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A cell-free organelle-based in vitro system for studying the peroxisomal protein import machinery

Nature Protocols volume 11, pages 2454–2469 (2016)Cite this article

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Abstract

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 35S-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.

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Figure 1: The PEX5-mediated protein import pathway.
Figure 2: Receptor- and cargo-centered in vitro import assays.

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Acknowledgements

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.

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  1. Tony A Rodrigues and Tânia Francisco: These authors contributed equally to this work.

Authors and Affiliations

  1. Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Porto, Portugal

    Tony A Rodrigues, Tânia Francisco, Ana F Dias, Ana G Pedrosa, Cláudia P Grou & Jorge E Azevedo

  2. Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal

    Tony A Rodrigues, Tânia Francisco, Ana F Dias, Ana G Pedrosa, Cláudia P Grou & Jorge E Azevedo

  3. Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto, Porto, Portugal

    Tony A Rodrigues, Ana F Dias, Ana G Pedrosa & Jorge E Azevedo

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  1. Tony A Rodrigues
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Contributions

T.A.R., T.F., C.P.G. and J.E.A. designed research. T.A.R., T.F., A.F.D. and A.G.P. performed the experiments. T.A.R., T.F., A.F.D., A.G.P., C.P.G. and J.E.A. wrote and edited the manuscript.

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Correspondence to Jorge E Azevedo.

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Rodrigues, T., Francisco, T., Dias, A. et al. A cell-free organelle-based in vitro system for studying the peroxisomal protein import machinery. Nat Protoc 11, 2454–2469 (2016). https://doi.org/10.1038/nprot.2016.147

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