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Suresh Subramani - One of the best experts on this subject based on the ideXlab platform.
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Redox-regulated Cargo Binding and Release by the Peroxisomal Targeting Signal Receptor, Pex5
The Journal of biological chemistry, 2013Co-Authors: Danielle Hagstrom, Soumi Guha Polley, Suresh SubramaniAbstract:In its role as a mobile receptor for Peroxisomal matrix cargo containing a Peroxisomal Targeting Signal called PTS1, the protein Pex5 shuttles between the cytosol and the peroxisome lumen. Pex5 binds PTS1 proteins in the cytosol via its C-terminal tetratricopeptide domains and delivers them to the peroxisome lumen, where the receptor·cargo complex dissociates. The cargo-free receptor is exported to the cytosol for another round of import. How cargo release and receptor recycling are regulated is poorly understood. We found that Pex5 functions as a dimer/oligomer and that its protein interactions with itself (homo-oligomeric) and with Pex8 (hetero-oligomeric) control the binding and release of cargo proteins. These interactions are controlled by a redox-sensitive amino acid, cysteine 10 of Pex5, which is essential for the formation of disulfide bond-linked Pex5 forms, for high affinity cargo binding, and for receptor recycling. Disulfide bond-linked Pex5 showed the highest affinity for PTS1 cargo. Upon reduction of the disulfide bond by dithiothreitol, Pex5 transitioned to a noncovalent dimer, concomitant with the partial release of PTS1 cargo. Additionally, dissipation of the redox balance between the cytosol and the peroxisome lumen caused an import defect. A hetero-oligomeric interaction between the N-terminal domain (amino acids 1-110) of Pex5 and a conserved motif at the C terminus of Pex8 further facilitates cargo release, but only under reducing conditions. This interaction is also important for the release of PTS1 proteins. We suggest a redox-regulated model for Pex5 function during the Peroxisomal matrix protein import cycle.
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Unique Requirements for Mono- and Polyubiquitination of the Peroxisomal Targeting Signal Co-receptor, Pex20
The Journal of biological chemistry, 2013Co-Authors: Xueqian Liu, Suresh SubramaniAbstract:In Pichia pastoris, the Peroxisomal Targeting Signal 2 (PTS2)-dependent Peroxisomal matrix protein import pathway requires the receptor, Pex7, and its co-receptor Pex20. A conserved lysine (Lys19) near the N terminus of Pex20 is required for its polyubiquitination and proteasomal degradation, whereas a conserved cysteine (Cys8) is essential for its recycling. In this study, we found that Cys8 is required for the DTT-sensitive mono- and diubiquitination of Pex20. We also show that the PTS2 cargo receptor, Pex7, is required for Pex20 polyubiquitination. Pex4, the E2 ubiquitin-conjugation enzyme, is required for monoubiquitination of Pex20. However, it is also necessary for polyubiquitination of Pex20, making its behavior distinct from the ubiquitination described for other PTS receptors. Unlike the roles of specific RING peroxins in Pex5 ubiquitination, we found that all the RING peroxins (Pex2, Pex10, and Pex12) are required as E3 ubiquitin ligases for Pex20 mono- and polyubiquitination. A model for Pex20 ubiquitination is proposed based on these observations. This is the first description of the complete ubiquitination pathway of Pex20, which provides a better understanding of the recycling and degradation of this PTS2 cargo co-receptor.
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isolation and characterization of pas2p a Peroxisomal membrane protein essential for peroxisome biogenesis in the methylotrophic yeast pichia pastoris
Journal of Biological Chemistry, 1996Co-Authors: Erik A C Wiemer, Marten Veenhuis, Georg H Luers, Klaas Nico Faber, Thibaut Jose Wenzel, Suresh SubramaniAbstract:The pas2 mutant of the methylotrophic yeast Pichia pastoris is characterized by a deficiency in peroxisome biogenesis. We have cloned the PpPAS2 gene by functional complementation and show that it encodes a protein of 455 amino acids with a molecular mass of 52 kDa. In a Pppas2 null mutant, import of both Peroxisomal Targeting Signal 1 (PTS1)- and PTS2-containing proteins is impaired as shown by biochemical fractionation and fluorescence microscopy. No morphologically distinguishable Peroxisomal structures could be detected by electron microscopy in Pppas2 null cells induced on methanol and oleate, suggesting that PpPas2p is involved in the early stages of peroxisome biogenesis. PpPas2p is a Peroxisomal membrane protein (PMP) and is resistant to extraction by 1 M NaCl or alkaline sodium carbonate, suggesting that it is a Peroxisomal integral membrane protein. Two hydrophobic domains can be distinguished which may be involved in anchoring PpPas2p to the Peroxisomal membrane. PpPas2p is homologous to the Saccharomyces cerevisiae Pas3p. The first 40 amino acids of PpPas2p, devoid of the hydrophobic domains, are sufficient to target a soluble fluorescent reporter protein to the Peroxisomal membrane, with which it associates tightly. A comparison with the membrane Peroxisomal Targeting Signal of PMP47 of Candida boidinii revealed a stretch of positively charged amino acids common to both sequences. The role of Peroxisomal membrane Targeting Signals and transmembrane domains in anchoring PMPs to the Peroxisomal membrane is discussed.
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The Pichia pastoris Peroxisomal protein PAS8p is the receptor for the C-terminal tripeptide Peroxisomal Targeting Signal.
The EMBO journal, 1995Co-Authors: Stanley R. Terlecky, William M. Nuttley, Dannel Mccollum, Elisabeth Sock, Suresh SubramaniAbstract:The Peroxisomal Targeting Signal 1 (PTS1), consisting of a C-terminal tripeptide (SKL and variants), directs polypeptides to the peroxisome matrix in evolutionarily diverse organisms. Previous studies in the methylotrophic yeast Pichia pastoris identified a 68 kDa protein, PAS8p, as a potential component of the PTS1 import machinery. We now report several new properties of this molecule which, taken together, show that it is the Peroxisomal PTS1 receptor. (i) PAS8p is localized to and tightly associated with the cytoplasmic side of the Peroxisomal membrane, (ii) peroxisomes of wild-type, but not of pas8 delta (null) mutant, P.pastoris cells bind a PTS1-containing peptide (CRYHLKPLQSKL), (iii) CRYHLKPLQSKL can be cross-linked to PAS8p after binding at the peroxisome membrane and (iv) purified PAS8p binds CRYHLKPLQSKL with high affinity (nanomolar dissociation constant). In addition, the tetratricopeptide repeat (TPR) domain of PAS8p is identified as the PTS1 binding region.
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Import of stably folded proteins into peroxisomes.
Molecular biology of the cell, 1995Co-Authors: Paul A. Walton, P E Hill, Suresh SubramaniAbstract:By virtue of their synthesis in the cytoplasm, proteins destined for import into peroxisomes are obliged to traverse the single membrane of this organelle. Because the Targeting Signal for most Peroxisomal matrix proteins is a carboxy-terminal tripeptide sequence (SKL or its variants), these proteins must remain import competent until their translation is complete. We sought to determine whether stably folded proteins were substrates for Peroxisomal import. Prefolded proteins stabilized with disulfide bonds and chemical cross-linkers were shown to be substrates for Peroxisomal import, as were mature folded and disulfide-bonded IgG molecules containing the Peroxisomal Targeting Signal. In addition, colloidal gold particles conjugated to proteins bearing the Peroxisomal Targeting Signal were translocated into the Peroxisomal matrix. These results support the concept that proteins may fold in the mammalian cytosol, before their import into the peroxisome, and that protein unfolding is not a prerequisite for Peroxisomal import.
Richard A. Rachubinski - One of the best experts on this subject based on the ideXlab platform.
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Distinct Roles for Peroxisomal Targeting Signal Receptors Pex5 and Pex7 in Drosophila.
Genetics, 2018Co-Authors: Francesca Di Cara, Richard A. Rachubinski, Andrew J. SimmondsAbstract:Peroxisomes are ubiquitous membrane-enclosed organelles involved in lipid processing and reactive oxygen detoxification. Mutations in human peroxisome biogenesis genes (Peroxin, PEX, or Pex) cause developmental disabilities and often early death. Pex5 and Pex7 are receptors that recognize different Peroxisomal Targeting Signals called PTS1 and PTS2, respectively, and traffic proteins to the Peroxisomal matrix. We characterized mutants of Drosophila melanogaster Pex5 and Pex7 and found that adult animals are affected in lipid processing. Pex5 mutants exhibited severe developmental defects in the embryonic nervous system and muscle, similar to what is observed in humans with PEX5 mutations, while Pex7 fly mutants were weakly affected in brain development, suggesting different roles for fly Pex7 and human PEX7. Of note, although no PTS2-containing protein has been identified in Drosophila, Pex7 from Drosophila can function as a bona fide PTS2 receptor because it can rescue Targeting of the PTS2-containing protein thiolase to peroxisomes in PEX7 mutant human fibroblasts.
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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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Tetratricopeptide repeat domain of Yarrowia lipolytica Pex5p is essential for recognition of the type 1 Peroxisomal Targeting Signal but does not confer full biological activity on Pex5p.
Biochemical Journal, 2000Co-Authors: Rachel K. Szilard, Richard A. RachubinskiAbstract:Peroxins are proteins required for peroxisome assembly and are encoded by the PEX genes. The Yarrowia lipolytica pex5-1 mutant fails to import a subset of Peroxisomal matrix proteins, including those with a type 1 Peroxisomal Targeting Signal (PTS1). Pex5p family members interact with a PTS1 through their characteristic tetratricopeptide repeat (TPR) domain. We used binding assays in vitro to investigate the nature of the association of Y. lipolytica Pex5p (YlPex5p) with the PTS1 Signal. A purified recombinant YlPex5p fusion protein interacted specifically, directly and autonomously with a protein terminating in a PTS1. Wild-type YlPex5p translated in vitro recognized functional PTS1s specifically. This activity is abrogated by the substitution of an aspartic residue for a conserved glycine residue in the TPR domain (G455D) of YlPex5p encoded by the pex5-1 allele. Deletion analysis demonstrated that an intact TPR domain of YlPex5p is necessary but not sufficient for both interaction with a PTS1 and functional complementation of a strain lacking YlPex5p.
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mutagenesis of the amino Targeting Signal of saccharomyces cerevisiae 3 ketoacyl coa thiolase reveals conserved amino acids required for import into peroxisomes in vivo
Journal of Biological Chemistry, 1994Co-Authors: J.r. Glover, Davidw . Andrews, Suresh Subramani, Richard A. RachubinskiAbstract:Abstract Saccharomyces cerevisiae Peroxisomal 3-ketoacyl-CoA thiolase is a soluble matrix protein that does not end in a consensus Peroxisomal Targeting Signal-1. The amino terminus of S. cerevisiae Peroxisomal thiolase is conserved in 6 of 11 residues with the amino terminus of rat thiolase B, shown to act as a Peroxisomal Targeting Signal-2 (Swinkels, B.W., Gould, S.J., Bodnar, A.G., Rachubinski, R.A., and Subramani, S. (1991) EMBO J. 10, 3255-3262). Unlike mammalian Peroxisomal thiolases, there is no extensive cleavage of S. cerevisiae thiolase upon import into peroxisomes. We demonstrate by in vivo expression that the amino-terminal 16 amino acids of S. cerevisiae thiolase are necessary and sufficient for Targeting to peroxisomes. This result implies that yeast, like mammalian cells, can target proteins to the Peroxisomal matrix by at least two different routes. We also demonstrate by targeted mutagenesis and in vivo expression of mutated thiolase genes that three amino acids conserved in the amino termini of all known thiolases are critical for efficient Targeting of S. cerevisiae thiolase to peroxisomes.
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Transport of microinjected alcohol oxidase from Pichia pastoris into vesicles in mammalian cells: involvement of the Peroxisomal Targeting Signal.
The Journal of cell biology, 1992Co-Authors: Paul A. Walton, Richard A. Rachubinski, Stephen J. Gould, Suresh Subramani, James R. FeramiscoAbstract:This report describes the microinjection of a purified Peroxisomal protein, alcohol oxidase, from Pichia pastoris into mammalian tissue culture cells and the subsequent transport of this protein into vesicular structures. Transport was into membrane-enclosed vesicles as judged by digitonin-permeabilization experiments. The transport was time and temperature dependent. Vesicles containing alcohol oxidase could be detected as long as 6 d after injection. Coinjection of synthetic peptides containing a consensus carboxyterminal tripeptide Peroxisomal Targeting Signal resulted in abolition of alcohol oxidase transport into vesicles in all cell lines examined. Double-label experiments indicated that, although some of the alcohol oxidase was transported into vesicles that contained other Peroxisomal proteins, the bulk of the alcohol oxidase did not appear to be transported to preexisting peroxisomes. While the inhibition of transport of alcohol oxidase by peptides containing the Peroxisomal Targeting Signal suggests a competition for some limiting component of the machinery involved in the sorting of proteins into peroxisomes, the organelles into which the majority of the protein is targeted appear to be unusual and distinct from endogenous peroxisomes by several criteria. Microinjected alcohol oxidase was transported into vesicles in normal fibroblasts and also in cell lines derived from patients with Zellweger syndrome, which are unable to transport proteins containing the ser-lys-leu-COOH Peroxisomal Targeting Signal into peroxisomes (Walton et al., 1992). The implications of this result for the mechanism of Peroxisomal protein transport are discussed.
Ben Distel - One of the best experts on this subject based on the ideXlab platform.
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The Peroxisomal Targeting Signal 1 in sterol carrier protein 2 is autonomous and essential for receptor recognition
BMC Biochemistry, 2011Co-Authors: Chris Williams, Ralf Erdmann, Nicole Schueller, Colin A Thompson, Marlene Van Den Berg, Simon D. Van Haren, Charles S. Bond, Ben Distel, Wolfgang Schliebs, Matthias WilmannsAbstract:Background The majority of Peroxisomal matrix proteins destined for translocation into the Peroxisomal lumen are recognised via a C-terminal Peroxisomal Target Signal type 1 by the cycling receptor Pex5p. The only structure to date of Pex5p in complex with a cargo protein is that of the C-terminal cargo-binding domain of the receptor with sterol carrier protein 2, a small, model Peroxisomal protein. In this study, we have tested the contribution of a second, ancillary receptor-cargo binding site, which was found in addition to the characterised Peroxisomal Target Signal type 1.
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The Peroxisomal Targeting Signal 1 in sterol carrier protein 2 is autonomous and essential for receptor recognition
BMC Biochemistry, 2011Co-Authors: Chris Williams, Ralf Erdmann, Nicole Schueller, Colin A Thompson, Marlene Van Den Berg, Simon D. Van Haren, Charles S. Bond, Ben Distel, Wolfgang Schliebs, Matthias WilmannsAbstract:Background: The majority of Peroxisomal matrix proteins destined for translocation into the Peroxisomal lumen are recognised via a C-terminal Peroxisomal Target Signal type 1 by the cycling receptor Pex5p. The only structure to date of Pex5p in complex with a cargo protein is that of the C-terminal cargo-binding domain of the receptor with sterol carrier protein 2, a small, model Peroxisomal protein. In this study, we have tested the contribution of a second, ancillary receptor-cargo binding site, which was found in addition to the characterised Peroxisomal Target Signal type 1. Results: To investigate the function of this secondary interface we have mutated two key residues from the ancillary binding site and analyzed the level of binding first by a yeast-two-hybrid assay, followed by quantitative measurement of the binding affinity and kinetics of purified protein components and finally, by in vivo measurements, to determine translocation capability. While a moderate but significant reduction of the interaction was found in binding assays, we were not able to measure any significant defects in vivo. Conclusions: Our data therefore suggest that at least in the case of sterol carrier protein 2 the contribution of the second binding site is not essential for Peroxisomal import. At this stage, however, we cannot rule out that other cargo proteins may require this ancillary binding site.
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Protein Quality Control in Peroxisomes: Ubiquitination of the Peroxisomal Targeting Signal Receptors
Handbook of Cell Signaling, 2010Co-Authors: Chris Williams, Ben DistelAbstract:Publisher Summary This chapter discusses the ubiquitination of the PTS (co-) receptors and addresses the role of each of the proteins involved in this process together with the implications of receptor ubiquitination on Peroxisomal matrix protein import. Proteins destined for the Peroxisomal matrix begin their journey in the cytosol, where they are synthesized on free polyribosomes. Peroxisomal sorting, like the sorting into other subcellular compartments, relies on Targeting Signals, in this case a Peroxisomal Targeting Signal (PTS). Ubiquitination is the attachment of ubiquitin to a substrate protein. Either mono- or poly-ubiquitination of Pex5p is required to remove the protein from the Peroxisomal membrane. In the absence of one of these pathways, the other is capable of taking over. After completion of the docking and PTS translocation steps, the membrane associated PTS (co-) receptor is mono-ubiquitinated by Pex4p, allowing recognition by the AAA proteins, Pex1p and Pex6p. The PTS (co-) receptor is then pulled out of the membrane and the ubiquitin is removed by the de-ubiquitinating enzyme. The PTS (co-) receptor is then free to partake in another round of PTS protein import. In the situation that no efficient recycling is possible, due to the absence of one of the peroxins involved in recycling or in certain PTS (co-) receptor mutants, PTS (co-) receptor poly-ubiquitination, mediated by Ubc4p, is observed. This modified form is then removed from the membrane and destroyed by the 26S proteasome, effectively removing the blockage.
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Recognition of Peroxisomal Targeting Signal type 1 by the import receptor Pex5p.
The Journal of biological chemistry, 2001Co-Authors: André Klein, Phil Barnett, Gina Bottger, Daphne Konings, Henk F. Tabak, Ben DistelAbstract:Abstract We have studied how Pex5p recognizes Peroxisomal Targeting Signal type 1 (PTS1)-containing proteins. A randomly mutagenized pex5 library was screened in a two-hybrid setup for mutations that disrupted the interaction with the PTS1 protein Mdh3p or for suppressor mutations that could restore the interaction with Mdh3p containing a mutation in its PTS1. All mutations localized in the tetratricopeptide repeat (TPR) domain of Pex5p. The Pex5p TPR domain was modeled based on the crystal structure of a related TPR protein. Mapping of the mutations on this structural model revealed that some of the loss-of-interaction mutations consisted of substitutions in α-helices of TPRs with bulky amino acids, probably resulting in local misfolding and thereby indirectly preventing binding of PTS1 proteins. The other loss-of-interaction mutations and most suppressor mutations localized in short, exposed, intra-repeat loops of TPR2, TPR3, and TPR6, which are predicted to mediate direct interaction with PTS1 amino acids. Additional site-directed mutants at conserved positions in intra-repeat loops underscored the importance of the loops of TPR2 and TPR3 for PTS1 interaction. Based on the mutational analysis and the structural model, we put forward a model as to how PTS1 proteins are selected by Pex5p.
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Analysis of the Carboxyl-terminal Peroxisomal Targeting Signal 1 in a Homologous Context in Saccharomyces cerevisiae
The Journal of biological chemistry, 1996Co-Authors: Ype Elgersma, Ben Distel, Arnold Vos, M. Van Den Berg, C. W. T. Van Roermund, P. Van Der Sluijs, Henk F. TabakAbstract:Abstract Most Peroxisomal matrix proteins contain a carboxyl-terminal tripeptide that directs them to peroxisomes. Within limits, these amino acids may be varied, without loss of function. The specificity of this Peroxisomal Targeting Signal (PTS1) is remarkable considering its small size and its relaxed consensus sequence. Moreover, several Peroxisomal proteins have a PTS1-like Signal that does not fit the reported consensus sequence. Because many of these PTS1 variants seem to be functional in a species-dependent or protein context-dependent manner, we investigated the PTS1 requirements in a homologous context, using Saccharomyces cerevisiae and endogenous Peroxisomal malate dehydrogenase (MDH3). Peroxisomal import of the MDH3-PTS1 variants was tested qualitatively by the ability to complement the Δmdh3 mutant and quantitatively by subcellular fractionation. We observed efficient import of MDH3 into peroxisomes with a large variety of PTS1 tripeptides. Many of these variants do not fit the observed PTS1 requirements for heterologously expressed proteins, which suggests that additional domains in the protein may be of decisive importance whether or not a certain PTS1 variant is recognized by the components of the Peroxisomal import machinery. Because we show that dimerization of MDH3 precedes import into the organelle, these domains are most likely conformational domains.
Andreas Hartig - One of the best experts on this subject based on the ideXlab platform.
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Prediction of Peroxisomal Targeting Signal 1 containing proteins from amino acid sequence.
Journal of molecular biology, 2003Co-Authors: Georg Neuberger, Andreas Hartig, Sebastian Maurer-stroh, Birgit Eisenhaber, Frank EisenhaberAbstract:Peroxisomal matrix proteins have to be imported into their target organelle post-translationally. The major translocation pathway depends on a C-terminal Targeting Signal, termed PTS1. Our previous analysis of sequence variability in the PTS1 motif revealed that, in addition to the known C-terminal tripeptide, at least nine residues directly upstream are important for Signal recognition in the PTS1-Pex5 receptor complex. The refined PTS1 motif description was implemented in a prediction tool composed of taxon-specific functions (metazoa, fungi, remaining taxa), capable of recognising potential PTS1s in query sequences. The composite score function consists of classical profile terms and additional terms penalising deviations from the derived physical property pattern over sequence segments. The prediction algorithm has been validated with a self-consistency and three different cross-validation tests. Additionally, we tested the tool on a large set of non-Peroxisomal negatives, on mutation data, and compared the prediction rate to the PTS1 component of the PSORT2 program. The sensitivity of our predictor in recognising documented PTS1 Signal containing proteins is close to 90% for reliable prediction. The predictor distinguishes even SKL-appended non-Peroxisomally targeted proteins such as a mouse dihydrofolate reductase-SKL construct. The corresponding rate of false positives is not worse than 0.8%; thus, the tool can be applied for large-scale unsupervised sequence database annotation. A scan of public protein databases uncovered a number of yet uncharacterised proteins for which the PTS1 Signal might be critical for biological function. The predicted presence of a PTS1 Signal implies Peroxisomal localisation in the absence of N-terminal Targeting sequences such as the mitochondrial import Signal.
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Motif refinement of the Peroxisomal Targeting Signal 1 and evaluation of taxon-specific differences.
Journal of molecular biology, 2003Co-Authors: Georg Neuberger, Andreas Hartig, Sebastian Maurer-stroh, Birgit Eisenhaber, Frank EisenhaberAbstract:Abstract Eukaryote peroxisomes, plant glyoxysomes and trypanosomal glycosomes belong to the microbody family of organelles that compartmentalise a variety of biochemical processes. The interaction between the PTS1 Signal and its cognate receptor Pex5 initiates the major import mechanism for proteins into the matrix of these organelles. Relying on the analysis of amino acid sequence variability of known PTS1-targeted proteins and PTS1-containing peptides that interact with Pex5 in the yeast two-hybrid assay, on binding site studies of the Pex5–ligand complex crystal structure, 3D models and sequences of Pex5 proteins from various taxa, we derived the requirements for a C-terminal amino acid sequence to interact productively with Pex5. We found evidence that, at least the 12 C-terminal residues of a given substrate protein are implicated in PTS1 Signal recognition. This motif can be structurally and functionally divided into three regions: (i) the C-terminal tripeptide, (ii) a region interacting with the surface of Pex5 (about four residues further upstream), and (iii) a polar, solvent-accessible and unstructured region with linker function (the remaining five residues). Specificity differences are confined to taxonomic subgroups (metazoa and fungi) and are connected with amino acid type preferences in region 1 and deviating hydrophobicity patterns in region 2.
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the difference in recognition of terminal tripeptides as Peroxisomal Targeting Signal 1 between yeast and human is due to different affinities of their receptor pex5p to the cognate Signal and to residues adjacent to it
Journal of Biological Chemistry, 1998Co-Authors: Guenther Lametschwandtner, Cécile Brocard, Marc Fransen, Johannes Berger, Paul P. Van Veldhoven, Andreas HartigAbstract:Abstract Pex5p is the receptor for the Peroxisomal Targeting Signal 1 (PTS1) that consists of a C-terminal tripeptide (consensus (S/A/C)(K/R/H)(L/M)). Hexadecapeptides recognized by Pex5p from Homo sapiens and Saccharomyces cerevisiaewere identified by screening a two-hybrid peptide library, and the Targeting ability of the peptides was demonstrated using the green fluorescent protein as reporter. The PTS1 receptors recognized in a species-specific manner a broad range of C-terminal tripeptides, and these are reported herein. In addition, residues upstream of the tripeptide influenced the strength of the interaction in the two-hybrid system as well as in an in vitro competition assay. In peptides interacting with the human protein, hydrophobic residues were found with high frequency especially at positions −2 and −5, whereas peptides interacting with S. cerevisiae Pex5p were more hydrophilic and frequently contained arginine at position −2. In instances where the terminal tripeptide deviated from the consensus, upstream residues exerted a greater influence on the ability of the hexadecapeptides to bind Pex5p.
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IDENTIFICATION AND ANALYSIS OF THE PLANT Peroxisomal Targeting Signal 1 RECEPTOR NTPEX5
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Friedrich Kragler, Andreas Hartig, Günther Lametschwandtner, James Christmann, John J. HaradaAbstract:Abstract Protein translocation into peroxisomes takes place via recognition of a Peroxisomal Targeting Signal present at either the extreme C termini (PTS1) or N termini (PTS2) of matrix proteins. In mammals and yeast, the Peroxisomal Targeting Signal receptor, Pex5p, recognizes the PTS1 consisting of -SKL or variants thereof. Although many plant Peroxisomal matrix proteins are transported through the PTS1 pathway, little is known about the PTS1 receptor or any other peroxisome assembly protein from plants. We cloned tobacco (Nicotiana tabacum) cDNAs encoding Pex5p (NtPEX5) based on the protein’s interaction with a PTS1-containing protein in the yeast two-hybrid system. Nucleotide sequence analysis revealed that the tobacco Pex5p contains seven tetratricopeptide repeats and that NtPEX5 shares greater sequence similarity with its homolog from humans than from yeast. Expression of NtPEX5 fusion proteins, consisting of the N-terminal part of yeast Pex5p and the C-terminal region of NtPEX5, in a Saccharomyces cerevisiae pex5 mutant restored protein translocation into peroxisomes. These experiments confirmed the identity of the tobacco protein as a PTS1 receptor and indicated that components of the Peroxisomal translocation apparatus are conserved functionally. Two-hybrid assays showed that NtPEX5 interacts with a wide range of PTS1 variants that also interact with the human Pex5p. Interestingly, the C-terminal residues of some of these peptides deviated from the established plant PTS1 consensus sequence. We conclude that there are significant sequence and functional similarities between the plant and human Pex5ps.
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The tetratricopeptide repeat-domain of the PAS10 protein of Saccharomyces cerevisiae is essential for binding the Peroxisomal Targeting Signal-SKL.
Biochemical and biophysical research communications, 1994Co-Authors: Cécile Brocard, Friedrich Kragler, Manuel M. Simon, T. Schuster, Andreas HartigAbstract:The PAS10 gene was found in a two-hybrid screen for the isolation of genes encoding proteins which interact with the C-terminal Peroxisomal Targeting Signal -SKL. The PAS10 protein is known to be involved in import of proteins into peroxisomes and to contain a tetratricopeptide repeat (TPR) domain. All TPR-containing proteins involved in diverse processes like mitosis or RNA-synthesis share the ability to interact with other proteins. Here we show that the PAS10 protein interacts in vivo with the C-terminal Peroxisomal Targeting Signal. The part essential for this interaction contains the complete tetratricopeptide repeat domain.
Stephen J. Gould - One of the best experts on this subject based on the ideXlab platform.
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Peroxisomal Targeting Signal 1 recognition by the tpr domains of human pex5
Nature Structural & Molecular Biology, 2000Co-Authors: Gregory J Gatto, Stephen J. Gould, Brian V Geisbrecht, Jeremy M BergAbstract:Many proteins contain Targeting Signals within their sequences that specify their delivery to particular organelles. The Peroxisomal Targeting Signal-1 (PTS1) is a C-terminal tripeptide that is sufficient to direct proteins into peroxisomes. The PTS1 sequence closely approximates Ser-Lys-Leu-COO−. PEX5, the receptor for PTS1, interacts with the Signal via a series of tetratricopeptide repeats (TPRs) within its C-terminal half. Here we report the crystal structure of a fragment of human PEX5 that includes all seven predicted TPR motifs in complex with a pentapeptide containing a PTS1 sequence. Two clusters of three TPRs almost completely surround the peptide, while a hinge region, previously identified as TPR4, forms a distinct structure that enables the two sets of TPRs to form a single binding site. This structure reveals the molecular basis for PTS1 recognition and demonstrates a novel mode of TPR–peptide interaction.
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A proposed model for the PEX5-Peroxisomal Targeting Signal-1 recognition complex.
Proteins, 2000Co-Authors: Gregory J Gatto, Stephen J. Gould, Brian V Geisbrecht, Jeremy M BergAbstract:The three-dimensional structure of a protein can greatly illuminate the relationship between its sequence and its function. However, in the absence of a set of experimentally derived coordinates, one often seeks a model of the protein of interest to guide future study. We describe the combined utilization of orthologous sequence information along with knowledge of the related structural fold to model the interaction between PEX5 and its ligand, the Peroxisomal Targeting Signal-1 (PTS1). With this model, we are able to identify residues within PEX5 that appear to be important for peptide recognition, as well as explain some of the sequence requirements of the PTS1. Specifically, our model highlights four asparagine residues as important for ligand backbone atom recognition, which, along with previously observed examples, suggests this as a general mechanism for the binding of extended polypeptides. Proteins 2000;38:241–246. © 2000 Wiley-Liss, Inc.
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pex13p is an sh3 protein of the peroxisome membrane and a docking factor for the predominantly cytoplasmic pts1 receptor
Journal of Cell Biology, 1996Co-Authors: Stephen J. Gould, Jennifer E Kalish, James C Morrell, Jonas Carlotto Bjorkman, Aaron J Urquhart, Denis I CraneAbstract:Import of newly synthesized PTS1 proteins into the peroxisome requires the PTS1 receptor (Pex5p), a predominantly cytoplasmic protein that cycles between the cytoplasm and peroxisome. We have identified Pex13p, a novel integral Peroxisomal membrane from both yeast and humans that binds the PTS1 receptor via a cytoplasmically oriented SH3 domain. Although only a small amount of Pex5p is bound to peroxisomes at steady state (< 5%), loss of Pex13p further reduces the amount of peroxisome-associated Pex5p by approximately 40-fold. Furthermore, loss of Pex13p eliminates import of Peroxisomal matrix proteins that contain either the type-1 or type-2 Peroxisomal Targeting Signal but does not affect Targeting and insertion of integral Peroxisomal membrane proteins. We conclude that Pex13p functions as a docking factor for the predominantly cytoplasmic PTS1 receptor.
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Transport of microinjected alcohol oxidase from Pichia pastoris into vesicles in mammalian cells: involvement of the Peroxisomal Targeting Signal.
The Journal of cell biology, 1992Co-Authors: Paul A. Walton, Richard A. Rachubinski, Stephen J. Gould, Suresh Subramani, James R. FeramiscoAbstract:This report describes the microinjection of a purified Peroxisomal protein, alcohol oxidase, from Pichia pastoris into mammalian tissue culture cells and the subsequent transport of this protein into vesicular structures. Transport was into membrane-enclosed vesicles as judged by digitonin-permeabilization experiments. The transport was time and temperature dependent. Vesicles containing alcohol oxidase could be detected as long as 6 d after injection. Coinjection of synthetic peptides containing a consensus carboxyterminal tripeptide Peroxisomal Targeting Signal resulted in abolition of alcohol oxidase transport into vesicles in all cell lines examined. Double-label experiments indicated that, although some of the alcohol oxidase was transported into vesicles that contained other Peroxisomal proteins, the bulk of the alcohol oxidase did not appear to be transported to preexisting peroxisomes. While the inhibition of transport of alcohol oxidase by peptides containing the Peroxisomal Targeting Signal suggests a competition for some limiting component of the machinery involved in the sorting of proteins into peroxisomes, the organelles into which the majority of the protein is targeted appear to be unusual and distinct from endogenous peroxisomes by several criteria. Microinjected alcohol oxidase was transported into vesicles in normal fibroblasts and also in cell lines derived from patients with Zellweger syndrome, which are unable to transport proteins containing the ser-lys-leu-COOH Peroxisomal Targeting Signal into peroxisomes (Walton et al., 1992). The implications of this result for the mechanism of Peroxisomal protein transport are discussed.
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Targeting efficiencies of various permutations of the consensus C-terminal tripeptide Peroxisomal Targeting Signal.
FEBS letters, 1992Co-Authors: Bart W Swinkels, Stephen J. Gould, Suresh SubramaniAbstract:Two types of peptide Signals are known to independently target proteins into the Peroxisomal matrix. One of these is a consensus C-terminal tripeptide which is conserved in many microbody proteins derived from diverse species. The second Signal is an N-terminal sequence found in a small subset of Peroxisomal proteins. We have tested 18 possible variants of the consensus tripeptide Targeting Signal for their ability to facilitate the transport of a cytosolic passenger protein, chloramphenicol acetyltransferase, into peroxisomes of monkey kidney cells. Our results reveal the presence of a hierarchy of preferred amino acid substitutions at each position of the tripeptide.