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Purification and Interaction Analysis of a Plant-Specific RAB5 Effector by In Vitro Pull-Down Assay

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Plant Endosomes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2177))

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Abstract

RAB GTPases regulate membrane traffic by interacting with effector proteins in the GTP-bound active form. RAB GTPases are highly conserved in a broad range of eukaryotic organisms, while land plants and some green algal species possess a plant-specific RAB5 group. A plant-specific RAB5 in Arabidopsis called ARA6 was shown to regulate a characteristic trafficking route, and participate in abiotic and biotic stress responses. The identification of ARA6 effectors is a powerful strategy to get insights into the molecular basis of ARA6 functions. Recently, we identified an ARA6 effector, PLANT-UNIQUE RAB5 EFFECTOR 2 (PUF2), and characterized its functions by biochemical means. PUF2 was hardly expressed as a recombinant protein in the bacterial system, but we solved this problem by optimizing the codon usage of PUF2 CDS to suite for expression in Escherichia coli. Here, we present the protocol we employed to purify PUF2 protein, and to test its nucleotide state-specific interaction with ARA6 by in vitro pull-down assay. This approach would be extended to analyze the molecular functions of other effector proteins of RAB GTPases.

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Acknowledgments

This work was financially supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to T.U., 24114003 and 24370019, and E.I., 15K18527 and 17K15144), a Grant-in-Aid for JSPS fellows (E.I., 2010649), the Mitsubishi Foundation, Yamada Science Foundation, Kato Memorial Bioscience Foundation, NIBB Collaborative Research Program (16-339, 17-302, 18-302 to E.I.), and the Building of Consortia for the Development of Human Research in Science and Technology, MEXT, Japan.

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Authors and Affiliations

  1. Department of Natural Sciences, International Christian University, Tokyo, Japan

    Emi Ito & Seung-won Choi

  2. Division of Cellular Dynamics, National Institute for Basic Biology, Okazaki, Aichi, Japan

    Takashi Ueda

  3. Department of Basic Biology, SOKENDAI, Myodaiji-cho, Okazaki, Aichi, Japan

    Takashi Ueda

  4. Institute for Global Leadership, Ochanomizu University, Tokyo, Japan

    Emi Ito

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  1. Emi Ito
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  2. Seung-won Choi
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  3. Takashi Ueda
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Correspondence to Takashi Ueda .

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Editors and Affiliations

  1. Department of Botany and Laboratory of Cell and Molecular Biology, University of Wisconsin-Madison, Madison, WI, USA

    Marisa S. Otegui

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Ito, E., Choi, Sw., Ueda, T. (2020). Purification and Interaction Analysis of a Plant-Specific RAB5 Effector by In Vitro Pull-Down Assay. In: Otegui, M. (eds) Plant Endosomes. Methods in Molecular Biology, vol 2177. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0767-1_15

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  • DOI: https://doi.org/10.1007/978-1-0716-0767-1_15

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