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The AAA ATPases PEX1•PEX6 extract PEX5, the peroxisomal protein shuttling receptor, from the peroxisomal membrane so that a new protein transport cycle can start. Extraction requires ubiquitination of PEX5 at residue 11 and involves a threading mechanism, but how exactly this occurs is unclear. We used a cell-free in vitro system and a variety of engineered PEX5 and ubiquitin molecules to challenge the extraction machinery. We show that PEX5 modified with a single ubiquitin is a substrate for extraction and extend previous findings proposing that neither the N- nor the C-terminus of PEX5 are required for extraction. Chimeric PEX5 molecules possessing a branched polypeptide structure at their C-terminal domains can still be extracted from the peroxisomal membrane thus suggesting that the extraction machinery can thread more than one polypeptide chain simultaneously. Importantly, we found that the PEX5-linked monoubiquitin is unfolded at a pre-extraction stage and, accordingly, an intra-molecularly cross-linked ubiquitin blocked extraction when conjugated to residue 11 of PEX5. Collectively, our data suggest that the PEX5-linked monoubiquitin is the extraction initiator and that the complete ubiquitin-PEX5 conjugate is threaded by PEX1•PEX6.

The 7th Open European Peroxisome Meeting (OEPM) was held on the 15th and 16th of October 2020 due to the Corona pandemic as ZOOM conference. The OEPM is a biannual meeting organized by European expert researchers in peroxisome biology. Previous meetings were held in Leuven, Belgium (2006), Lunteren, The Netherlands (2010), Dijon, France (2012), Neuss, Germany (2014), Vienna, Austria (2016), and Groningen, The Netherlands (2018). For the 2020 meeting nearly 200 participants registered from 10 European countries, as well as Israel, Canada, the USA, Japan, Russia, and South Korea. At the OEPM, it is tradition that only young peroxisome researchers (PhD students or junior Post-docs) present their work to an international audience. At this year’s meeting, 46 talks were presented in eight sessions. During the meeting, the OEPM 2020 Peroxisome Research Young Investigator Award (sponsored by Ruhr-University of Bochum) was awarded. This award is given to the early career researcher, who was first author of the best publication in the peroxisome field over the last 2 years. At this meeting, the award was given to Victoria Riccio from the group of Peter Kim at the University of Toronto (Canada). The awarded paper describes that the deubiquitinating enzyme USP30 prevents pexophagy by counteracting the action of the peroxisomal E3 ubiquitin ligase PEX2. They also show that USP30 can rescue the peroxisome loss observed in some disease-causing peroxisome mutations, pointing to a potential therapeutic target (Riccio et al. 2019). Victoria Riccio received the prize during the meeting and presented the Young Investigator Award lecture about the paper. Members of the jury were Myriam Baes (Leuven), Mustapha Cherkaoui-Malki (Dijon), Ralf Erdmann (Bochum), Marc Fransen (Leuven), Aurora Pujol (Barcelona), Sigrun Reumann (Hamburg), Maya Schuldiner (Rehovot), Ron Wanders (Amsterdam), Bettina Warscheid (Freiburg), Hans Waterham (Amsterdam), and Einat Zalckvar (Rehovot). Iulia Revenco (KU Leuven, Belgium) and Marc Pilegaard Pedersen (University of Groningen, The Netherlands) presented the Marie Sklodowska-Curie Innovative European Training Network ‘PerICo’ (Peroxisome Interactions and Communication) that is coordinated by Prof. Dr. Ida van der Klei (University of Groningen). PerICo brings together ten full partners and five partner organizations, from seven European countries, together with Israel and Canada, and fosters the education of 15 PhD-students in projects aimed at uncovering how peroxisomes participate in cellular interactions and signaling. In the evening session, Andrew Longenecker presented ‘Diego’s story’, the case of his son Diego, who suffers from a PEX10 Zellweger Spectrum Disorder (ZSD), a peroxisome biogenesis disorder (PBD). In his presentation, he reported on a non-profit PBD-project (DiegoZSDResearch@gmail.com) that he and his wife created to engage with the research community and other parents. Peroxisomes are ubiquitous cell organelles of eukaryotic cells. They are surrounded by a single membrane and harbor a highly variable enzyme composition that determines the function of these multi-purpose organelles. Common functions of peroxisomes include β-oxidation of fatty acids and containment of pathways that produce hydrogen peroxide, which is then detoxified by catalase. Examples for species-specific peroxisomal pathways are alpha-oxidation of branched-chain fatty acids and synthesis of plasmalogens in humans, and contribution to the synthesis of penicillin in fungi or bile acids in humans (Wanders and Waterham 2006). Defects in peroxisomal enzymes or biogenesis of the organelle result in peroxisomal disorders, like X-linked Adrenoleukodystrophy or the Zellweger syndrome, respectively (Waterham et al. 2016). There is an increasing interest in peroxisome research, which is fueled by the fact that major questions concerning the biology of these fascinating organelles are still unanswered. Such as the import of folded, even oligomeric proteins into peroxisomes with the underlying mechanism still being unresolved (Baker et al. 2016; Walter and Erdmann 2019). Our knowledge on the biogenesis, dynamics (fission, transport), degradation by autophagy (pexophagy), etc. is still scarce, leaving room for exciting discoveries (Costello and Schrader 2018; Islinger et al. 2018; Mast et al. 2020; Mahalingam et al. 2020). Moreover, peroxisomes function in concert with other organelles, including metabolic interaction and signal transduction. In this context, an emerging theme is the investigation of physical contacts of peroxisomes and mitochondria, lipid droplets, vacuoles (lysosomes), and the ER via contact sites (Shai et al. 2016; Islinger et al. 2018; Farre et al. 2019; Schrader et al. 2020). Moreover, glycosomes, as peroxisomes of trypanosomatid parasites are called (due to the localization of glycolytic enzymes) have been identified as suitable targets for novel leads compounds for therapy of diseases like African sleeping sickness, Chagas Disease and Leishmaniasis (Dawidowski et al. 2017). Another reason for the steadily increasing interest is the perception that the cellular duties of these fascinating organelles go well beyond their undoubted metabolic functions. Peroxisomes turned out to have important non-metabolic roles, such as their function as signaling platform in antiviral response and thus their contribution to innate immunity (Ferreira et al. 2019), and their contribution to cooperative cell defense mechanisms against oxidative stress (Fujiki et al. 2020). Moreover, peroxisomes are critical contributors to ageing, longevity (Deori et al. 2018) and age-related disorders, including Alzheimer’s disease and cancer (Berger et al. 2016; Islinger et al. 2018; Dahabieh et al. 2018). Below is an overview of the sessions and talks of this very successful and inspiring OEPM. The order of talks at the meeting were essentially ordered alphabetically.

Peroxisomes, single-membrane intracellular organelles, play an important role in various metabolic pathways. The translocation of proteins from the cytosol to peroxisomes depends on peroxisome import receptor proteins and defects in peroxisome transport result in a wide spectrum of peroxisomal disorders. Here, we report a large consanguineous family with autosomal recessive congenital cataracts and developmental defects. Genome-wide linkage analysis localized the critical interval to chromosome 12p with a maximum two-point LOD score of 4.2 (θ = 0). Next-generation exome sequencing identified a novel homozygous missense variant (c.653 T > C; p.F218S) in peroxisomal biogenesis factor 5 (PEX5), a peroxisome import receptor protein. This missense mutation was confirmed by bidirectional Sanger sequencing. It segregated with the disease phenotype in the family and was absent in ethnically matched control chromosomes. The lens-specific knockout mice of Pex5 recapitulated the cataractous phenotype. In vitro import assays revealed a normal capacity of the mutant PEX5 to enter the peroxisomal Docking/Translocation Module (DTM) in the presence of peroxisome targeting signal 1 (PTS1) cargo protein, be monoubiquitinated and exported back into the cytosol. Importantly, the mutant PEX5 protein was unable to form a stable trimeric complex with peroxisomal biogenesis factor 7 (PEX7) and a peroxisome targeting signal 2 (PTS2) cargo protein and, therefore, failed to promote the import of PTS2 cargo proteins into peroxisomes. In conclusion, we report a novel missense mutation in PEX5 responsible for the defective import of PTS2 cargo proteins into peroxisomes resulting in congenital cataracts and developmental defects.

Despite having a membrane that is impermeable to all but the smallest of metabolites, peroxisomes acquire their newly synthesized (cytosolic) matrix proteins in an already folded conformation. In some cases, even oligomeric proteins have been reported to translocate the organelle membrane. The protein sorting machinery that accomplishes this feat must be rather flexible and, unsurprisingly, several of its key components have large intrinsically disordered domains. Here, we provide an overview on these domains and their interactions trying to infer their functional roles in this protein sorting pathway.

In contrast to many protein translocases that use ATP or GTP hydrolysis as the driving force to transport proteins across biological membranes, the peroxisomal matrix protein import machinery relies on a regulated self-assembly mechanism for this purpose and uses ATP hydrolysis only to reset its components. The ATP-dependent protein complex in charge of resetting this machinery—the Receptor Export Module (REM)—comprises two members of the “ATPases Associated with diverse cellular Activities” (AAA+) family, PEX1 and PEX6, and a membrane protein that anchors the ATPases to the organelle membrane. In recent years, a large amount of data on the structure/function of the REM complex has become available. Here, we discuss the main findings and their mechanistic implications. This work was financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project PTDC/BEX-BCM/2311/2014 (POCI-01-0145-FEDER-016613) and the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274). This work is a result of the project NORTE-01-0145-FEDER-000008—Porto Neurosciences and Neurologic Disease Research Initiative at I3S, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). A.B.-B., A.G.P., M.J.F., T.F. and T.A.R. are supported by Fundação para a Ciência e Tecnologia, Programa Operacional Potencial Humano do QREN, and Fundo Social Europeu.

Here we describe a protocol to dissect the peroxisomal matrix protein import pathway using a cell-free in vitro system. The system relies on a postnuclear supernatant (PNS), which is prepared from rat/mouse liver, to act as a source of peroxisomes and cytosolic components. A typical in vitro assay comprises the following steps: (i) incubation of the PNS with an in vitro-synthesized 35 S-labeled reporter protein; (ii) treatment of the organelle suspension with a protease that degrades reporter proteins that have not associated with peroxisomes; and (iii) SDS-PAGE/autoradiography analysis. To study transport of proteins into peroxisomes, it is possible to use organelle-resident proteins that contain a peroxisomal targeting signal (PTS) as reporters in the assay. In addition, a receptor (PEX5L/S or PEX5L.PEX7) can be used to report the dynamics of shuttling proteins that mediate the import process. Thus, different but complementary perspectives on the mechanism of this pathway can be obtained. We also describe strategies to fortify the system with recombinant proteins to increase import yields and block specific parts of the machinery at a number of steps. The system recapitulates all the steps of the pathway, including mono-ubiquitination of PEX5L/S at the peroxisome membrane and its ATP-dependent export back into the cytosol by PEX1/PEX6. An in vitro import(/export) experiment can be completed in 24 h. We thank M. Fransen, Katholieke Universiteit-Leuven, for critical comments on the manuscript and for the plasmid encoding histidine-tagged PEX19. We thank P. van Veldhoven, Katholieke Universiteit-Leuven, and P. Maciel, Universidade do Minho, for the expression plasmids encoding prePHYH and GST-Ub, respectively. This work was funded by FEDER—Fundo Europeu de Desenvolvimento Regional through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, Portugal’s FCT—Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia e Inovação in the framework of the projects ‘The molecular mechanism of protein import into peroxisomes’ (FCOMP-01-0124-FEDER-019731-PTDC/BIA-BCM/118577/2010), ‘Institute for Research and Innovation in Health Sciences’ (POCI-01-0145-FEDER-007274) and ‘The molecular mechanisms of peroxisome biogenesis’ (PTDC /BEX-BC M/2311/2014) and Norte 2020—Programa Operacional Regional do Norte, under the application of the ‘Porto Neurosciences and Neurologic Disease Research Initiative at i3S (NORTE-01-0145-FEDER-000008)’, awarded to J.E.A. T.A.R., T.F., A.F.D. and C.P.G. were supported by Fundação para a Ciência e a Tecnologia, Programa Operacional Potencial Humano do QREN and Fundo Social Europeu.

PEX1 and PEX6 are two members of the ATPases associated with diverse cellular activities (AAA) family and the core components of the receptor export module of the peroxisomal matrix protein import machinery. Their role is to extract monoubiquitinated PEX5, the peroxisomal protein-shuttling receptor, from the peroxisomal membrane docking/translocation module (DTM), so that a new cycle of protein transportation can start. Recent data have shown that PEX1 and PEX6 form a heterohexameric complex that unfolds substrates by processive threading. However, whether the natural substrate of the PEX1-PEX6 complex is monoubiquitinated PEX5 (Ub-PEX5) itself or some Ub-PEX5-interacting component(s) of the DTM remains unknown. In this work, we used an established cell-free in vitro system coupled with photoaffinity cross-linking and protein PEGylation assays to address this problem. We provide evidence suggesting that DTM-embedded Ub-PEX5 interacts directly with both PEX1 and PEX6 through its ubiquitin moiety and that the PEX5 polypeptide chain is globally unfolded during the ATP-dependent extraction event. These findings strongly suggest that DTM-embedded Ub-PEX5 is a bona fide substrate of the PEX1-PEX6 complex. The authors thank Britta Moellers (Ruhr-Universität Bochum, Germany) for providing plasmids and recombinant protein for the generation of the anti-PEX6 antibody. This work was funded by FEDER (Fundo Europeu de Desenvolvimento Regional), through COMPETE 2020 –Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through Fundação para a Ciência e Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Inovação in the framework of the projects “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274) and “The molecular mechanisms of peroxisome biogenesis” (PTDC/BEX-BCM/2311/2014), and through Norte 2020 – Programa Operacional Regional do Norte, under the application of the “Porto Neurosciences and Neurologic Disease Research Initiative at i3S” (NORTE-01-0145-FEDER-000008). A.G.P, T.F., D.B., A.F.D., A.B.B. and T.A.R. are supported by Fundação para a Ciência e Tecnologia, Programa Operacional Potencial Humano do QREN and Fundo Social Europeu.

A remarkable property of the machinery for import of peroxisomal matrix proteins is that it can accept already folded proteins as substrates. This import involves binding of newly synthesized proteins by cytosolic peroxisomal biogenesis factor 5 (PEX5) followed by insertion of the PEX5– cargo complex into the peroxisomal membrane at the docking/translocation module (DTM). However, how these processes occur remains largely unknown. Here, we used truncated PEX5 molecules to probe the DTM architecture. We found that the DTM can accommodate a larger number of truncated PEX5 molecules comprising amino acid residues 1–197 than full-length PEX5 molecules. A shorter PEX5 version (PEX5(1–125)) still interacted correctly with the DTM; however, this species was largely accessible to exoge-nously added proteinase K, suggesting that this protease can access the DTM occupied by a small PEX5 protein. Interestingly, the PEX5(1–125)–DTM interaction was inhibited by a polypeptide comprising PEX5 residues 138 – 639. Apparently, the DTM can recruit soluble PEX5 through interactions with different PEX5 domains, suggesting that the PEX5–DTM interactions are to some degree fuzzy. Finally, we found that the interaction between PEX5 and PEX14, a major DTM component, is stable at pH 11.5. Thus, there is no reason to assume that the hitherto intriguing resistance of DTM-bound PEX5 to alkaline extraction reflects its direct contact with the peroxisomal lipid bilayer. Collectively, these results suggest that the DTM is best described as a large cavity-forming protein assembly into which cytosolic PEX5 can enter to release its cargo. This work was supported in part by Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020-Operational Pro-gramme for Competitiveness and Internationalization (POCI), Portugal 2020 and by Portuguese funds through Fundação para a Ciência e a Tec-nologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the projects “Institute for Research and Innovation in Health Sciences” (Grant POCI-01-0145-FEDER-007274) and “The molecular mechanisms of peroxisome biogenesis” (Grant PTDC/BEX-BCM/2311/2014) and through Norte 2020-Programa Operacional Regional do Norte under the application of the “Porto Neurosciences and Neurologic Disease Research Initiative at i3S” (Grant NORTE-01-0145-FEDER-000008). The authors declare that they have no conflicts of interest with the contents of this article.

Protease protection assays are powerful tools to determine the topology of organelle proteins. Their simplicity, together with the fact that they are particularly suited to characterize endogenous proteins, are their major advantages and the reason why these assays have been in use for so many years. Here, we provide a detailed protocol to use with mammalian peroxisomes. Suggestions on how these assays can be controlled, and how to identify some technical pitfalls, are also presented.

PEX5 is the shuttling receptor for newly synthesized peroxisomal matrix proteins. Alone, or with the help of an adaptor protein, this receptor binds peroxisomal matrix proteins in the cytosol and transports them to the peroxisomal membrane docking/translocation module (DTM). The interaction between cargo-loaded PEX5 and the DTM ultimately results in its insertion into the DTM with the concomitant translocation of the cargo protein across the organelle membrane. PEX5 is not consumed in this event; rather it is dislocated back into the cytosol so that it can promote additional rounds of protein transportation. Remarkably, the data collected in recent years indicate that dislocation is preceded by monoubiquitination of PEX5 at a conserved cysteine residue. This mandatory modification is not the only type of ubiquitination occurring at the DTM. Indeed, several findings suggest that defective receptors jamming the DTM are polyubiquitinated and targeted to the proteasome for degradation. This work was funded by FEDER funds through the Operational Competitiveness Programme e COMPETE and by National Funds through FCT e Fundação para a Ciência e a Tecnologia under the project FCOMP-01-0124-FEDER-019731 (PTDC/BIA-BCM/118577/2010). T.F., T.A.R., M.P.P. and C.P.G. are supported by Fundação para a Ciência e a Tecnologia, Programa Operacional Potencial Humano do QREN, and Fundo Social Europeu. A.F.C. is supported by Programa Ciência, funded by Programa Operacional Potencial Humano do QREN, Tipologia 4.2, Promoção do Emprego Científico, by Fundo Social Europeu and by National Funds from Ministério da Ciência, Tecnologia e Ensino Superior.

Girão et al. use structure-guided functional mutants of CLASP2 to show that recognition of growing microtubule plus-ends through EB–protein interaction and the ability to associate with curved microtubule protofilaments through TOG2 and TOG3 domains promote growth and stabilization of kinetochore–microtubules required for poleward flux., CLASPs are conserved microtubule plus-end–tracking proteins that suppress microtubule catastrophes and independently localize to kinetochores during mitosis. Thus, CLASPs are ideally positioned to regulate kinetochore–microtubule dynamics required for chromosome segregation fidelity, but the underlying mechanism remains unknown. Here, we found that human CLASP2 exists predominantly as a monomer in solution, but it can self-associate through its C-terminal kinetochore-binding domain. Kinetochore localization was independent of self-association, and driving monomeric CLASP2 to kinetochores fully rescued normal kinetochore–microtubule dynamics, while partially sustaining mitosis. CLASP2 kinetochore localization, recognition of growing microtubule plus-ends through EB–protein interaction, and the ability to associate with curved microtubule protofilaments through TOG2 and TOG3 domains independently sustained normal spindle length, timely spindle assembly checkpoint satisfaction, chromosome congression, and faithful segregation. Measurements of kinetochore–microtubule half-life and poleward flux revealed that CLASP2 regulates kinetochore–microtubule dynamics by integrating distinctive microtubule-binding properties at the kinetochore–microtubule interface. We propose that kinetochore CLASP2 suppresses microtubule depolymerization and detachment by binding to curved protofilaments at microtubule plus-ends., Graphical Abstract

Protease protection assays are powerful tools to determine the topology of organelle proteins. Their simplicity, together with the fact that they are particularly suited to characterize endogenous proteins, are their major advantages and the reason why these assays have been in use for so many years. Here, we provide a detailed protocol to use with mammalian peroxisomes. Suggestions on how these assays can be controlled, and how to identify some technical pitfalls, are also presented.

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. We would like to thank Dr. Marc Fransen (KU Leuven) for his critical reading of the manuscript. This work was financed by FEDER - Fundo Europeu de Desenvolvimento Regional, funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministerio da Ciência, Tecnologia e Inovação in the framework of the projects “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274) and “Themolecular mechanisms of peroxisome biogenesis” (PTDC/BEX-BCM/2311/2014), and through Norte 2020–Programa Operacional Regional do Norte, under the application of the “Porto Neurosciences and Neurologic Disease Research Initiative at i3S (NORTE-01-0145-FEDER-000008).” T.F., T.A.R., A.F.D., A.B.B., and D.B. were supported by Fundação para a Ciência e a Tecnologia, Programa Operacional Potencial Humano do QREN and Fundo Social Europeu.

O presente estudo visa analisar o desempenho financeiro das PME hoteleiras portuguesas na região do Algarve e da Madeira. Para o estudo elaborado, utilizou-se como metodologia o modelo de dados em painel recorrendo à base de dados Amadeus. A amostra é constituída por 365 PME hoteleiras portuguesas com dados referentes ao período 2011-2015. Os resultados do presente estudo indicam que para a região do Algarve o endividamento de curto prazo e o endividamento de longo prazo apresentam uma relação negativa com a rendibilidade dos ativos. A idade da empresa apresenta uma relação positiva. As variáveis dimensão, tangibilidade e crescimento do ativo não são estatisticamente significativas. Para a região da Madeira os resultados do presente estudo indicam que o endividamento de curto prazo e tangibilidade apresentam uma relação negativa com a rendibilidade dos ativos. A dimensão apresenta uma relação positiva as variáveis endividamento de longo prazo, idade e crescimento do ativo não são estatisticamente significativas.

Peroxin 5 (PEX5), the peroxisomal protein shuttling receptor, binds newly synthesized peroxisomal matrix proteins in the cytosol and promotes their translocation across the organelle membrane. During the translocation step, PEX5 itself becomes inserted into the peroxisomal docking/translocation machinery. PEX5 is then monoubiquitinated at a conserved cysteine residue and extracted back into the cytosol in an ATP-dependent manner. We have previously shown that the ubiquitin-PEX5 thioester conjugate (Ub-PEX5) released into the cytosol can be efficiently disrupted by physiological concentrations of glutathione, raising the possibility that a fraction of Ub-PEX5 is nonenzymatically deubiquitinated in vivo. However, data suggesting that Ub-PEX5 is also a target of a deubiquitinase were also obtained in that work. Here, we used an unbiased biochemical approach to identify this enzyme. Our results suggest that ubiquitin-specific protease 9X (USP9X) is by far the most active deubiquitinase acting on Ub-PEX5, both in female rat liver and HeLa cells. We also show that USP9X is an elongated monomeric protein with the capacity to hydrolyze thioester, isopeptide, and peptide bonds. The strategy described here will be useful in identifying deubiquitinases acting on other ubiquitin conjugates.

Newly synthesized peroxisomal matrix proteins are targeted to the organelle by PEX5. PEX5 has a dual role in this process. First, it acts as a soluble receptor recognizing these proteins in the cytosol. Subsequently, at the peroxisomal docking/translocation machinery, PEX5 promotes their translocation across the organelle membrane. Despite significant advances made in recent years, several aspects of this pathway remain unclear. Two important ones regard the formation and disruption of the PEX5-cargo protein interaction in the cytosol and at the docking/translocation machinery, respectively. Here, we provide data on the interaction of PEX5 with catalase, a homotetrameric enzyme in its native state. We found that PEX5 interacts with monomeric catalase yielding a stable protein complex; no such complex was detected with tetrameric catalase. Binding of PEX5 to monomeric catalase potently inhibits its tetramerization, a property that depends on domains present in both the N- and C-terminal halves of PEX5. Interestingly, the PEX5-catalase interaction is disrupted by the N-terminal domain of PEX14, a component of the docking/translocation machinery. One or two of the seven PEX14-binding diaromatic motifs present in the N-terminal half of PEX5 are probably involved in this phenomenon. These results suggest the following: 1) catalase domain(s) involved in the interaction with PEX5 are no longer accessible upon tetramerization of the enzyme; 2) the catalase-binding interface in PEX5 is not restricted to its C-terminal peroxisomal targeting sequence type 1-binding domain and also involves PEX5 N-terminal domain(s); and 3) PEX14 participates in the cargo protein release step.

In the field of intracellular protein sorting, peroxisomes are most famous by their capacity to import oligomeric proteins. The data supporting this remarkable property are abundant and, understandably, have inspired a variety of hypothetical models on how newly synthesized (cytosolic) proteins reach the peroxisome matrix. However, there is also accumulating evidence suggesting that many peroxisomal oligomeric proteins actually arrive at the peroxisome still as monomers. In support of this idea, recent data suggest that PEX5, the shuttling receptor for peroxisomal matrix proteins, is also a chaperone/holdase, binding newly synthesized peroxisomal proteins in the cytosol and blocking their oligomerization. Here we review the data behind these two different perspectives and discuss their mechanistic implications on this protein sorting pathway. This article is part of a Special Issue entitled: Peroxisomes edited by Ralf Erdmann. This work was supported by national funds through FCT – Fundação para a Ciência e a Tecnologia/MEC-Ministério da Educação e Ciência and when applicable co-funded by FEDER funds within the partnership agreement PT2020 related with the research unit number 4293; and by the project FCOMP-01-0124-FEDER-019731 (PTDC/BIABCM/118577/2010) funded by national funds through FCT and co-funded by Fundo Europeu de Desenvolvimento Regional (FEDER) through the Operation- alCompetitiveness Programme(COMPETE).A. F.D., T.F., T.A.R. and C. P. G. were supported by FCT, Programa Operacional Potencial Humano (POPH) do Quadro de Referência Estratégico Nacional (QREN), and Fundo Social Europeu (FSE).

Newly synthesized peroxisomal proteins containing a cleavable type 2 targeting signal (PTS2) are transported to the peroxisome by a cytosolic PEX5-PEX7 complex. There, the trimeric complex becomes inserted into the peroxisomal membrane docking/translocation machinery (DTM), a step that leads to the translocation of the cargo into the organelle matrix. Previous work suggests that PEX5 is retained at the DTM during all the steps occurring at the peroxisome but whether the same applies to PEX7 was unknown. By subjecting different pre-assembled trimeric PEX5-PEX7-PTS2 complexes to in vitro co-import/export assays we found that the export competence of peroxisomal PEX7 is largely determined by the PEX5 molecule that transported it to the peroxisome. This finding suggests that PEX7 is also retained at the DTM during the peroxisomal steps and implies that cargo proteins are released into the organelle matrix by DTM-embedded PEX7. The release step does not depend on PTS2 cleavage. Rather, our data suggest that insertion of the trimeric PEX5-PEX7-PTS2 protein complex into the DTM is probably accompanied by conformational alterations in PEX5 to allow release of the PTS2 protein into the organelle matrix. This work was funded by FEDER funds through the Operational Competitiveness Programme, COMPETE,and by National Funds through FCT, Fundacao para a Ciencia e a Tecnologia, under the project FCOMP-01-0124-FEDER-022718 (Pest-C/SAU/LA0002/2011) and FCOMP-01-0124-FEDER-019731 (PTDC/BIABCM/118577/2010). T.A.R. and C.P.G were supported by Fundacao para a Ciencia e a Tecnologia,Programa Operacional Potencial Humano do QREN and Fundo Social Europeu.

Peroxisomal 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. This work was funded by FEDER funds through the Operational Competitiveness Programme, COMPETE, and by National Funds through FCT, Fundação para a Ciência e a Tecnologia, under the project FCOMP-01-0124-FEDER-022718 (PEst-C/SAU/LA0002/2011) and FCOMP-01-0124-FEDER-019731(PTDC/BIABCM/118577/2010).T.A.R.,I.S.A.,T.F., and C.P.G. were supported by Fundação para a Ciência e a Tecnologia, Programa Operacional Potencial Humano do QREN, and Fundo Social Europeu. M.F. was supported by FWO-Vlaanderen (Onderzoeksproject G.0754.09) and KU Leuven (OT/09/045).

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and post-translationally targeted to the organelle by the soluble factor PEX5. Besides a role as a receptor, and probably as a chaperone, PEX5 also holds the key to the matrix of the organelle. Indeed, the available data suggest that PEX5 itself pushes these proteins across the peroxisomal membrane using as driving force the strong protein–protein interactions that it establishes with components of the peroxisomal membrane docking/translocation module (DTM). In recent years, much has been learned on how this transport system is reset and kept fine-tuned. Notably, this involves covalent modification of PEX5 with ubiquitin. Two types of PEX5 ubiquitination have been characterized: monoubiquitination at a conserved cysteine, a mandatory event for the extraction of PEX5 from the DTM; and polyubiquitination, probably the result of a quality control mechanism aiming at clearing the DTM from entangled PEX5 molecules. Monoubiquitination of PEX5 is transient in nature and the factors that reverse this modification have recently been identified.

Background: How the soluble receptor PEX5 delivers its cargoes to the peroxisome remains largely unknown. Results: Cargo translocation occurs after docking of the receptor at the peroxisome and before any ATP-dependent step. Conclusion: Translocation is concomitant with PEX5 insertion into the docking/translocation machinery. Significance: These results support a model in which cargoes are pushed across the peroxisomal membrane by PEX5. Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and post-translationally targeted to the organelle by PEX5, the peroxisomal shuttling receptor. The pathway followed by PEX5 during this process is known with reasonable detail. After recognizing cargo proteins in the cytosol, the receptor interacts with the peroxisomal docking/translocation machinery, where it gets inserted; PEX5 is then monoubiquitinated, extracted back to the cytosol and, finally, deubiquitinated. However, despite this information, the exact step of this pathway where cargo proteins are translocated across the organelle membrane is still ill-defined. In this work, we used an in vitro import system to characterize the translocation mechanism of a matrix protein possessing a type 1 targeting signal. Our results suggest that translocation of proteins across the organelle membrane occurs downstream of a reversible docking step and upstream of the first cytosolic ATP-dependent step (i.e. before ubiquitination of PEX5), concomitantly with the insertion of the receptor into the docking/translocation machinery.

Newly synthesized peroxisomal matrix proteins are targeted to the organelle by PEX5, the peroxisomal cycling receptor. Over the last few years, valuable data on the mechanism of this process have been obtained using a PEX5-centered in vitro system. The data gathered until now suggest that cytosolic PEX5·cargo protein complexes dock at the peroxisomal docking/translocation machinery, where PEX5 becomes subsequently inserted in an ATP-independent manner. This PEX5 species is then monoubiquitinated at a conserved cysteine residue, a mandatory modification for the next step of the pathway, the ATP-dependent dislocation of the ubiquitin-PEX5 conjugate back into the cytosol. Finally, the ubiquitin moiety is removed, yielding free PEX5. Despite its usefulness, there are many unsolved mechanistic aspects that cannot be addressed with this in vitro system and that call for a cargo protein-centered perspective instead. Here we describe a robust peroxisomal in vitro import system that provides this perspective. The data obtained with it suggest that translocation of a cargo protein across the peroxisomal membrane, including its release into the organelle matrix, occurs prior to PEX5 ubiquitination.

The peroxisomal protein import machinery displays remarkable properties. Be it its capacity to accept already folded proteins as substrates, its complex architecture or its energetics, almost every aspect of this machinery seems unique. The list of unusual properties is still growing as shown by the recent finding that one of its central components, Pex5p, is transiently monoubiquitinated at a cysteine residue. However, the data gathered in recent years also suggest that the peroxisomal import machinery is not that exclusive and similarities with p97/Cdc48-mediated processes and with multisubunit RING-E3 ligases are starting to emerge. Here, we discuss these data trying to distill the principles by which this complex machinery operates.

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and transported by the shuttling receptor PEX5 to the peroxisomal membrane docking/translocation machinery, where they are translocated into the organelle matrix. Under certain experimental conditions this protein import machinery has the remarkable capacity to accept already oligomerized proteins, a property that has heavily influenced current models on the mechanism of peroxisomal protein import. However, whether or not oligomeric proteins are really the best and most frequent clients of this machinery remain unclear. In this work, we present three lines of evidence suggesting that the peroxisomal import machinery displays a preference for monomeric proteins. First, in agreement with previous findings on catalase, we show that PEX5 binds newly synthesized (monomeric) acyl-CoA oxidase 1 (ACOX1) and urate oxidase (UOX), potently inhibiting their oligomerization. Second, in vitro import experiments suggest that monomeric ACOX1 and UOX are better peroxisomal import substrates than the corresponding oligomeric forms. Finally, we provide data strongly suggesting that although ACOX1 lacking a peroxisomal targeting signal can be imported into peroxisomes when co-expressed with ACOX1 containing its targeting signal, this import pathway is inefficient. This work was funded by Fundo Europeu de Desenvolvimento Regional (FEDER) through the Operational Competitiveness Programme (COMPETE); by National Funds through Fundação para a Ciência e a Tecnologia (FCT) under the project FCOMP-01–0124-FEDER-022718 (PEst-C/SAU/LA0002/2011) and FCOMP-01–0124-FEDER-019731 (PTDC/BIABCM/118577/2010); by Portuguese National Mass Spectrometry Network (RNEM) through the project REDE/1504/REM/2005; and by Química Orgânica, Produtos Naturais e Agroalimentares (QOPNA) research unit funds provided by FCT, European Union, QREN, FEDER and COMPETE under the projects PEst-C/QUI/UI0062/2013 and FCOMP-01–0124-FEDER-037296. M.O.F., T.F., T.A.R., M.P.P. and C.P.G. were supported by FCT, Programa Operacional Potencial Humano (POPH) do Quadro de Referência Estratégico Nacional (QREN) and Fundo Social Europeu. The work done in Leuven was supported by grants from the ‘Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (Onderzoeksproject G.0754.09)’ and the KU Leuven (OT/14/100).