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Rab GTPases pp 331–354Cite as

Quantitative Bead-Based Flow Cytometry for Assaying Rab7 GTPase Interaction with the Rab-Interacting Lysosomal Protein (RILP) Effector Protein

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1298))

Abstract

Rab7 facilitates vesicular transport and delivery from early endosomes to late endosomes as well as from late endosomes to lysosomes. The role of Rab7 in vesicular transport is dependent on its interactions with effector proteins, among them Rab-interacting lysosomal protein (RILP), which aids in the recruitment of active Rab7 (GTP-bound) onto dynein–dynactin motor complexes to facilitate late endosomal transport on the cytoskeleton. Here we detail a novel bead-based flow cytometry assay to measure Rab7 interaction with the Rab-interacting lysosomal protein (RILP) effector protein and demonstrate its utility for quantitative assessment and studying drug–target interactions. The specific binding of GTP-bound Rab7 to RILP is readily demonstrated and shown to be dose-dependent and saturable enabling K d and B max determinations. Furthermore, binding is nearly instantaneous and temperature-dependent. In a novel application of the assay method, a competitive small molecule inhibitor of Rab7 nucleotide binding (CID 1067700 or ML282) is shown to inhibit the Rab7–RILP interaction. Thus, the assay is able to distinguish that the small molecule, rather than incurring the active conformation, instead ‘locks’ the GTPase in the inactive conformation. Together, this work demonstrates the utility of using a flow cytometry assay to quantitatively characterize protein–protein interactions involving small GTPases and which has been adapted to high-throughput screening. Further, the method provides a platform for testing small molecule effects on protein–protein interactions, which can be relevant to drug discovery and development.

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Acknowledgements

This work was generously supported by National Science Foundation (MCB0956027) and the National Institutes of Health (R21NS7740241) to AWN and (P30CA1181000, U54MH074425, and U54MH084690) to LAS. DS was supported as a visiting MARC scholar (T34 GM008395, PI Zavala, CSUN) and as a summer intern (ASERT IRACDA K12 GM088021, PI Wandinger-Ness). We thank Ms. Janet Kelly for administrative support. We also acknowledge Elsa Romero and Patricia Jim for technical support. Small molecule screening was performed in the NMMLSC/UNMCMD and flow cytometry assays were conducted in the Flow Cytometry Shared Resource Center supported by the University of New Mexico Cancer Center (P30 CA11810).

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Authors and Affiliations

  1. Department of Pathology, University of New Mexico School of Medicine, Albuquerque, NM, 87131, USA

    Jacob O. Agola, Peter C. Simons, Tione Buranda, Larry A. Sklar & Angela Wandinger-Ness

  2. Cancer Center, University of New Mexico School of Medicine, Albuquerque, NM, 87131, USA

    Jacob O. Agola, Daniel F. Cimino, Peter C. Simons, Tione Buranda, Larry A. Sklar & Angela Wandinger-Ness

  3. Center for Micro-Engineered Materials, Department of Chemical and Biological Engineering, School of Engineering, University of New Mexico, Albuquerque, NM, 87131, USA

    Jacob O. Agola

  4. Department of Biology, California State University, Northridge, CA, 91330, USA

    Daniel Sivalingam

  5. Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095, USA

    Daniel Sivalingam

  6. Department of Cell Biology and Physiology, University of New Mexico School of Medicine, Albuquerque, NM, 87131, USA

    Daniel F. Cimino

  7. Center for Molecular Discovery, University of New Mexico School of Medicine, Albuquerque, NM, 87131, USA

    Peter C. Simons & Larry A. Sklar

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  1. Jacob O. Agola
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  2. Daniel Sivalingam
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Correspondence to Angela Wandinger-Ness .

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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, Oklahoma, USA

    Guangpu Li

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Agola, J.O. et al. (2015). Quantitative Bead-Based Flow Cytometry for Assaying Rab7 GTPase Interaction with the Rab-Interacting Lysosomal Protein (RILP) Effector Protein. In: Li, G. (eds) Rab GTPases. Methods in Molecular Biology, vol 1298. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2569-8_28

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  • DOI: https://doi.org/10.1007/978-1-4939-2569-8_28

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2568-1

  • Online ISBN: 978-1-4939-2569-8

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