A Cargo-centered Perspective on the PEX5 Receptor-mediated Peroxisomal Protein Import Pathway

Francisco, Tânia; Rodrigues, Tony A.; Freitas, Marta O.; Grou, Cláudia P.; Carvalho, Andreia F.; Sá-Miranda, Clara; Pinto, Manuel P.; Azevedo, Jorge E.

doi:10.1074/jbc.m113.487140

Cited by 47 publications

(80 citation statements)

References 64 publications

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“…3D, compare lanes 1 and 5 with lanes 3 and 7). As shown before, this recombinant protein completely blocks the PEX5-mediated protein import pathway (36). Interestingly, when the protease treatment was omitted, cleaved PEX7 was also detected in the supernatant of the import assays but only under import-permissive conditions (Fig.…”

Section: Fig 3 Pex7 Becomes Transiently Exposed To the Organelle Matrmentioning

confidence: 85%

“…2A showing that PEX7 import is not blocked in assays containing apyrase, a condition previously shown to block PEX5L monoubiquitination (35,36), also suggest that import of PEX7, like import of prethiolase, occurs upstream of PEX5L monoubiquitination. Additional data supporting this conclusion are presented in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…ATP or AMP-PNP were used at a 3 mM final concentration. NTP depletion from both PNS and reticulocyte lysates using apyrase (grade VII; Sigma) was done exactly as described previously (36). Where indicated, import reaction mixtures were supplemented with recombinant PEX5 proteins [PEX5L, PEX5L(N526K), ⌬C1PEX5L, ⌬C1PEX5L(C11A), or ⌬C1PEX5S; 30 nM final concentrations], GST-Ub or bovine ubiquitin (10 M), and recombinant p-PHYH or m-PHYH (140 nM final concentration).…”

Section: Plasmidsmentioning

confidence: 99%

“…Following this docking event, PEX5 gets inserted into the DTM, acquiring a transmembrane topology (33,34), a step that results in the translocation of the cargo protein across the organelle membrane and its release into the peroxisomal lumen (35,36). Interestingly, none of these steps require cytosolic ATP (35)(36)(37). PEX5 is then extracted from the DTM through a two-step mechanism.…”

mentioning

confidence: 99%

“…After binding a cargo protein in the cytosol, PEX5 interacts with the peroxisomal docking/ translocation machinery (DTM) (29), a peroxisomal membrane protein complex comprising PEX13, PEX14, and the RING peroxins PEX2, PEX10, and PEX12 (30)(31)(32). Following this docking event, PEX5 gets inserted into the DTM, acquiring a transmembrane topology (33,34), a step that results in the translocation of the cargo protein across the organelle membrane and its release into the peroxisomal lumen (35,36). Interestingly, none of these steps require cytosolic ATP (35)(36)(37).…”

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confidence: 99%

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A PEX7-Centered Perspective on the Peroxisomal Targeting Signal Type 2-Mediated Protein Import Pathway

Rodrigues

Alencastre

Francisco

et al. 2014

Molecular and Cellular Biology

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dPeroxisomal matrix proteins are synthesized on cytosolic ribosomes and transported to the organelle by shuttling receptors. Matrix proteins containing a type 1 signal are carried to the peroxisome by PEX5, whereas those harboring a type 2 signal are transported by a PEX5-PEX7 complex. The pathway followed by PEX5 during the protein transport cycle has been characterized in detail. In contrast, not much is known regarding PEX7. In this work, we show that PEX7 is targeted to the peroxisome in a PEX5-and cargo-dependent manner, where it becomes resistant to exogenously added proteases. Entry of PEX7 and its cargo into the peroxisome occurs upstream of the first cytosolic ATP-dependent step of the PEX5-mediated import pathway, i.e., before monoubiquitination of PEX5. PEX7 passing through the peroxisome becomes partially, if not completely, exposed to the peroxisome matrix milieu, suggesting that cargo release occurs at the trans side of the peroxisomal membrane. Finally, we found that export of peroxisomal PEX7 back into the cytosol requires export of PEX5 but, strikingly, the two export events are not strictly coupled, indicating that the two proteins leave the peroxisome separately. P eroxisomal matrix proteins are synthesized on cytosolic ribosomes and posttranslationally targeted to the organelle via one of two peroxisomal targeting sequences (PTSs): (i) the PTS type 1 (PTS1), a small peptide frequently ending with the sequence SKL located at the C terminus of the vast majority of matrix proteins (1, 2), and (ii) the PTS2, a degenerated nonapeptide present at the amino terminus of a few matrix proteins (3-5). In contrast to the PTS1, the PTS2 is generally cleaved when the protein reaches the organelle matrix (5-7). In mammals and many other organisms, both PTS1 and PTS2 proteins are transported to the organelle by PEX5, the peroxisomal shuttling receptor (8-11). The interaction of PEX5 with PTS1 proteins is direct (12-16), whereas the interaction between PEX5 and PTS2 proteins requires the adaptor protein PEX7 (17-19). Interestingly, not all PEX5 proteins in a mammalian cell are capable of binding PEX7. This is due to alternative splicing of the PEX5 transcript, which yields two major isoforms of the receptor, PEX5S and PEX5L. In contrast to PEX5L, PEX5S is not able to bind PEX7 because it lacks an internal 37-amino-acid domain (8, 10). The situation in yeasts is different. While these organisms also use PEX5 to target PTS1 proteins to the peroxisome, import of PTS2 proteins is promoted by PEX7 and a species-specific member of the so-called PEX20 family (19-23), a group of proteins that have no mammalian counterpart but that display functional similarities with the N-terminal half of PEX5L (17,19,24).The pathway followed by PEX5 during the protein transport process is reasonably known (25-28). After binding a cargo protein in the cytosol, PEX5 interacts with the peroxisomal docking/ translocation machinery (DTM) (29), a peroxisomal membrane protein complex comprising PEX13, PEX14, and the RING peroxins PEX2, PE...

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Section: Fig 3 Pex7 Becomes Transiently Exposed To the Organelle Matrmentioning

confidence: 85%

Section: Resultsmentioning

confidence: 99%

Section: Plasmidsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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A PEX7-Centered Perspective on the Peroxisomal Targeting Signal Type 2-Mediated Protein Import Pathway

Rodrigues

Alencastre

Francisco

et al. 2014

Molecular and Cellular Biology

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Protein Import into Peroxisomes: The Principles and Methods of Studying

Fujiki

Okumoto

Honsho

2015

Encyclopedia of Life Sciences

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Peroxisomes are essential intracellular organelles that involve many metabolic processes, such as β‐oxidation of very long‐chain fatty acids and synthesis of plasmalogen and bile acids as well as generation and degradation of hydrogen peroxide. These peroxisomal functions are fulfilled by strictly and spatiotemporally regulated compartmentation of the proteins catalysing these reactions. Defects in peroxisomal protein import results in inherited peroxisome biogenesis disorders in humans. Peroxisomal matrix and membrane proteins are synthesised on free ribosomes but transported into peroxisomes by distinct pathways determined by specific targeting signals and their receptors. The mechanism by which this is achieved has been clarified by identification of many PEX genes and the products named peroxins, the essential factors for peroxisome biogenesis. This article introduces several basic methods to investigate protein import into peroxisomes. Key Concepts Peroxisome plays an essential role in various metabolic pathways. Deficiency of peroxisomes in human causes severe foetal genetic diseases, peroxisome‐deficient disorders. Functions and the integrity of peroxisomes are archived by proper import of both matrix and membrane proteins into peroxisomes. Peroxisomal proteins are encoded by nuclear DNA, synthesised in cytosolic free ribosomes and post‐translationally imported into pre‐existing peroxisomes. Peroxisomal matrix and membrane proteins are translocated into peroxisomes via different pathways in a manner dependent on their distinct targeting signals. Many of peroxins encoded by PEX genes are responsible factors for peroxisome biogenesis and are involved in the import of peroxisomal proteins. Various experimental approaches have elucidated the molecular mechanisms underlying peroxisome biogenesis. Post‐translational import of peroxisomal proteins into isolated peroxisomes can be reproduced in vitro . Semi‐permeabilised cells, in which only the plasma membrane is selectively permeabilised, are used to investigate peroxisomal protein import, as with living cells.

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Protein transport into peroxisomes: Knowns and unknowns

et al. 2017

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Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and rapidly transported into the organelle by a complex machinery. The data gathered in recent years suggest that this machinery operates through a syringe-like mechanism, in which the shuttling receptor PEX5 - the "plunger" - pushes a newly synthesized protein all the way through a peroxisomal transmembrane protein complex - the "barrel" - into the matrix of the organelle. Notably, insertion of cargo-loaded receptor into the "barrel" is an ATP-independent process, whereas extraction of the receptor back into the cytosol requires its monoubiquitination and the action of ATP-dependent mechanoenzymes. Here, we review the main data behind this model.

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A Cargo-centered Perspective on the PEX5 Receptor-mediated Peroxisomal Protein Import Pathway

Cited by 47 publications

References 64 publications

A PEX7-Centered Perspective on the Peroxisomal Targeting Signal Type 2-Mediated Protein Import Pathway

A PEX7-Centered Perspective on the Peroxisomal Targeting Signal Type 2-Mediated Protein Import Pathway

Protein Import into Peroxisomes: The Principles and Methods of Studying

Protein transport into peroxisomes: Knowns and unknowns

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