Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
STAR Protoc
2023 Mar 17;41:102111. doi: 10.1016/j.xpro.2023.102111.
Show Gene links
Show Anatomy links
Cell-free reconstitution of peroxisomal matrix protein import using Xenopus egg extract.
Skowyra ML
,
Rapoport TA
.
???displayArticle.abstract???
Peroxisomes are vital metabolic organelles whose matrix enzymes are imported from the cytosol in a folded state by the soluble receptor PEX5. The import mechanism has been challenging to decipher because of the lack of suitable in vitro systems. Here, we present a protocol for reconstituting matrix protein import using Xenopus egg extract. We describe how extract is prepared, how to replace endogenous PEX5 with recombinant versions, and how to perform and interpret a peroxisomal import reaction using a fluorescent cargo. For complete details on the use and execution of this protocol, please refer to Skowyra and Rapoport (2022).1.
Antonenkov,
The behavior of peroxisomes in vitro: mammalian peroxisomes are osmotically sensitive particles.
2004, Pubmed
Antonenkov,
The behavior of peroxisomes in vitro: mammalian peroxisomes are osmotically sensitive particles.
2004,
Pubmed
Antoniou,
Optimization of Soluble Expression and Purification of Recombinant Human Rhinovirus Type-14 3C Protease Using Statistically Designed Experiments: Isolation and Characterization of the Enzyme.
2017,
Pubmed
Bartoszewska,
The significance of peroxisomes in secondary metabolite biosynthesis in filamentous fungi.
2011,
Pubmed
Behari,
The carboxyl terminus of isocitrate lyase is not essential for import into glyoxysomes in an in vitro system.
1993,
Pubmed
Blok,
Unique double-ring structure of the peroxisomal Pex1/Pex6 ATPase complex revealed by cryo-electron microscopy.
2015,
Pubmed
Brickner,
Protein transport into higher plant peroxisomes. In vitro import assay provides evidence for receptor involvement.
1997,
Pubmed
Brocard,
Peroxisome targeting signal 1: is it really a simple tripeptide?
2006,
Pubmed
Cannon-Carlson,
Modification of the Laemmli sodium dodecyl sulfate-polyacrylamide gel electrophoresis procedure to eliminate artifacts on reducing and nonreducing gels.
1997,
Pubmed
Carvalho,
The N-terminal half of the peroxisomal cycling receptor Pex5p is a natively unfolded domain.
2006,
Pubmed
Debelyy,
Ubp15p, a ubiquitin hydrolase associated with the peroxisomal export machinery.
2011,
Pubmed
Deming,
Study of apoptosis in vitro using the Xenopus egg extract reconstitution system.
2006,
Pubmed
,
Xenbase
Feng,
Structure and function of the peroxisomal ubiquitin ligase complex.
2022,
Pubmed
Feng,
A peroxisomal ubiquitin ligase complex forms a retrotranslocation channel.
2022,
Pubmed
Ferdous,
Optimization of E. Coli Tip-Sonication for High-Yield Cell-Free Extract using Finite Element Modeling.
2021,
Pubmed
Field,
Xenopus extract approaches to studying microtubule organization and signaling in cytokinesis.
2017,
Pubmed
,
Xenbase
Francisco,
Ubiquitin in the peroxisomal protein import pathway.
2014,
Pubmed
Francisco,
A cargo-centered perspective on the PEX5 receptor-mediated peroxisomal protein import pathway.
2013,
Pubmed
Fujiki,
Peroxisome biogenesis and human peroxisome-deficiency disorders.
2016,
Pubmed
Fujiki,
Post-translational import of fatty acyl-CoA oxidase and catalase into peroxisomes of rat liver in vitro.
1985,
Pubmed
Gatto,
Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5.
2000,
Pubmed
Gerhart,
Cell cycle dynamics of an M-phase-specific cytoplasmic factor in Xenopus laevis oocytes and eggs.
1984,
Pubmed
,
Xenbase
Gillespie,
Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins.
2012,
Pubmed
,
Xenbase
Grou,
Identification of ubiquitin-specific protease 9X (USP9X) as a deubiquitinase acting on ubiquitin-peroxin 5 (PEX5) thioester conjugate.
2012,
Pubmed
Grou,
Members of the E2D (UbcH5) family mediate the ubiquitination of the conserved cysteine of Pex5p, the peroxisomal import receptor.
2008,
Pubmed
Gusseck,
A molecular approach to fertilization. I. Disulfide bonds in Xenopus laevis jelly coat and a molecular hypothesis for fertilization.
1971,
Pubmed
,
Xenbase
Jansen,
Comparative Genomics of Peroxisome Biogenesis Proteins: Making Sense of the PEX Proteins.
2021,
Pubmed
Kalel,
Unraveling of the Structure and Function of Peroxisomal Protein Import Machineries.
2018,
Pubmed
Kao,
Peroxisome Function, Biogenesis, and Dynamics in Plants.
2018,
Pubmed
Kunze,
The type-2 peroxisomal targeting signal.
2020,
Pubmed
Lambert,
FPbase: a community-editable fluorescent protein database.
2019,
Pubmed
Lismont,
Peroxisomal Hydrogen Peroxide Metabolism and Signaling in Health and Disease.
2019,
Pubmed
Lodhi,
Peroxisomes: a nexus for lipid metabolism and cellular signaling.
2014,
Pubmed
Léon,
Uniqueness of the mechanism of protein import into the peroxisome matrix: transport of folded, co-factor-bound and oligomeric proteins by shuttling receptors.
2006,
Pubmed
Maller,
Pioneering the Xenopus oocyte and egg extract system.
2012,
Pubmed
,
Xenbase
Miyata,
Shuttling mechanism of peroxisome targeting signal type 1 receptor Pex5: ATP-independent import and ATP-dependent export.
2005,
Pubmed
Mukai,
The cytosolic peroxisome-targeting signal (PTS)-receptors, Pex7p and Pex5pL, are sufficient to transport PTS2 proteins to peroxisomes.
2019,
Pubmed
Okumoto,
Peroxisomal Membrane and Matrix Protein Import Using a Semi-Intact Mammalian Cell System.
2017,
Pubmed
Pedrosa,
Peroxisomal monoubiquitinated PEX5 interacts with the AAA ATPases PEX1 and PEX6 and is unfolded during its dislocation into the cytosol.
2018,
Pubmed
Pushparaj,
Revisiting the Micropipetting Techniques in Biomedical Sciences: A Fundamental Prerequisite in Good Laboratory Practice.
2020,
Pubmed
Rapoport,
Structural and Mechanistic Insights into Protein Translocation.
2017,
Pubmed
Rodrigues,
A cell-free organelle-based in vitro system for studying the peroxisomal protein import machinery.
2016,
Pubmed
Romano,
Peroxisome protein import recapitulated in Xenopus egg extracts.
2019,
Pubmed
,
Xenbase
Schneider,
NIH Image to ImageJ: 25 years of image analysis.
2012,
Pubmed
Skowyra,
PEX5 translocation into and out of peroxisomes drives matrix protein import.
2022,
Pubmed
,
Xenbase
Tamaoki,
Morphological, biochemical, transcriptional and epigenetic responses to fasting and refeeding in intestine of Xenopus laevis.
2016,
Pubmed
,
Xenbase
Taylor,
A defined methodology for reliable quantification of Western blot data.
2013,
Pubmed
Terlecky,
Quantitative analysis of peroxisomal protein import in vitro.
2001,
Pubmed
Terlecky,
In vitro analysis of peroxisomal protein import.
2002,
Pubmed
Walter,
Current Advances in Protein Import into Peroxisomes.
2019,
Pubmed
Wanders,
The physiological functions of human peroxisomes.
2023,
Pubmed
Wang,
Roles of the TRAPP-II Complex and the Exocyst in Membrane Deposition during Fission Yeast Cytokinesis.
2016,
Pubmed
Wendland,
Cytosol-dependent peroxisomal protein import in a permeabilized cell system.
1993,
Pubmed
Wiedemann,
Mitochondrial Machineries for Protein Import and Assembly.
2017,
Pubmed
Wühr,
Deep proteomics of the Xenopus laevis egg using an mRNA-derived reference database.
2014,
Pubmed
,
Xenbase
Yamashita,
Mitotic phosphorylation of Pex14p regulates peroxisomal import machinery.
2020,
Pubmed