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Deconvolution of Multiple Rab Binding Domains Using the Batch Yeast 2-Hybrid Method DEEPN

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Rab GTPases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2293))

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Abstract

A hallmark of functionally significant interactions between Rab proteins and their targets is whether that binding depends on the type of nucleotide bound to the Rab GTPase. A system that can directly compare those sets of interactions mediated by a Rab in its GTP-bound conformation versus its GDP bound conformation would provide a direct route to finding biologically relevant partners. Comprehensive large-scale yeast 2-hybrid assays allow a potential method to compare one interactome against another provided that the same set of potentially interacting partners is interrogated between samples. Here we describe the use of such a yeast 2-hybrid system that lends itself toward comparing pairs of Rab mutants, locked in either their GTP or GDP conformation. Importantly, using a complex library of protein fragments as potential binding (“prey”) partners, identification of interacting proteins as well as the domain(s) mediating those interactions can be determined using a series of sequence analyses and binary validation experiments.

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Authors and Affiliations

  1. Molecular Physiology and Biophysics, Carver College of Medicine, University of Iowa, Iowa City, IA, USA

    Tabitha A. Peterson & Robert C. Piper

Authors
  1. Tabitha A. Peterson
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  2. Robert C. Piper
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Correspondence to Robert C. Piper .

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Editors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Peggy and Charles Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

    Guangpu Li

  2. Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago, Chicago, IL, USA

    Nava Segev

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Peterson, T.A., Piper, R.C. (2021). Deconvolution of Multiple Rab Binding Domains Using the Batch Yeast 2-Hybrid Method DEEPN . In: Li, G., Segev, N. (eds) Rab GTPases. Methods in Molecular Biology, vol 2293. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1346-7_9

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  • DOI: https://doi.org/10.1007/978-1-0716-1346-7_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1345-0

  • Online ISBN: 978-1-0716-1346-7

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