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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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peroxisome biogenesis disorders molecular basis for impaired peroxisomal membrane assembly in metabolic functions and biogenesis of peroxisomes in health and disease
Biochimica et Biophysica Acta, 2012Co-Authors: Yukio Fujiki, Yuichi Yagita, Takashi MatsuzakiAbstract:Peroxisome is a single-membrane organelle in eukaryotes. The functional importance of peroxisomes in humans is highlighted by peroxisome-deficient peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome (ZS). Gene defects of Peroxins required for both membrane assembly and matrix protein import are identified: ten mammalian pathogenic Peroxins for ten complementation groups of PBDs, are required for matrix protein import; three, Pex3p, Pex16p and Pex19p, are shown to be essential for peroxisome membrane assembly and responsible for the most severe ZS in PBDs of three complementation groups 12, 9, and 14, respectively. Patients with severe ZS with defects of PEX3, PEX16, and PEX19 tend to carry severe mutation such as nonsense mutations, frameshifts and deletions. With respect to the function of these three Peroxins in membrane biogenesis, two distinct pathways have been proposed for the import of peroxisomal membrane proteins in mammalian cells: a Pex19p- and Pex3p-dependent class I pathway and a Pex19p- and Pex16p-dependent class II pathway. In class II pathway, Pex19p also forms a soluble complex with newly synthesized Pex3p as the chaperone for Pex3p in the cytosol and directly translocates it to peroxisomes. Pex16p functions as the peroxisomal membrane receptor that is specific to the Pex3p-Pex19p complexes. A model for the import of peroxisomal membrane proteins is suggested, providing new insights into the molecular mechanisms underlying the biogenesis of peroxisomes and its regulation involving Pex3p, Pex19p, and Pex16p. Another model suggests that in Saccharomyces cerevisiae peroxisomes likely emerge from the endoplasmic reticulum.
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characterization of the interaction between recombinant human Peroxin pex3p and pex19p identification of trp 104 in pex3p as a critical residue for the interaction
Journal of Biological Chemistry, 2008Co-Authors: Yasuhiko Sato, Yukio Fujiki, Yuji Matsuzono, Hiroyuki Shibata, Hiroaki Nakano, Yoshinori Kashiwayama, Yuji Kobayashi, Tsuneo Imanaka, Hiroaki KatoAbstract:Abstract Proteins required for peroxisome biogenesis are termed Peroxins. The Peroxin Pex3p is a peroxisomal membrane protein (PMP), involved in peroxisomal membrane biogenesis. It acts as a docking receptor for another Peroxin Pex19p, which is a specific carrier protein for newly synthesized PMPs. Here we have determined the physicochemical properties and binding manners of Pex3p-Pex19p interaction, in terms of the affinity, the stoichiometry, and the binding site in Pex3p. The cytosolic domain of human Pex3p was overproduced, using an Escherichia coli expression system and was highly purified by two chromatography steps. Gel filtration chromatography analyses and intrinsic tryptophan fluorescence titrations revealed that a one-to-one complex is formed between monomeric Pex3p and monomeric Pex19p. The tryptophan fluorescence spectrum of Pex3p showed a large 18-nm blue shift of the maximum emission wavelength by the binding of Pex19p. This result indicates that either one or two tryptophan residues of Pex3p (Trp-104 and Trp-224) are directly involved in binding to Pex19p. We investigated the binding activities of the wild-type and tryptophan mutants of Pex3p by pull-down assays and surface plasmon resonance analyses. As a result, the wild-type and the W104A and W104F mutants showed KD values of 3.4 nm, 1080 nm, and 66.2 nm, respectively. The affinity differences with mutation affected their peroxisome restoring activities in pex3 ZPG208 cells. These findings suggest that the indole ring of Trp-104 directly interacts with Pex19p to facilitate the specific peroxisomal translocation of the Pex19p-PMP complexes.
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functional domain mapping of Peroxin pex19p interaction with pex3p is essential for function and translocation
Journal of Cell Science, 2006Co-Authors: Yuji Matsuzono, Takashi Matsuzaki, Yukio FujikiAbstract:The Peroxin Pex19p functions in peroxisomal membrane assembly. Here we mapped functional domains of human Pex19p comprising 299 amino acids. Pex19p mutants deleted in the C-terminal CAAx farnesylation motif, the C-terminal 38 amino acid residues and the N-terminal 11 residues, maintained peroxisome-restoring activity in pex19 cells. The sequence 12-261 was essential for re-establishing peroxisome activity. Pex19p was partly localized to peroxisomes but mostly localized in the cytosol. Pex19p interacted with multiple membrane proteins, including the other two membrane biogenesis Peroxins, Pex3p and Pex16p, those involved in matrix protein import such as Pex14p, Pex13p, Pex10p, and Pex26p, peroxisome morphogenesis factor Pex11pbeta, and a PMP70 peroxisome-targeting signal region at residues 1-123. In yeast two-hybrid assays, Pex10p and Pex11pbeta interacted only with full-length Pex19p. Of various truncated Pex19p variants active in translocating to peroxisomes, the mutants with the shortest sequence (residues 12-73 and 40-131) were localized to peroxisomes and competent in binding to Pex3p. Furthermore, membrane Peroxins were initially discernible in a cytosolic staining pattern in pex19 cells only when co-expressed with Pex19p and were then localized to peroxisomes in a temporally differentiated manner. Pex19p probably functions as a chaperone for membrane proteins and transports them to peroxisomes by anchoring to Pex3p using residues 12-73 and 40-131.
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functional domains and dynamic assembly of the Peroxin pex14p the entry site of matrix proteins
Journal of Biological Chemistry, 2006Co-Authors: Ryota Itoh, Yukio FujikiAbstract:Abstract The 41-kDa membrane-anchored Peroxin Pex14p functions as the peroxisome targeting signal (PTS) receptor-mediated, initial import site for matrix proteins. We here identify the functional domains of Pex14p involved in the assembly of import site subcomplexes. The minimal region of Pex14p required for restoring impaired protein import in pex14 Chinese hamster ovary cell mutant lies at residues 21-260 in the primary sequence. A highly conserved N-terminal region, encompassing residues 21-70, interacts with the PTS1 receptor Pex5p, Pex13p, and Pex19p that is essential for membrane biogenesis. N-terminal residues 21-140, including a hydrophobic segment at 110-138, function as a topo-genic sequence. Site-directed mutagenesis, size fractionation, and chemical cross-linking analyses demonstrate that the coiled-coil domain at residues 156-197 regulates homodimerization of Pex14p. Moreover, AXXXA and GXXXG motifs in the transmembrane segment mediate homomeric oligomerization of Pex14p, giving rise to assembly of high molecular mass complexes and thereby assuring Pex13p-dependent localization of Pex14p to peroxisomes. Pex5p, Pex13p, and Pex19p bind to Pex14p homo-oligomers with different molecular masses, whereas cargo-unloaded Pex5p apparently disassembles Pex14p homo-oligomers. Thus, Pex14p most likely forms several distinct Peroxin complexes involved in peroxisomal matrix protein import.
Armando Jardim - One of the best experts on this subject based on the ideXlab platform.
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the hydrophobic region of the leishmania Peroxin 14 requirements for association with a glycosome mimetic membrane
Biochemical Journal, 2017Co-Authors: Terry K Smith, Louisphilippe Leroux, Anwar Hasil Kottarampatel, Amanda Davidsen, Christian Salesse, Elodie Boisselier, Armando JardimAbstract:Protein import into the Leishmania glycosome requires docking of the cargo loaded Peroxin 5(PEX5) receptor to the Peroxin 14(PEX14) bound to the glycosome surface. To examine the LdPEX14-membrane interaction, we purified L. donovani promastigote glycosomes and determined the phospholipid and fatty acid composition. These membranes contained predominately phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol modified primarily with C18 and C22 unsaturated fatty acid. Using large unilamellar vesicles (LUVs) with a lipid composition mimicking the glycosomal membrane in combination with sucrose density centrifugation and fluorescence activated cell sorting technique we established that the LdPEX14 membrane binding activity was dependent on a predicted transmembrane helix found within residues 149-179. Monolayer experiments showed that the incorporation of phosphatidylglycerol and phospholipids with unsaturated fatty acids, which increase membrane fluidity and favor a liquid expanded phase, facilitated the penetration of LdPEX14 into biological membranes. Moreover, we demonstrated that the binding of LdPEX5 receptor or LdPEX5-PTS1 receptor-cargo complex was contingent on the presence of LdPEX14 at the surface of LUVs.
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the hydrophobic region of the leishmania Peroxin 14 requirements for association with a glycosome mimetic membrane
Biochemical Journal, 2017Co-Authors: Normand Cyr, Louisphilippe Leroux, Anwar Hasil Kottarampatel, Amanda Davidsen, Christian Salesse, Terry K Smith, Elodie Boisselier, Armando JardimAbstract:Protein import into the Leishmania glycosome requires docking of the cargo-loaded Peroxin 5 (PEX5) receptor to the Peroxin 14 (PEX14) bound to the glycosome surface. To examine the LdPEX14-membrane interaction, we purified L. donovani promastigote glycosomes and determined the phospholipid and fatty acid composition. These membranes contained predominately phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol (PG) modified primarily with C18 and C22 unsaturated fatty acid. Using large unilamellar vesicles (LUVs) with a lipid composition mimicking the glycosomal membrane in combination with sucrose density centrifugation and fluorescence-activated cell sorting technique, we established that the LdPEX14 membrane-binding activity was dependent on a predicted transmembrane helix found within residues 149-179. Monolayer experiments showed that the incorporation of PG and phospholipids with unsaturated fatty acids, which increase membrane fluidity and favor a liquid expanded phase, facilitated the penetration of LdPEX14 into biological membranes. Moreover, we demonstrated that the binding of LdPEX5 receptor or LdPEX5-PTS1 receptor-cargo complex was contingent on the presence of LdPEX14 at the surface of LUVs.
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the leishmania donovani Peroxin 14 binding domain accommodates a high degeneracy in the pentapeptide motifs present on Peroxin 5
Biochimica et Biophysica Acta, 2015Co-Authors: Hamed Hojjat, Armando JardimAbstract:Abstract Background The glycosome is a unique organelle found in Kinetoplastids known to compartmentalize vital metabolic pathways including glycolysis, β-fatty acid oxidation and purine salvage. Organelle biogenesis depends on a network of proteins for trafficking and translocation of nascent protein into the glycosome. The interaction of the proteins LdPEX14 and LdPEX5 at the glycosome membrane is crucial for targeting proteins into this organelle. Methods Deletion mutagenesis, pull-down, and bacterial two hybrid assay were used to map the LdPEX5 domain bound by LdPEX14. ELISA assays, ITC, intrinsic fluorescence and size exclusion chromatography to monitor binding and structural changes associated with the LdPEX5–LdPEX14 interaction. Results and conclusions The LdPEX14 binding site was mapped to residues 280–300 on LdPEX5, a region containing the pentapeptide motif W 293 AQEY 297 . Deletion of this region abolished the LdPEX5–LdPEX14 interaction. Intrinsic fluorescence spectroscopy suggests that the stabilization of the LdPEX5–LdPEX14 complex is dependent on W293 docking into a hydrophobic pocket within the binding domain of ldpex14. Studies using a panel of synthetic peptides suggest a critical role for Y297 and to a lesser extent E296 in stabilizing the LdPEX5–LdPEX14 association. General significance We show that the LdPEX14 binding site is more promiscuous and in contrast to other eukaryotic systems will accommodate a more degenerate pentapeptide motif with the sequences WXXXW or FXXXF, findings which may be exploited for potential drug design.
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identification of leishmania donovani Peroxin 14 residues required for binding the Peroxin 5 receptor proteins
Biochemical Journal, 2015Co-Authors: Hamed Hojjat, Armando JardimAbstract:Trafficking of peroxisomal targeting signal 1 (PTS1) proteins to the Leishmania glycosome is dependent on the docking of the LdPEX5 receptor to LdPEX14 on the glycosomal membrane. A combination of deletion and random mutagenesis was used to identify residues in the LdPEX14 N-terminal region that are critical for mediating the LdPEX5–LdPEX14 interaction. These studies highlighted residues 35–75 on ldpex14 as the core domain required for binding LdPEX5. Single point mutation within this core domain generally did not affect the ldpex5-(203–391)–ldpex14-(1–120) interaction; notable exceptions were substitutions at Phe 40 , Val 46 or Phe 57 which completely abolished or increased the apparent K d value for ldpex5-(203–391) binding 30-fold. Biochemical studies revealed that these point mutations did not alter either the secondary or quaternary structure of LdPEX14 and indicated that the latter residues were critical for stabilizing the LdPEX5–LdPEX14 interaction.
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Analysis of the Leishmania Peroxin 7 interactions with Peroxin 5, Peroxin 14 and PTS2 ligands
The Biochemical journal, 2014Co-Authors: Ana Victoria C. Pilar, Hamed Hojjat, Rona Strasser, James Mclean, Elizabeth Quinn, Normand Cyr, Anwer Hasil Kottarampatel, Armando JardimAbstract:LPEX7 (Leishmania Peroxin 7) is essential for targeting newly synthesized proteins with a PTS2 (peroxisome-targeting signal type 2) import signal into the glycosome. In the present paper, we describe the biophysical characterization of a functional LPEX7 isolated from Escherichia coli inclusion bodies. Pull-down assays showed that LPEX7 binds the interacting partners LdPEX5 (Leishmania donovani Peroxin 5) and LdPEX14, but, more importantly, this receptor can specifically bind PTS2 cargo proteins in the monomeric and dimeric states. However, in the absence of interacting partners, LPEX7 preferentially adopts a tetrameric structure. Mapping studies localized the LdPEX5- and LdPEX14-binding sites to the N-terminal portion of LPEX7. Deletion of the first 52 residues abolished LdPEX14 association without altering the LdPEX5 interaction. Intrinsic fluorescence techniques suggested that each LPEX7 subunit has a single unique binding site for each of the respective interacting partners LdPEX5, LdPEX14 and PTS2 cargo proteins. Extrinsic fluorescence studies with ANS (8-anilinonaphthalene-1-sulfonic acid) demonstrated that LPEX7 contains a surface-exposed hydrophobic region(s) that was not altered by the binding of a PTS2 protein or LdPEX5. However, in the presence of these ligands, the accessibility of the hydrophobic domain was dramatically restricted, suggesting that both ligands are necessary to induce notable conformational changes in LPEX7. In contrast, binding of LdPEX14 did not alter the hydrophobic domain on LPEX7. It is possible that the hydrophobic surfaces on LPEX7 may be a crucial characteristic for the shuttling of this receptor in and out of the glycosome.
Richard A. Rachubinski - One of the best experts on this subject based on the ideXlab platform.
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a small molecule inhibitor of pex3 pex19 interaction disrupts glycosome biogenesis and causes lethality in trypanosoma brucei
Frontiers in Cell and Developmental Biology, 2021Co-Authors: Hiren Banerjee, Paul Lapointe, Gary Eitzen, Richard A. RachubinskiAbstract:Trypanosomatid parasites, including Trypanosoma and Leishmania, are infectious zoonotic agents for a number of severe diseases such as African sleeping sickness and American trypanosomiasis (Chagas disease) that affect millions of people, mostly in the emergent world. The glycosome is a specialized member of the peroxisome family of organelles found in trypanosomatids. These organelles compartmentalize essential enzymes of the glycolytic pathway, making them a prime target for drugs that can kill these organisms by interfering with either their biochemical functions or their formation. Glycosome biogenesis, like peroxisome biogenesis, is controlled by a group of proteins called Peroxins (Pex). Pex3 is an early acting Peroxin that docks Pex19, the receptor for peroxisomal membrane proteins, to initiate biogenesis of peroxisomes from the endoplasmic reticulum. Identification of Pex3 as the essential master regulator of glycosome biogenesis has implications in developing small molecule inhibitors that can impede Pex3-Pex19 interaction. Low amino acid sequence conservation between trypanosomatid Pex3 and human Pex3 (HsPex3) would aid in the identification of small molecule inhibitors that selectively interfere with the trypanosomatid Pex3-Pex19 interaction. We tested a library of pharmacologically active compounds in a modified yeast two-hybrid assay and identified a compound that preferentially inhibited the interaction of Trypanosoma brucei Pex3 and Pex19 versus HsPex3 and Pex19. Addition of this compound to either the insect or bloodstream form of T. brucei disrupted glycosome biogenesis, leading to mislocalization of glycosomal enzymes to the cytosol and lethality for the parasite. Our results show that preferential disruption of trypanosomal Pex3 function by small molecule inhibitors could help in the accelerated development of drugs for the treatment of trypanosomiases.
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A Small Molecule Inhibitor of Pex3–Pex19 Interaction Disrupts Glycosome Biogenesis and Causes Lethality in Trypanosoma brucei
'Frontiers Media SA', 2021Co-Authors: Hiren Banerjee, Paul Lapointe, Gary Eitzen, Richard A. RachubinskiAbstract:Trypanosomatid parasites, including Trypanosoma and Leishmania, are infectious zoonotic agents for a number of severe diseases such as African sleeping sickness and American trypanosomiasis (Chagas disease) that affect millions of people, mostly in the emergent world. The glycosome is a specialized member of the peroxisome family of organelles found in trypanosomatids. These organelles compartmentalize essential enzymes of the glycolytic pathway, making them a prime target for drugs that can kill these organisms by interfering with either their biochemical functions or their formation. Glycosome biogenesis, like peroxisome biogenesis, is controlled by a group of proteins called Peroxins (Pex). Pex3 is an early acting Peroxin that docks Pex19, the receptor for peroxisomal membrane proteins, to initiate biogenesis of peroxisomes from the endoplasmic reticulum. Identification of Pex3 as the essential master regulator of glycosome biogenesis has implications in developing small molecule inhibitors that can impede Pex3–Pex19 interaction. Low amino acid sequence conservation between trypanosomatid Pex3 and human Pex3 (HsPex3) would aid in the identification of small molecule inhibitors that selectively interfere with the trypanosomatid Pex3–Pex19 interaction. We tested a library of pharmacologically active compounds in a modified yeast two-hybrid assay and identified a compound that preferentially inhibited the interaction of Trypanosoma brucei Pex3 and Pex19 versus HsPex3 and Pex19. Addition of this compound to either the insect or bloodstream form of T. brucei disrupted glycosome biogenesis, leading to mislocalization of glycosomal enzymes to the cytosol and lethality for the parasite. Our results show that preferential disruption of trypanosomal Pex3 function by small molecule inhibitors could help in the accelerated development of drugs for the treatment of trypanosomiases
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Supplemental Material for Di Cara, Rachubinski and Simmonds, 2018
2018Co-Authors: Francesca Di Cara, Richard A. Rachubinski, Andrew SimmondsAbstract:Supplemental Material for Di Cara et al. 2018 Distinct Roles for Peroxisomal Targeting Signal Receptors Pex5 and Pex7 in DrosophilaFigure S1 - Additional quantification of larval size, cell proliferation, Non-esterified fatty acid levels and locomotory activity for Peroxin 7 mutant flies. Figure S2 Comparison of the Drosophila Peroxin7 protein sequence to human, zebrafish, Arabidobsis and yeast.
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pex11 related proteins in peroxisome dynamics a role for the novel Peroxin pex27p in controlling peroxisome size and number in saccharomyces cerevisiae
Molecular Biology of the Cell, 2003Co-Authors: Yuen Yi C Tam, Jennifer J. Smith, Marcello Marelli, Juan C Torresguzman, Franco J Vizeacoumar, John D Aitchison, Richard A. RachubinskiAbstract:Transcriptome profiling identified the gene PEX25 encoding Pex25p, a peroxisomal membrane Peroxin required for the regulation of peroxisome size and maintenance in Saccharomyces cerevisiae. Pex25p is related to a protein of unknown function encoded by the open reading frame, YOR193w, of the S. cerevisiae genome. Yor193p is a peripheral peroxisomal membrane protein that exhibits high sequence similarity not only to Pex25p but also to the peroxisomal membrane Peroxin Pex11p. Unlike Pex25p and Pex11p, Yor193p is constitutively expressed in wild-type cells grown in oleic acid-containing medium, the metabolism of which requires intact peroxisomes. Cells deleted for the YOR193w gene show a few enlarged peroxisomes. Peroxisomes are greatly enlarged in cells harboring double deletions of the YOR193w and PEX25 genes, the YOR193w and PEX11 genes, and the PEX25 and PEX11 genes. Yeast two-hybrid analyses showed that Yor193p interacts with Pex25p and itself, Pex25p interacts with Yor193p and itself, and Pex11p interacts only with itself. Overexpression of YOR193w, PEX25, or PEX11 led to peroxisome proliferation and the formation of small peroxisomes. Our data suggest a role for Yor193p, renamed Pex27p, in controlling peroxisome size and number in S. cerevisiae.
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rna interference of peroxisome related genes in c elegans a new model for human peroxisomal disorders
Physiological Genomics, 2002Co-Authors: Oleh I Petriv, Richard A. Rachubinski, David B Pilgrim, Vladimir I TitorenkoAbstract:RNA-mediated interference (RNAi) for the posttranscriptional silencing of genes was used to evaluate the importance of various peroxisomal enzymes and Peroxins for the development of Caenorhabditis...
Ralf Erdmann - One of the best experts on this subject based on the ideXlab platform.
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the novel Peroxin pex37 the pxmp2 family joins the peroxisomal fission machinery
FEBS Journal, 2020Co-Authors: Harald W Platta, Ralf ErdmannAbstract:Peroxisomes can undergo fission during cell division, followed by their segregation between mother and daughter cells. Despite species-specific variations in the molecular composition of the fission machinery, the central mechanistic factors can be assigned to two groups: the Pex11 family and the dynamin-related protein family. In a recent study, Singh et al. describe the involvement of a member of the Pxmp2-related protein family in peroxisome fission: the novel Peroxin Pex37.
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a combined approach of quantitative interaction proteomics and live cell imaging reveals a regulatory role for endoplasmic reticulum er reticulon homology proteins in peroxisome biogenesis
Molecular & Cellular Proteomics, 2013Co-Authors: Christine David, Andreas Schummer, Alexandra Laernsack, Sophie Melchior, Sebastian Wiese, Ralf Erdmann, Silke Oeljeklaus, Bettina Warscheid, Johannes Koch, Cécile BrocardAbstract:Peroxisome biogenesis initiates at the endoplasmic reticulum (ER) and maturation allows for the formation of metabolically active organelles. Yet, peroxisomes can also multiply by growth and division. Several proteins, called Peroxins, are known to participate in these processes but little is known about their organization to orchestrate peroxisome proliferation. Here, we demonstrate that regulation of peroxisome proliferation relies on the integrity of the tubular ER network. Using a dual track SILAC-based quantitative interaction proteomics approach, we established a comprehensive network of stable as well as transient interactions of the Peroxin Pex30p, an integral membrane protein. Through association with merely ER resident proteins, in particular with proteins containing a reticulon homology domain, and with other Peroxins, Pex30p designates peroxisome contact sites at ER subdomains. We show that Pex30p traffics through the ER and segregates in punctae to which peroxisomes specifically append, and we ascertain its transient interaction with all subunits of the COPI coatomer complex suggesting the involvement of a vesicle-mediated transport. We establish that the membrane protein Pex30p facilitates the connection of peroxisomes to the ER. Taken together, our data indicate that Pex30p-containing protein complexes act as focal points from which peroxisomes can form and that the tubular ER architecture organized by the reticulon homology proteins Rtn1p, Rtn2p and Yop1p controls this process.
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protein import machineries of peroxisomes
Biochimica et Biophysica Acta, 2011Co-Authors: Robert Rucktaschel, Wolfgang Girzalsky, Ralf ErdmannAbstract:Peroxisomes are a class of structurally and functionally related organelles present in almost all eukaryotic cells. The importance of peroxisomes for human life is highlighted by severe inherited diseases which are caused by defects of Peroxins, encoded by PEX genes. To date 32 Peroxins are known to be involved in different aspects of peroxisome biogenesis. This review addresses two of these aspects, the translocation of soluble proteins into the peroxisomal matrix and the biogenesis of the peroxisomal membrane. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.
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farnesylation of pex19p is required for its structural integrity and function in peroxisome biogenesis
Journal of Biological Chemistry, 2009Co-Authors: Robert Rucktaschel, Sven Thoms, Vadim Sidorovitch, Andre Halbach, Markos Pechlivanis, Rudolf Volkmer, Kirill Alexandrov, Jurgen Kuhlmann, Hanspeter Rottensteiner, Ralf ErdmannAbstract:The conserved CaaX box Peroxin Pex19p is known to be modified by farnesylation. The possible involvement of this lipid modification in peroxisome biogenesis, the degree to which Pex19p is farnesylated, and its molecular function are unknown or controversial. We resolve these issues by first showing that the complete pool of Pex19p is processed by farnesyltransferase in vivo and that this modification is independent of peroxisome induction or the Pex19p membrane anchor Pex3p. Furthermore, genomic mutations of PEX19 prove that farnesylation is essential for proper matrix protein import into peroxisomes, which is supposed to be caused indirectly by a defect in peroxisomal membrane protein (PMP) targeting or stability. This assumption is corroborated by the observation that mutants defective in Pex19p farnesylation are characterized by a significantly reduced steady-state concentration of prominent PMPs (Pex11p, Ant1p) but also of essential components of the peroxisomal import machinery, especially the RING Peroxins, which were almost depleted from the importomer. In vivo and in vitro, PMP recognition is only efficient when Pex19p is farnesylated with affinities differing by a factor of 10 between the non-modified and wild-type forms of Pex19p. Farnesylation is likely to induce a conformational change in Pex19p. Thus, isoprenylation of Pex19p contributes to substrate membrane protein recognition for the topogenesis of PMPs, and our results highlight the importance of lipid modifications in protein-protein interactions.
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function of the ubiquitin conjugating enzyme pex4p and the aaa Peroxin complex pex1p pex6p in peroxisomal matrix protein transport
The Enzymes, 2007Co-Authors: Harald W Platta, Sven Thoms, Wolfh Kunau, Ralf ErdmannAbstract:Publisher Summary This chapter discusses the enzymatically catalyzed mechanisms underlying the transport of matrix proteins across the peroxisomal membrane into the lumen of the organelle, a process that involves most of the known Peroxins. The chapter focuses on the basic experimental evidence concerning the functional roles of Pex4p and AAA Peroxins in Pex5p recycling and matrix protein import to combine and discuss them in a unified model. Ubiquitin-conjugating enzymes play a central role in the process of ubiquitination and function to bridge the first, nonspecific step of ubiquitin activation by E1 with the transfer of activated ubiquitin to target-proteins by substrate-specific E3 enzymes. Pex4p/Ubc10p is a ubiquitin-conjugating enzyme essential for peroxisomal biogenesis. Pex4p contains the catalytically relevant active site Cys residue of ubiquitin-conjugating enzymes within the core Ubc fold, while AAAs are mechanoenzymes that manipulate the structure of substrate proteins, and thereby unfold them or disassemble protein complexes. The energy dependence of peroxisomal protein import is caused by the cycle of the peroxisomal targeting signal (PTS) receptors. A model is emerging in which the previously disparate roles of Pex4p and the AAA Peroxins are combined in a concerted reaction sequence. However, it will be a challenge to elucidate the way ATP-dependent receptor dislocation is mechanistically linked to the import of folded proteins.
Normand Cyr - One of the best experts on this subject based on the ideXlab platform.
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the hydrophobic region of the leishmania Peroxin 14 requirements for association with a glycosome mimetic membrane
Biochemical Journal, 2017Co-Authors: Normand Cyr, Louisphilippe Leroux, Anwar Hasil Kottarampatel, Amanda Davidsen, Christian Salesse, Terry K Smith, Elodie Boisselier, Armando JardimAbstract:Protein import into the Leishmania glycosome requires docking of the cargo-loaded Peroxin 5 (PEX5) receptor to the Peroxin 14 (PEX14) bound to the glycosome surface. To examine the LdPEX14-membrane interaction, we purified L. donovani promastigote glycosomes and determined the phospholipid and fatty acid composition. These membranes contained predominately phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol (PG) modified primarily with C18 and C22 unsaturated fatty acid. Using large unilamellar vesicles (LUVs) with a lipid composition mimicking the glycosomal membrane in combination with sucrose density centrifugation and fluorescence-activated cell sorting technique, we established that the LdPEX14 membrane-binding activity was dependent on a predicted transmembrane helix found within residues 149-179. Monolayer experiments showed that the incorporation of PG and phospholipids with unsaturated fatty acids, which increase membrane fluidity and favor a liquid expanded phase, facilitated the penetration of LdPEX14 into biological membranes. Moreover, we demonstrated that the binding of LdPEX5 receptor or LdPEX5-PTS1 receptor-cargo complex was contingent on the presence of LdPEX14 at the surface of LUVs.
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Analysis of the Leishmania Peroxin 7 interactions with Peroxin 5, Peroxin 14 and PTS2 ligands
The Biochemical journal, 2014Co-Authors: Ana Victoria C. Pilar, Hamed Hojjat, Rona Strasser, James Mclean, Elizabeth Quinn, Normand Cyr, Anwer Hasil Kottarampatel, Armando JardimAbstract:LPEX7 (Leishmania Peroxin 7) is essential for targeting newly synthesized proteins with a PTS2 (peroxisome-targeting signal type 2) import signal into the glycosome. In the present paper, we describe the biophysical characterization of a functional LPEX7 isolated from Escherichia coli inclusion bodies. Pull-down assays showed that LPEX7 binds the interacting partners LdPEX5 (Leishmania donovani Peroxin 5) and LdPEX14, but, more importantly, this receptor can specifically bind PTS2 cargo proteins in the monomeric and dimeric states. However, in the absence of interacting partners, LPEX7 preferentially adopts a tetrameric structure. Mapping studies localized the LdPEX5- and LdPEX14-binding sites to the N-terminal portion of LPEX7. Deletion of the first 52 residues abolished LdPEX14 association without altering the LdPEX5 interaction. Intrinsic fluorescence techniques suggested that each LPEX7 subunit has a single unique binding site for each of the respective interacting partners LdPEX5, LdPEX14 and PTS2 cargo proteins. Extrinsic fluorescence studies with ANS (8-anilinonaphthalene-1-sulfonic acid) demonstrated that LPEX7 contains a surface-exposed hydrophobic region(s) that was not altered by the binding of a PTS2 protein or LdPEX5. However, in the presence of these ligands, the accessibility of the hydrophobic domain was dramatically restricted, suggesting that both ligands are necessary to induce notable conformational changes in LPEX7. In contrast, binding of LdPEX14 did not alter the hydrophobic domain on LPEX7. It is possible that the hydrophobic surfaces on LPEX7 may be a crucial characteristic for the shuttling of this receptor in and out of the glycosome.
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leishmania donovani Peroxin 14 undergoes a marked conformational change following association with Peroxin 5
Journal of Biological Chemistry, 2008Co-Authors: Normand Cyr, Rona Strasser, Kleber P Madrid, Mark R P Aurousseau, Ron M Finn, Juan Ausio, Armando JardimAbstract:The import of PTS1 proteins into the glycosome or peroxisome requires binding of a PTS1-laden PEX5 receptor to the membrane-associated protein PEX14 to facilitate translocation of PTS1 proteins into the lumen of these organelles. Quaternary structure analysis of protozoan parasite Leishmania donovani PEX14 (LdPEX14) revealed that this protein forms a homomeric complex with a size > 670 kDa. Moreover, deletion mapping indicated that disruption of LdPEX14 oligomerization correlated with the elimination of the hydrophobic region and coiled-coil motif present in LdPEX14. Analysis of the LdPEX5-LdPEX14 interaction by isothermal titration calorimetry revealed a molar binding stoichiometry of 1:4 (LdPEX5: LdPEX14) and an in-solution dissociation constant (K(d)) of approximately 74 nm. Calorimetry, circular dichroism, intrinsic fluorescence, and analytical ultracentrifugation experiments showed that binding of LdPEX5 resulted in a dramatic conformational change in the LdPEX14 oligomeric complex that involved the reorganization of the hydrophobic segment in LdPEX14. Finally, limited tryptic proteolysis assays established that in the presence of LdPEX5, LdPEX14 became more susceptible to proteolytic degradation consistent with this protein interaction triggering a significant conformational change in the recombinant and native LdPEX14 structures. These structural changes provide essential clues to how LdPEX14 functions in the translocation of folded proteins across the glycosomal membrane.