Biomedical subjects

Marc Fransen

Publications and source records attributed to Marc Fransen.

12 recordsLinked to original sources

PubMed2006

Import of peroxisomal membrane proteins: the interplay of Pex3p- and Pex19p-mediated interactions.

In contrast to the molecular mechanisms underlying import of peroxisomal matrix proteins, those involving the transport of membrane proteins remain rather elusive. At present, two targeting routes for peroxisomal membrane proteins (PMPs) have been depicted: class I PMPs are targeted from the cytoplasm directly to the peroxisome membrane, and class II PMPs are sorted indirectly to peroxisomes via the endoplasmic reticulum (ER). In addition, three peroxins--Pex3p, Pex16p, and Pex19p - have been identified as essential factors for PMP assembly in several species including humans: Pex19p is a predominantly cytoplasmic protein that shows a broad PMP-binding specificity; Pex3p serves as the membrane-anchoring site for Pex19p; and Pex16p - a protein absent in most yeasts--is thought to provide the initial scaffold for recruiting the protein import machinery required for peroxisome membrane biogenesis. Remarkably, the function of Pex16p does not appear to be conserved between different species. In addition, significant disagreement exists about whether Pex19p has a chaperone-like role in the cytosol or at the peroxisome membrane and/or functions as a cycling import receptor for newly synthesized PMPs. Here we review the recent progress made in our understanding of the role of two key players in PMP biogenesis, Pex3p and Pex19p.

Endoplasmic Reticulum↗

PubMed2006

The import competence of a peroxisomal membrane protein is determined by Pex19p before the docking step.

Biogenesis of the mammalian peroxisomal membrane requires the action of Pex3p and Pex16p, two proteins present in the organelle membrane, and Pex19p, a protein that displays a dual subcellular distribution (peroxisomal and cytosolic). Pex19p interacts with most peroxisomal intrinsic membrane proteins, but whether this property reflects its role as an import receptor for this class of proteins or a chaperone-like function in the assembly/disassembly of peroxisomal membrane proteins has been the subject of much controversy. Here, we describe an in vitro system particularly suited to address this issue. It is shown that insertion of a reporter protein into the peroxisomal membrane is a Pex3p-dependent process that does not require ATP/GTP hydrolysis. The system can be programmed with recombinant versions of Pex19p, allowing us to demonstrate that Pex19p-cargo protein complexes formed in the absence of peroxisomes are the substrates for the peroxisomal docking/insertion machinery. Data suggesting that cargo-loaded Pex19p displays a much higher affinity for Pex3p than Pex19p alone are also provided. These results suggest that soluble Pex19p participates in the targeting of newly synthesized peroxisomal membrane proteins to the organelle membrane and support the existence of a cargo-induced peroxisomal targeting mechanism for Pex19p.

Adenosine Triphosphate↗

PubMed2006

Targeting signals in peroxisomal membrane proteins.

Peroxisomal membrane proteins (PMPs) are encoded by the nuclear genome and translated on cytoplasmic ribosomes. Newly synthesized PMPs can be targeted directly from the cytoplasm to peroxisomes or travel to peroxisomes via the endoplasmic reticulum (ER). The mechanisms responsible for the targeting of these proteins to the peroxisomal membrane are still rather poorly understood. However, it is clear that the trafficking of PMPs to peroxisomes depends on the presence of cis-acting targeting signals, called mPTSs. These mPTSs show great variability both in the identity and number of requisite residues. An emerging view is that mPTSs consist of at least two functionally distinct domains: a targeting element, which directs the newly synthesized PMP from the cytoplasm to its target membrane, and a membrane-anchoring sequence, which is required for the permanent insertion of the protein into the peroxisomal membrane. In this review, we summarize our knowledge of the mPTSs currently identified.

Amino Acid Sequence↗

PubMed2006

Localization of a portion of the liver isoform of fatty-acid-binding protein (L-FABP) to peroxisomes.

The liver isoform of fatty-acid-binding protein (L-FABP) facilitates the cellular uptake, transport and metabolism of fatty acids and is also involved in the regulation of gene expressions and cell differentiation. Consistent with these functions, L-FABP is predominantly present in the cytoplasm and to a lesser extent in the nucleus; however, a significant portion of this protein has also been detected in fractions containing different organelles. More recent observations, notably on L-FABP-deficient mice, indicated a possible direct involvement of L-FABP in the peroxisomal oxidation of long-chain fatty acids. In order to clarify the links between L-FABP and peroxisomal lipid metabolism, we reinvestigated the subcellular distribution of the protein. Analytical subcellular fractionation by a method preserving the intactness of isolated peroxisomes, two-dimensional gel electrophoresis of peroxisomal matrix proteins combined with MS analysis, and immunoelectron microscopy of liver sections demonstrate the presence of L-FABP in the matrix of peroxisomes as a soluble protein. Peroxisomal L-FABP was highly inducible by clofibrate. The induction of L-FABP was accompanied by a marked increase in the binding capacity of peroxisomal matrix proteins for oleic acid and cis-parinaric acid. The peroxisomal beta-oxidation of palmitoyl-CoA and acyl-CoA thioesterase activity were stimulated by L-FABP, indicating that the protein modulates the function of peroxisomal lipid-metabolizing enzymes. The possible role of intraperoxisomal L-FABP in lipid metabolism is discussed.

Animals↗

PubMed2005

The N-terminal half of the peroxisomal cycling receptor Pex5p is a natively unfolded domain.

Targeting of most newly synthesised peroxisomal matrix proteins to the organelle requires Pex5p, the so-called PTS1 receptor. According to current models of peroxisomal biogenesis, Pex5p interacts with these proteins in the cytosol, transports them to the peroxisomal membrane and catalyses their translocation across the membrane. Presently, our knowledge on the structural details behind the interaction of Pex5p with the cargo proteins is reasonably complete. In contrast, information regarding the structure of the Pex5p N-terminal half (a region containing its peroxisomal targeting domain) is still limited. We have recently observed that the Stokes radius of this Pex5p domain is anomalously large, suggesting that this portion of the protein is either a structured elongated domain or that it adopts a low compactness conformation. Here, we address this issue using a combination of biophysical and biochemical approaches. Our results indicate that the N-terminal half of Pex5p is best described as a natively unfolded pre-molten globule-like domain. The implications of these findings on the mechanism of protein import into the peroxisome are discussed.

Amino Acids↗

PubMed2005

Pex5p, the peroxisomal cycling receptor, is a monomeric non-globular protein.

In mammals, targeting of newly synthesized peroxisomal matrix proteins to the organelle requires Pex5p, the peroxisomal cycling receptor. Pex5p is a multidomain protein involved in a complex network of transient protein-protein interactions. Besides interacting directly with most peroxisomal proteins en route to the organelle, Pex5p has also binding domains for several components of the peroxisomal docking/translocation machinery. However, our knowledge of how binding of a cargo protein to Pex5p influences its properties is still rather limited. Here, we describe a protease assay particularly useful for identifying and characterizing protein-protein interactions involving human Pex5p. Binding of a PTS1-containing peptide/protein to Pex5p as well as the interaction of this peroxin with the Src homology domain 3 of Pex13p could be easily demonstrated using this assay. To address the possible effects of these Pex5p-interacting peptides/proteins on the assumed quaternary structure of Pex5p, we have analyzed the hydrodynamic properties of human Pex5p using size exclusion chromatography, sucrose gradient centrifugation, and sedimentation equilibrium centrifugation. Our results show that Pex5p is a monomeric protein with an abnormal shape. The implications of these findings on current models of protein translocation across the peroxisomal membrane are discussed.

Centrifugation↗

PubMed2005

Analysis of human Pex19p's domain structure by pentapeptide scanning mutagenesis.

Pex19p, a primarily cytosolic protein, is essential for the biogenesis of numerous peroxisomal membrane proteins (PMPs); however, its precise function is unclear. Pex19p might function as a PMP-specific chaperone, a cycling PMP-receptor protein, a PMP membrane insertion factor, or an association/dissociation factor of membrane-associated protein complexes. Alternatively, Pex19p might act as a multifunctional peroxin and participate in a number of these activities. Here, we have employed transposon mutagenesis to generate a library of human pex19 alleles coding for Pex19p variants containing random in-frame pentapeptide insertions. A total of 87 different variants were characterized to identify functionally important regions. These studies revealed that Pex19p has a tripartite domain structure consisting of: (i) an amino-terminal domain that binds to Pex3p and is essential for docking at the peroxisome membrane; (ii) a central domain that competes with Pex5p and Pex13p for binding to Pex14p and may play a role in the assembly of PTS-receptor docking complexes; and (iii) a carboxy-terminal domain that interacts with multiple PMPs including Pex3p, Pex11pbeta, Pex12p, Pex13p, Pex16p, and Pex26p. Whether the latter interactions constitute the chaperone or transport functions (or both), remains to be determined. Finally, our observation that Pex19p contains two distinct binding sites for Pex3p suggests that the peroxin may bind PMPs in multiple places and for multiple purposes.

Amino Acid Sequence↗

PubMed2004

The N terminus of the peroxisomal cycling receptor, Pex5p, is required for redirecting the peroxisome-associated peroxin back to the cytosol.

Most newly synthesized peroxisomal matrix proteins are transported to the organelle by Pex5p, a remarkable multidomain protein involved in an intricate network of transient protein-protein interactions. Presently, our knowledge regarding the structure/function of amino acid residues 118 to the very last residue of mammalian Pex5p is quite vast. Indeed, the cargo-protein receptor domain as well as the binding sites for several peroxins have all been mapped to this region of Pex5p. In contrast, structural/functional data regarding the first 117 amino acid residues of Pex5p are still scarce. Here we show that a truncated Pex5p lacking the first 110 amino acid residues (DeltaN110-Pex5p) displays exactly the peroxisomal import properties of the full-length peroxin implying that this N-terminal domain is involved neither in cargo-protein binding nor in the docking/translocation step of the Pex5p-cargo protein complex at the peroxisomal membrane. However, the ATP-dependent export step of DeltaN110-Pex5p from the peroxisomal membrane is completely blocked, a phenomenon that was also observed for a Pex5p version lacking just the first 17 amino acid residues but not for a truncated protein comprising amino acid residues 1-324 of Pex5p. By exploring the unique properties of DeltaN110-Pex5p, the effect of temperature on the import/export kinetics of Pex5p was characterized. Our data indicate that the export step of Pex5p from the peroxisomal compartment (in contrast with its insertion into the organelle membrane) is highly dependent on the temperature.

Adenosine Triphosphatases↗

PubMed2004

Potential role for Pex19p in assembly of PTS-receptor docking complexes.

Human Pex19p binds a broad spectrum of peroxisomal membrane proteins (PMPs). It has been proposed that this peroxin may: (i) act as a cycling PMP receptor protein, (ii) facilitate the insertion of newly synthesized PMPs into the peroxisomal membrane, or (iii) function as a chaperone to associate and/or dissociate complexes comprising integral PMPs already in the peroxisomal membrane. We previously demonstrated that human Pex19p binds peroxisomal integral membrane proteins at regions distinct from their sorting sequences. Here we demonstrate that a mutant of Pex13p that fails to bind to Pex19p nevertheless targets to and integrates into the peroxisomal membrane. In addition, through in vitro biochemical analysis, we show that Pex19p competes with Pex5p and Pex13p for binding to Pex14p, supporting a role for this peroxin in regulating assembly/disassembly of membrane-associated protein complexes. To further examine the molecular mechanism underlying this competition, six evolutionarily conserved amino acids in the Pex5p/Pex13p/Pex19p binding domain of Pex14p were subjected to site-directed mutagenesis and the corresponding mutants functionally analyzed. Our results indicate that the physically overlapping binding sites of Pex14p for Pex5p, Pex13p, and Pex19p are functionally distinct, suggesting that competition occurs through induction of structural changes, rather than through direct competition. Importantly, we also found that amino acid substitutions resulting in a strongly reduced binding affinity for Pex13p affect the peroxisomal localization of Pex14p.

Animals↗

PubMed2002

Functional studies on human Pex7p: subcellular localization and interaction with proteins containing a peroxisome-targeting signal type 2 and other peroxins.

Pex7p is a WD40-containing protein involved in peroxisomal import of proteins containing an N-terminal peroxisome-targeting signal (PTS2). The interaction of human recombinant Pex7p expressed in different hosts/systems with its PTS2 ligand and other peroxins was analysed using various experimental approaches. Specific binding of human Pex7p to PTS2 could be demonstrated only when Pex7p was formed in vitro by a coupled transcription/translation system or synthesized in vivo in Chinese hamster ovary K1 cells transfected with a construct coding for a Pex7p-green fluorescent protein (GFP) fusion protein. Apparently, no cofactors are required and only monomeric Pex7p binds to PTS2. The interaction is reduced upon cysteine alkylation and is impaired upon truncation of the N-terminus of Pex7p. Interaction of Pex7p with other peroxins could not be demonstrated in bacterial or yeast two-hybrid screens, or in pull-down binding assays. The GFP fusion proteins, tagged at either the N- or C-terminus, were able to restore PTS2 import in rhizomelic chondrodysplasia punctata fibroblasts, and Pex7p-GFP was located both in the lumen of peroxisomes and in the cytosol.

Animals↗

PubMed2002

Analysis of mammalian peroxin interactions using a non-transcription-based bacterial two-hybrid assay.

In recent years, substantial progress has been made in the identification of proteins involved in peroxisome biogenesis. However, with the exception of the peroxisome-targeting signal receptors and the receptor docking proteins, the function of most of these proteins, called peroxins, remains largely unknown. One step toward elucidating the function of a protein is to identify its interacting partners. We have used a non-transcription-based bacterial two-hybrid system to analyze the interactions among a set of 12 mammalian peroxins and a yeast protein three-hybrid system to investigate whether proteins that interact with the same peroxin and have overlapping binding sites cooperate or compete for this site. Here we report a detailed interaction map of these peroxins and demonstrate that (i) farnesylation, and not the CAAX motif, of Pex19p strongly enhances its affinity for Pex13p; (ii) the CAAXmotif, and not farnesylation, of Pex19p strongly enhances its affinity for Pex11pbeta; and (iii) the C(3)HC(4) RING (really interesting new gene) finger domain of Pex12p does not alter the binding properties of Pex5p for the C-terminal peroxisome-targeting signal PTS1. Finally, we show that the Pex5p-Pex13p interaction is bridged by Pex14p and that the latter molecule exists predominantly as a dimer in vivo. Collectively, as demonstrated in this work with peroxins, these results indicate that the bacterial two-hybrid system is an attractive complementary approach to the conventional transcription-based yeast two-hybrid system for studying protein-protein interactions.

Animals↗

PubMedDate not supplied

Trypanosoma brucei glycosomal ABC transporters: identification and membrane targeting.

Trypanosomes contain unique peroxisome-like organelles designated glycosomes which sequester enzymes involved in a variety of metabolic processes including glycolysis. We identified three ABC transporters associated with the glycosomal membrane of Trypanosoma brucei. They were designated GAT1-3 for Glycosomal ABC Transporters. These polypeptides are so-called half-ABC transporters containing only one transmembrane domain and a single nucleotide-binding domain, like their homologues of mammalian and yeast peroxisomes. The glycosomal localization was shown by immunofluorescence microscopy of trypanosomes expressing fusion constructs of the transporters with Green Fluorescent Protein. By expression of fluorescent deletion constructs, the glycosome-targeting determinant of two transporters was mapped to different fragments of their respective primary structures. Interestingly, these fragments share a short sequence motif and contain adjacent to it one--but not the same--of the predicted six transmembrane segments of the transmembrane domain. We also identified the T. brucei homologue of peroxin PEX19, which is considered to act as a chaperonin and/or receptor for cytosolically synthesized proteins destined for insertion into the peroxisomal membrane. By using a bacterial two-hybrid system, it was shown that glycosomal ABC transporter fragments containing an organelle-targeting determinant can interact with both the trypanosomatid and human PEX19, despite their low overall sequence identity. Mutated forms of human PEX19 that lost interaction with human peroxisomal membrane proteins also did not bind anymore to the T. brucei glycosomal transporter. Moreover, fragments of the glycosomal transporter were targeted to the peroxisomal membrane when expressed in mammalian cells. Together these results indicate evolutionary conservation of the glycosomal/peroxisomal membrane protein import mechanism.

ATP-Binding Cassette Transporters↗