminal side by unknown parasite proteases, giving rise to the 72kDa form which remains functional for PTS1 binding. The relevance of these observations is discussed.
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Characterization of the role of the receptors PEX5 and PEX7 in the import of proteins into glycosomes of Trypanosoma brucei.
Biochimica et Biophysica Acta, 2007Co-Authors: Nathalie Galland, Véronique Hannaert, Emilie Verplaetse, Patrick Van Der Smissen, Fabian Demeure, Didier Vertommen, Pierre J Courtoy, Paul A M MichelsAbstract:Peroxins 5 and 7 are receptors for protein import into the peroxisomal matrix. We studied the involvement of these peroxins in the biogenesis of glycosomes in the protozoan parasite Trypanosoma brucei. Glycosomes are peroxisome-like organelles in which a major part of the glycolytic pathway is sequestered. We here report the characterization of the T. brucei homologue of PEX7 and provide several data strongly suggesting that it can bind to PEX5. Depletion of PEX5 or PEX7 by RNA interference had a severe effect on the growth of both the bloodstream-form of the parasite, that relies entirely on glycolysis for its ATP supply, and the procyclic form representative of the parasite living in the tsetse-fly midgut and in which also other metabolic pathways play a prominent role. The role of the two receptors in import of glycosomal matrix proteins with different types of peroxisome/glycosome-targeting signals (PTS) was analyzed by immunofluorescence and subcellular fractionation studies. Knocking down the expression of either receptor gene resulted, in procyclic cells, in the mislocalization of proteins with both a type 1 or 2 targeting motif (PTS1, PTS2) located at the C- and N-termini, respectively, and proteins with a sequence-internal signal (I-PTS) to the cytosol. Electron microscopy confirmed the apparent integrity of glycosomes in these procyclic cells. In bloodstream-form trypanosomes, PEX7 depletion seemed to affect only the subcellular distribution of PTS2-proteins. Western blot analysis suggested that, in both life-cycle stages of the trypanosome, the levels of both receptors are controlled in a coordinated fashion, by a mechanism that remains to be determined. The observation that both PEX5 and PEX7 are essential for the viability of the parasite indicates that the respective branches of the glycosome-import pathway in which each receptor acts might be interesting drug targets.
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analysis of the sequence motifs responsible for the interactions of peroxins 14 and 5 which are involved in glycosome biogenesis in trypanosoma brucei
Biochemistry, 2003Co-Authors: Jungwoo Choe, Juliette Moyersoen, Claudia Roach, Tyan L Carter, Paul A M MichelsAbstract:Glycosome biogenesis in trypanosomatids occurs via a process that is homologous to peroxisome biogenesis in other eukaryotes. Glycosomal matrix proteins are synthesized in the cytosol and imported posttranslationally. The import process involves a series of protein-protein interactions starting by recognition of glycosomal matrix proteins by a receptor in the cytosol. Most proteins to be imported contain so-called PTS-1 or PTS-2 targeting sequences recognized by, respectively, the receptor proteins PEX5 and PEX7. PEX14, a protein associated with the peroxisomal membrane, has been identified as a component of the docking complex and a point of convergence of the PEX5- and PEX7-dependent import pathways. In this paper, the strength of the interactions between Trypanosoma brucei PEX14 and PEX5 was studied by a fluorescence assay, using (i) a panel of N-terminal regions of TbPEX14 protein variants and (ii) a series of different peptides derived from TbPEX5, each containing one of the three WXXXF/Y motifs present in this receptor protein. On the PEX14 side, the N-terminal region of TbPEX14 including residues 1-84 appeared to be responsible for TbPEX5 binding. The results from PEX14 mutants identified specific residues in the N-terminal region of TbPEX14 involved in PEX5 binding and showed that in particular hydrophobic residues F35 and F52 are critical. On the PEX5 side, 13-mer peptides incorporating the first or the third WXXXF/Y motif bind to PEX14 with an affinity in the nanomolar range. However, the second WXXXF/Y motif peptide did not show any detectable affinity. Studies using variants of second and third motif peptides suggest that the alpha-helical content of the peptides as well as the charge of a residue at position 9 in the motif may be important for PEX14 binding. Assays with 7-, 10-, 13-, and 16-mer third motif peptides showed that 16-mers and 13-mers have comparable binding affinity for PEX14, whereas 10-mers and 7-mers have about 10- and 100-fold lower affinity than the 16-mers, respectively. The low sequence identities of PEX14 and PEX5 between parasite and its human host, and the vital importance of proper glycosome biogenesis to the parasite, render these peroxins highly promising drug targets.
Wolfgang Schliebs - One of the best experts on this subject based on the ideXlab platform.
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membrane interactions of the peroxisomal proteins PEX5 and pex14
Frontiers in Cell and Developmental Biology, 2021Co-Authors: Stefan Gaussmann, Wolfgang Schliebs, Ralf Erdmann, Mohanraj Gopalswamy, Maike Eberhardt, Maren Reuter, Peijian Zou, Michael SattlerAbstract:Human PEX5 and PEX14 are essential components of the peroxisomal translocon, which mediates import of cargo enzymes into peroxisomes. PEX5 is a soluble receptor for cargo enzymes comprised of an N-terminal intrinsically disordered domain (NTD) and a C-terminal tetratricopeptide (TPR) domain, which recognizes peroxisomal targeting signal 1 (PTS1) peptide motif in cargo proteins. The PEX5 NTD harbors multiple WF peptide motifs (WxxxF/Y or related motifs) that are recognized by a small globular domain in the NTD of the membrane-associated protein PEX14. How the PEX5 or PEX14 NTDs bind to the peroxisomal membrane and how the interaction between the two proteins is modulated at the membrane is unknown. Here, we characterize the membrane interactions of the PEX5 NTD and PEX14 NTD in vitro by membrane mimicking bicelles and nanodiscs using NMR spectroscopy and isothermal titration calorimetry. The PEX14 NTD weakly interacts with membrane mimicking bicelles with a surface that partially overlaps with the WxxxF/Y binding site. The PEX5 NTD harbors multiple interaction sites with the membrane that involve a number of amphipathic α-helical regions, which include some of the WxxxF/Y-motifs. The partially formed α-helical conformation of these regions is stabilized in the presence of bicelles. Notably, ITC data show that the interaction between the PEX5 and PEX14 NTDs is largely unaffected by the presence of the membrane. The PEX5/PEX14 interaction exhibits similar free binding enthalpies, where reduced binding enthalpy in the presence of bicelles is compensated by a reduced entropy loss. This demonstrates that docking of PEX5 to PEX14 at the membrane does not reduce the overall binding affinity between the two proteins, providing insights into the initial phase of PEX5-PEX14 docking in the assembly of the peroxisome translocon.
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Editorial Manager(tm) for Molecular Cell Manuscript Draft Manuscript Number: MC-D-06-00911R2 Title: Recognition of a functional peroxisome type 1 target by the dynamic import receptor PEX5p Article Type: Research Manuscript
2014Co-Authors: Will A. Stanley, Wolfgang Schliebs, Michael Sattler, Nicole Schuller, Petri Kursula, Matthias WilmannsAbstract:Abstract: Peroxisomes require the translocation of folded and functional target proteins of various sizes across the peroxisomal membrane. We have investigated the structure and function of the principal import receptor PEX5p, which recognizes targets bearing a C-terminal peroxisomal targeting signal type 1. Crystal structures of the receptor in the presence and absence of a peroxisomal target, sterol carrier protein 2, reveal major structural changes from an open, snail-like conformation into a closed, circular conformation. These changes are caused by a long loop C-terminal to the seven-fold tetratricopeptide repeat segments. Mutations in residues of this loop lead to defects in peroxisomal import in human fibroblasts. The structure of the receptor/cargo complex demonstrates that the primary receptor binding site of the cargo is structurally and topologically autonomous, enabling the cargo to retain its native structure and function. * C) Manuscript Recognition of a functional peroxisome type 1 target by the dynamic import receptor PEX5
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structural basis for competitive interactions of pex14 with the import receptors PEX5 and pex19
The EMBO Journal, 2009Co-Authors: Christian Neufeld, Ralf Erdmann, Alexander Neuhaus, Hamed Kooshapur, Tobias Madl, Fabian V Filipp, Bernd Simon, Nicole Schuller, Christine David, Wolfgang SchliebsAbstract:Protein import into peroxisomes depends on a complex and dynamic network of protein–protein interactions. Pex14 is a central component of the peroxisomal import machinery and binds the soluble receptors PEX5 and Pex19, which have important function in the assembly of peroxisome matrix and membrane, respectively. We show that the N-terminal domain of Pex14, Pex14(N), adopts a three-helical fold. PEX5 and Pex19 ligand helices bind competitively to the same surface in Pex14(N) albeit with opposite directionality. The molecular recognition involves conserved aromatic side chains in the PEX5 WxxxF/Y motif and a newly identified F/YFxxxF sequence in Pex19. The Pex14–PEX5 complex structure reveals molecular details for a critical interaction in docking PEX5 to the peroxisomal membrane. We show that mutations of Pex14 residues located in the PEX5/Pex19 binding region disrupt PEX5 and/or Pex19 binding in vitro. The corresponding full-length Pex14 variants are impaired in peroxisomal membrane localisation in vivo, showing that the molecular interactions mediated by the N-terminal domain modulate peroxisomal targeting of Pex14.
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functional similarity between the peroxisomal pts2 receptor binding protein pex18p and the n terminal half of the pts1 receptor PEX5p
Molecular and Cellular Biology, 2004Co-Authors: Antje Schafer, Wolf H. Kunau, Daniela Kerssen, Marten Veenhuis, Wolfgang SchliebsAbstract:Within the extended receptor cycle of peroxisomal matrix import, the function of the import receptor PEX5p comprises cargo recognition and transport. While the C-terminal half (PEX5p-C) is responsible for PTS1 binding, the contribution of the N-terminal half of PEX5p (PEX5p-N) to the receptor cycle has been less clear. Here we demonstrate, using different techniques, that in Saccharomyces cerevisiae PEX5p-N alone facilitates the import of the major matrix protein Fox1p. This finding suggests that PEX5p-N is sufficient for receptor docking and cargo transport into peroxisomes. Moreover, we found that PEX5p-N can be functionally replaced by Pex18p, one of two auxiliary proteins of the PTS2 import pathway. A chimeric protein consisting of Pex18p (without its Pex7p binding site) fused to PEX5p-C is able to partially restore PTS1 protein import in a PEX5 deletion strain. On the basis of these results, we propose that the auxiliary proteins of the PTS2 import pathway fulfill roles similar to those of the N-terminal half of PEX5p in the PTS1 import pathway.
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the di aromatic pentapeptide repeats of the human peroxisome import receptor PEX5 are separate high affinity binding sites for the peroxisomal membrane protein pex14
Journal of Biological Chemistry, 2001Co-Authors: Jurgen Saidowsky, Gabriele Dodt, Kristina Kirchberg, Wolfgang Nastainczyk, Wolf H. Kunau, Albrecht Wegner, Wolfgang SchliebsAbstract:Abstract PEX5 functions as a mobile import receptor for peroxisomal matrix proteins with a peroxisomal targeting signal 1 (PTS1). A critical step within the PTS1-import pathway is the interaction between PEX5 and the peroxisome membrane-associated protein PEX14. Based on two-hybrid analyses in mammalian cells and complementary in vitro binding assays, we demonstrate that the evolutionarily conserved pentapeptide repeat motifs, WX(E/D/Q/A/S)(E/D/Q)(F/Y), in PEX5 bind to PEX14 with high affinity. The results obtained indicate that each of the seven di-aromatic pentapeptides of human PEX5 interacts separately at the same binding site in the N terminus of PEX14 with equilibrium dissociation constants in the low nanomolar range. Mutational analysis of the PEX14-binding motifs reveals that the conserved aromatic amino acids at position 1 or 5 are essential for high affinity binding. We propose that the side chains of the aromatic amino acids are in close proximity as part of an amphipathic α-helix and together form hydrophobic anchors for binding PEX5 to individual PEX14 molecules.
Kanji Okumoto - One of the best experts on this subject based on the ideXlab platform.
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identification of peroxisomal protein complexes with pts receptors PEX5 and pex7 in mammalian cells
Sub-cellular biochemistry, 2018Co-Authors: Kanji Okumoto, Non Miyata, Yukio FujikiAbstract:PEX5 and Pex7 are cytosolic receptors for peroxisome targeting signal type-1 (PTS1) and type-2 (PTS2), respectively, and play a pivotal role in import of peroxisomal matrix proteins. Recent advance in mass spectrometry analysis has facilitated comprehensive analysis of protein-protein interaction network by a combination with immunoprecipitation or biochemical purification. In this chapter, we introduce several findings obtained by these methods applied to mammalian cells. Exploring PEX5-binding partners in mammalian cells revealed core components comprising the import machinery complex of matrix proteins and a number of PTS1-type cargo proteins. Biochemical purification of the PEX5-export stimulating factor from rat liver cytosol fraction identified Awp1, providing further insight into molecular mechanisms of the export step of mono-ubiquitinated PEX5. Identification of DDB1 (damage-specific DNA-binding protein 1), a component of CRL4 (Cullin4A-RING ubiquitin ligase) E3 complex, as a Pex7-interacting protein revealed that quality control of Pex7 by CRL4A is important for PTS2 protein import by preventing the accumulation of dysfunctional Pex7. Furthermore, analysis of binding partners of an intraperoxisomal processing enzyme, trypsin-domain containing 1 (Tysnd1), showed a protein network regulating peroxisomal fatty acid β-oxidation.
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localization of protein kinase ndr2 to peroxisomes and its role in ciliogenesis
Journal of Biological Chemistry, 2017Co-Authors: Tomoaki Nagai, Yukio Fujiki, Kanji Okumoto, Moe Masukawa, Yuta Homma, Kensaku MizunoAbstract:Abstract Nuclear Dbf2-related (NDR) kinases, comprising NDR1 and NDR2, are serine/threonine kinases that play crucial roles in the control of cell proliferation, apoptosis, and morphogenesis. We recently showed that NDR2, but not NDR1, is involved in primary cilium formation; however, the mechanism underlying their functional difference in ciliogenesis is unknown. To address this issue, we examined their subcellular localization. Despite their close sequence similarity, NDR2 exhibited punctate localization in the cytoplasm, whereas NDR1 was diffusely distributed within the cell. Notably, NDR2 puncta mostly co-localized with the peroxisome marker proteins, catalase and CFP-SKL (cyan fluorescent protein carrying the C-terminal typical peroxisome-targeting signal type-1 (PTS1) sequence, Ser-Lys-Leu). NDR2 contains the PTS1-like sequence, Gly-Lys-Leu, at the C-terminal end, whereas the C-terminal end of NDR1 is Ala-Lys. An NDR2 mutant lacking the C-terminal Leu, NDR2(ΔL), exhibited almost diffuse distribution in cells. Additionally, NDR2, but neither NDR1 nor NDR2(ΔL), bound to the PTS1 receptor PEX5p. Together, these findings indicate that NDR2 localizes to the peroxisome by using the C-terminal GKL sequence. Intriguingly, topology analysis of NDR2 suggests that NDR2 is exposed to the cytosolic surface of the peroxisome. The expression of wild-type NDR2, but not NDR2(ΔL), recovered the suppressive effect of NDR2 knockdown on ciliogenesis. Furthermore, knockdown of peroxisome biogenesis factor genes (PEX1 or PEX3 ) partially suppressed ciliogenesis. These results suggest that the peroxisomal localization of NDR2 is implicated in its function to promote primary cilium formation.
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PEX5p stabilizes pex14p a study using a newly isolated PEX5 cho cell mutant zpeg101
Biochemical Journal, 2013Co-Authors: Ryuichi Natsuyama, Kanji Okumoto, Yukio FujikiAbstract:PEX5p [PTS (peroxisome-targeting signal) type 1 receptor] plays an essential role in peroxisomal matrix protein import. In the present study, we isolated a novel PEX5 -deficient CHO (Chinese-hamster ovary) cell mutant, termed ZPEG101, showing typical peroxisomal import defects of both PTS1 and PTS2 proteins. ZPEG101 is distinct from other known PEX5 CHO mutants in its PEX5p expression. An undetectable level of PEX5p in ZPEG101 results in unstable Pex14p, which is due to inefficient translocation to the peroxisomal membrane. All of the mutant phenotypes of ZPEG101 are restored by expression of wild-type PEX5pL, a longer form of PEX5p, suggesting a role for PEX5p in sustaining the levels of Pex14p in addition to peroxisomal matrix protein import. Complementation analysis using various PEX5p mutants revealed that in the seven pentapeptide WXXXF/Y motifs in PEX5pL, known as the multiple binding sites for Pex14p, the fifth motif is an auxiliary binding site for Pex14p and is required for Pex14p stability. Furthermore, we found that PEX5p–Pex13p interaction is essential for the import of PTS1 proteins as well as catalase, but not for that of PTS2 proteins. Therefore ZPEG101 with no PEX5p would be a useful tool for investigating PEX5p function and delineating the mechanisms underlying peroxisomal matrix protein import.
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awp1 zfand6 functions in PEX5 export by interacting with cys monoubiquitinated PEX5 and pex6 aaa atpase
Traffic, 2012Co-Authors: Non Miyata, Kanji Okumoto, Satoru Mukai, Masafumi Noguchi, Yukio FujikiAbstract:During biogenesis of the peroxisome, a subcellular organelle, the peroxisomal-targeting signal 1 (PTS1) receptor PEX5 functions as a shuttling receptor for PTS1-containing peroxisomal matrix proteins. However, the precise mechanism of receptor shuttling between peroxisomes and cytosol remains elusive despite the identification of numerous peroxins involved in this process. Herein, a new factor was isolated by a combination of biochemical fractionation and an in vitro PEX5 export assay, and was identified as AWP1/ZFAND6, a ubiquitin-binding NF-κB modulator. In the in vitro PEX5 export assay, recombinant AWP1 stimulated PEX5 export and an anti-AWP1 antibody interfered with PEX5 export. AWP1 interacted with Pex6 AAA ATPase, but not with Pex1-Pex6 complexes. Preferential binding of AWP1 to the cysteine-ubiquitinated form of PEX5 rather than to unmodified PEX5 was mediated by the AWP1 A20 zinc-finger domain. Inhibition of AWP1 by RNA interference had a significant effect on PTS1-protein import into peroxisomes. Furthermore, in AWP1 knock-down cells, PEX5 stability was decreased, similar to fibroblasts from patients defective in Pex1, Pex6 and Pex26, all of which are required for PEX5 export. Taken together, these results identify AWP1 as a novel cofactor of Pex6 involved in the regulation of PEX5 export during peroxisome biogenesis.
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molecular anatomy of the peroxin pex12p ring finger domain is essential for pex12p function and interacts with the peroxisome targeting signal type 1 receptor PEX5p and a ring peroxin pex10p
Journal of Biological Chemistry, 2000Co-Authors: Kanji Okumoto, Isao Abe, Yukio FujikiAbstract:The three peroxin genes, PEX12, PEX2, and PEX10, encode peroxisomal integral membrane proteins with RING finger at the C-terminal part and are responsible for human peroxisome biogenesis disorders. Mutation analysis in PEX12 of Chinese hamster ovary cell mutants revealed a homozygous nonsense mutation at residue Trp263Ter in ZP104 cells and a pair of heterozygous nonsense mutations, Trp170Ter and Trp114Ter, in ZP109. This result and domain mapping of Pex12p showed that RING finger is essential for peroxisome-restoring activity of Pex12p but not necessary for targeting to peroxisomes. The N-terminal region of Pex12p, including amino acid residues at positions 17-76, was required for localization to peroxisomes, while the sequence 17-76 was not sufficient for peroxisomal targeting. Peroxins interacting with RING finger of Pex2p, Pex10p, and Pex12p were investigated by yeast two-hybrid as well as in vitro binding assays. The RING finger of Pex12p bound to Pex10p and the PTS1-receptor PEX5p. Pex10p also interacted with Pex2p and PEX5p in vitro. Moreover, Pex12p was co-immunoprecipitated with Pex10p from CHO-K1 cells, where PEX5p was not associated with the Pex12p-Pex10p complex. This observation suggested that PEX5p does not bind to, or only transiently interacts with, Pex10p and Pex12p when Pex10p and Pex12p are in the oligomeric complex in peroxisome membranes. Hence, the RING finger peroxins are most likely to be involved in PEX5p-mediated matrix protein import into peroxisomes.