Abstract
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.
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Acknowledgments
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 Tecnologia)/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), UIDB/04293/2020, and “Investigating the molecular mechanisms of mammalian pexophagy” (EXPL/BIA-MOL/1664/2021). MJF, TAR, AGP, and TF are supported by Fundação para a Ciência e Tecnologia, Programa Operacional Potencial Humano do QREN and Fundo Social Europeu.
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Ferreira, M.J., Rodrigues, T.A., Pedrosa, A.G., Francisco, T., Azevedo, J.E. (2023). A Cell-Free In Vitro Import System for Peroxisomal Proteins Containing a Type 2 Targeting Signal (PTS2). In: Schrader, M. (eds) Peroxisomes. Methods in Molecular Biology, vol 2643. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3048-8_23
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DOI: https://doi.org/10.1007/978-1-0716-3048-8_23
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