RESUMO
PEX5, the peroxisomal protein shuttling receptor, binds newly synthesized proteins in the cytosol and transports them to the organelle. During its stay at the peroxisomal protein translocon, PEX5 is monoubiquitinated at its cysteine 11 residue, a mandatory modification for its subsequent ATP-dependent extraction back into the cytosol. The reason why a cysteine and not a lysine residue is the ubiquitin acceptor is unknown. Using an established rat liver-based cell-free in vitro system, we found that, in contrast to wild-type PEX5, a PEX5 protein possessing a lysine at position 11 is polyubiquitinated at the peroxisomal membrane, a modification that negatively interferes with the extraction process. Wild-type PEX5 cannot retain a polyubiquitin chain because ubiquitination at cysteine 11 is a reversible reaction, with the E2-mediated deubiquitination step presenting faster kinetics than PEX5 polyubiquitination. We propose that the reversible nonconventional ubiquitination of PEX5 ensures that neither the peroxisomal protein translocon becomes obstructed with polyubiquitinated PEX5 nor is PEX5 targeted for proteasomal degradation.
Assuntos
Cisteína , Lisina , Animais , Ratos , Proteínas de Transporte/metabolismo , Cisteína/metabolismo , Lisina/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos/química , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Transporte Proteico , UbiquitinaçãoRESUMO
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.
Assuntos
Catarata/genética , Mutação de Sentido Incorreto , Sinais de Orientação para Peroxissomos , Receptor 1 de Sinal de Orientação para Peroxissomos/genética , Peroxissomos/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico Ativo , Catarata/congênito , Catarata/metabolismo , Cromossomos Humanos Par 12 , Consanguinidade , Feminino , Ligação Genética , Humanos , Cristalino/metabolismo , Masculino , Camundongos , Camundongos Knockout , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Proteína Sequestossoma-1/metabolismo , Sequenciamento do ExomaRESUMO
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.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Citosol/metabolismo , Proteínas de Membrana/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Mapas de Interação de Proteínas , Humanos , Modelos Moleculares , Receptor 1 de Sinal de Orientação para Peroxissomos/química , Peroxissomos/metabolismo , Transporte Proteico , Desdobramento de Proteína , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
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.
Assuntos
Peroxissomos/metabolismo , Transporte Proteico/fisiologia , Animais , Humanos , Receptor 2 de Sinal de Orientação para Peroxissomos/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Transdução de Sinais/fisiologia , Ubiquitinação/fisiologiaRESUMO
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.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Peroxissomos/metabolismo , ATPases Associadas a Diversas Atividades Celulares/química , Animais , Humanos , Receptor 1 de Sinal de Orientação para Peroxissomos/química , Transporte ProteicoRESUMO
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 exogenously 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.
Assuntos
Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Modelos Biológicos , Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Transporte Biológico , Endopeptidase K/metabolismo , Deleção de Genes , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Mutação , Mutação de Sentido Incorreto , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , SolubilidadeRESUMO
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.
Assuntos
Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Células Eucarióticas/química , Células Eucarióticas/metabolismo , Regulação da Expressão Gênica , Humanos , Receptor 2 de Sinal de Orientação para Peroxissomos , Receptor 1 de Sinal de Orientação para Peroxissomos , Peroxissomos/química , Plantas/química , Plantas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
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.
Assuntos
Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais , Trifosfato de Adenosina/metabolismo , Animais , Proteínas de Transporte/metabolismo , Citosol/metabolismo , Humanos , Camundongos , Modelos Biológicos , Receptor 1 de Sinal de Orientação para Peroxissomos , Sinais Direcionadores de Proteínas , Transporte Proteico , Frações Subcelulares/metabolismo , TemperaturaRESUMO
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.
Assuntos
Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina/metabolismo , Animais , Citosol/enzimologia , Ativação Enzimática/fisiologia , Ésteres/metabolismo , Feminino , Células HEK293 , Células HeLa , Humanos , Hidrólise , Fígado/enzimologia , Masculino , Receptor 1 de Sinal de Orientação para Peroxissomos , Coelhos , Ratos , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/isolamento & purificação , Especificidade por Substrato/fisiologia , Ubiquitina Tiolesterase/isolamento & purificaçãoRESUMO
Cell-free in vitro systems are invaluable tools to study the molecular mechanisms of protein translocation across biological membranes. We have been using such a strategy to dissect the mechanism of the mammalian peroxisomal matrix protein import machinery. Here, we provide a detailed protocol to import proteins containing a peroxisomal targeting signal type 2 (PTS2) into the organelle. The in vitro system consists of incubating a 35S-labeled reporter protein with a post-nuclear supernatant from rat/mouse liver. At the end of the incubation, the organelle suspensions are generally treated with an aggressive protease to degrade reporter proteins that did not enter peroxisomes, and the organelles are isolated by centrifugation and analyzed by SDS-PAGE and autoradiography. This in vitro system is particularly suited to characterize the functional consequences of PEX5 and PEX7 mutations found in patients affected with a peroxisomal biogenesis disorder.
Assuntos
Transtornos Peroxissômicos , Sinais de Orientação para Peroxissomos , Ratos , Camundongos , Animais , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Transporte Proteico , Peroxissomos/metabolismo , Transtornos Peroxissômicos/metabolismo , Mamíferos/metabolismoRESUMO
The AAA ATPases PEX1â¢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 PEX1â¢PEX6.
Assuntos
Proteínas de Membrana , Receptor 1 de Sinal de Orientação para Peroxissomos , Peroxissomos , Ubiquitina , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Membrana/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Peroxissomos/metabolismo , Transporte Proteico , Ubiquitina/metabolismo , Ubiquitinação , Humanos , Sistema Livre de CélulasRESUMO
Despite intensive research on peroxisome biochemistry, the role of glutathione in peroxisomal redox homeostasis has remained a matter of speculation for many years, and only recently has this issue started to be experimentally addressed. Here, we summarize and compare data from several organisms on the peroxisome-glutathione topic. It is clear from this comparison that the repertoire of glutathione-utilizing enzymes in peroxisomes of different organisms varies widely. In addition, the available data suggest that the kinetic connectivity between the cytosolic and peroxisomal pools of glutathione may also be different in different organisms, with some possessing a peroxisomal membrane that is promptly permeable to glutathione whereas in others this may not be the case. However, regardless of the differences, the picture that emerges from all these data is that glutathione is a crucial component of the antioxidative system that operates inside peroxisomes in all organisms.
Assuntos
Glutationa , Peroxissomos , Peroxissomos/metabolismo , Glutationa/metabolismo , Antioxidantes/metabolismo , Oxirredução , HomeostaseRESUMO
Despite the large amounts of H2O2 generated in mammalian peroxisomes, cysteine residues of intraperoxisomal proteins are maintained in a reduced state. The biochemistry behind this phenomenon remains unexplored, and simple questions such as "is the peroxisomal membrane permeable to glutathione?" or "is there a thiol-disulfide oxidoreductase in the organelle matrix?" still have no answer. We used a cell-free in vitro system to equip rat liver peroxisomes with a glutathione redox sensor. The organelles were then incubated with glutathione solutions of different redox potentials and the oxidation/reduction kinetics of the redox sensor was monitored. The data suggest that the mammalian peroxisomal membrane is promptly permeable to both reduced and oxidized glutathione. No evidence for the presence of a robust thiol-disulfide oxidoreductase in the peroxisomal matrix could be found. Also, prolonged incubation of organelle suspensions with glutaredoxin 1 did not result in the internalization of the enzyme. To explore a potential role of glutathione in intraperoxisomal redox homeostasis we performed kinetic simulations. The results suggest that even in the absence of a glutaredoxin, glutathione is more important in protecting cysteine residues of matrix proteins from oxidation by H2O2 than peroxisomal catalase itself.
Assuntos
Peroxissomos , Proteína Dissulfeto Redutase (Glutationa) , Ratos , Animais , Dissulfeto de Glutationa/metabolismo , Peroxissomos/metabolismo , Cisteína/metabolismo , Proteína Dissulfeto Redutase (Glutationa)/análise , Proteína Dissulfeto Redutase (Glutationa)/metabolismo , Peróxido de Hidrogênio/metabolismo , Glutationa/metabolismo , Oxirredução , Proteínas/metabolismo , Mamíferos/metabolismo , HomeostaseRESUMO
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.
Assuntos
Catalase/química , Catalase/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Multimerização Proteica/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/farmacologia , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Animais , Concentração Inibidora 50 , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Camundongos , Receptor 1 de Sinal de Orientação para Peroxissomos , Peroxissomos/efeitos dos fármacos , Peroxissomos/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Coelhos , Receptores Citoplasmáticos e Nucleares/químicaRESUMO
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.
Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , Peroxissomos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Humanos , Domínios Proteicos , Transporte Proteico , Transdução de SinaisRESUMO
PEX13 and PEX14 are two core components of the so-called peroxisomal docking/translocation module, the transmembrane hydrophilic channel through which newly synthesized peroxisomal proteins are translocated into the organelle matrix. The two proteins interact with each other and with PEX5, the peroxisomal matrix protein shuttling receptor, through relatively well characterized domains. However, the topologies of these membrane proteins are still poorly defined. Here, we subjected proteoliposomes containing PEX13 or PEX14 and purified rat liver peroxisomes to protease-protection assays and analyzed the protected protein fragments by mass spectrometry, Edman degradation and western blotting using antibodies directed to specific domains of the proteins. Our results indicate that PEX14 is a bona fide intrinsic membrane protein with a Nin -Cout topology, and that PEX13 adopts a Nout -Cin topology, thus exposing its carboxy-terminal Src homology 3 [SH3] domain into the organelle matrix. These results reconcile several enigmatic findings previously reported on PEX13 and PEX14 and provide new insights into the organization of the peroxisomal protein import machinery. ENZYMES: Trypsin, EC3.4.21.4; Proteinase K, EC3.4.21.64; Tobacco etch virus protease, EC3.4.22.44.
Assuntos
Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Peroxissomos/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , Animais , Lipossomos/metabolismo , Masculino , Proteínas de Membrana/genética , Transporte Proteico , Ratos , Ratos Wistar , Proteínas Recombinantes/genética , Proteínas Repressoras/genéticaRESUMO
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.