Quantitative bead-based flow cytometry for assaying Rab7 GTPase interaction with the Rab-interacting lysosomal protein (RILP) effector protein

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.

Fig. 1

GSH bead-based flow cytometry assay configurations for quantitative measurements of GTPase-effector protein binding. (a) Assay design for detecting Rab7 and Rab-interacting lysosomal protein (RILP) effector protein interaction based on detection of bound fluorescent BODIPY-GTP. GST-RILP (Rab binding domain of RILP only) is immobilized on 13 μm Superdex beads coated with GSH and incubated with purified His-tagged Rab7 complexed to fluorescent BODIPY-GTP. Flow cytometry detection is based on bead-associated fluorescence when His-Rab7-BODIPY-GTP binds to RILP. (b) Assay design for detecting Rab7 and Rab-interacting lysosomal protein (RILP) protein interaction based on detection of GFP-tagged Rab7. GST-RILP (Rab-binding domain of RILP only) is immobilized on 13 μm Superdex beads coated with GSH and incubated with GFP- tagged Rab7 complexed to nonhydrolyzable GTP-γ-S. Flow cytometry detection is based on bead-associated fluorescence when GFP-Rab7-GTP-γ-S binds to RILP

Fig. 2

Quantitative measurements of GTPase-effector protein binding. (a) Flow cytometry-based measurement of total Rab7 binding to RILP by detecting fluorescent BODIPY-GTP shows binding is saturable, quantitative, and specific. Rab7 binding to GSH beads coated with GST alone or without any protein coating was minimal. (b) Specific binding of Rab7-RILP with unwanted nonspecific background binding subtracted

Fig. 3

Quantitative measurements of Rab7 GTPase-RILP effector binding is rapid and nucleotide specific. (a) Flow cytometry-based measurement of the long-term kinetics of Rab7 binding to RILP by detecting fluorescent GFP-Rab7 shows binding is rapid and dependent on Rab7 being GTP bound. GFP-Rab7 prebound to nonhydrolyzable GTP-γ-S is nearly instantaneous and stable over 120 min. Addition of GDP results in displacement of GTP-γ-S from Rab7 and dissociation of GFP-Rab7GDP complex detected as a loss of bead-associated fluorescence. There is no binding of GFP-Rab7 in the GDP-bound state to RILP. (b) Data from panel (a) were replotted starting at the 30 min time point to allow determination of the dissociation rate of GFP-Rab7-GDP from RILP. Data was fitted to single-phase exponential decay function using PRISM software yielding a dissociation rate of 0.020 ± 0.003 min⁻¹

Fig. 4

Flow cytometry-based measurements show Rab7 GTPase-RILP effector binding is temperature dependent and sensitive to nucleotide hydrolysis. (a–c) Flow cytometry-based measurement of His-Rab7 binding to RILP by detecting fluorescent BODIPY-GTP. (a) Dose-dependent His-Rab7 binding is temperature dependent and negatively affected by GTP hydrolysis at higher temperature (b and c). (b) A kinetic temperature-shift experiment shows His-Rab7 binding to RILP increases steadily at 4 and 22 °C, but decreases rapidly upon shift to 37 °C, likely due to GTP hydrolysis and dissociation of Rab7 from RILP. (c) Data in (b) were replotted starting at the 75 min time point to allow determination of the dissociation rate. Data were fitted to a two-phase exponential decay function using PRISM software yielding a dissociation rate of 0.014 ± 0.003 min⁻¹ for the slow phase and 0.0436 ± 0.053 min^-1 for the fast phase. The rate constant value deduced for the fast phase is statistically close to that measured for the dissociation of GFP-Rab7-GDP from RILP in Fig. 2a, supporting the conclusion that 37 °C stimulates Rab7 GTPase hydrolysis of BODIPY-GTP and consequent dissociation from RILP

Fig. 5

GSH bead-based flow cytometry assays for quantitative measurements of Rab7 guanine nucleotide binding and dissociation kinetics. (a) Assay design for detecting nucleotide binding and dissociation kinetics on Rab7 based on detection of bound fluorescent BODIPY-GTP. GST-Rab7 is immobilized on 13 μm Superdex beads coated with GSH and detection is based on fluorescent BODIPY-GTP binding. (b) BODIPY-GTP (100 nM final) was added to GST-Rab7 immobilized on GSH beads suspended in 300 μl of buffer (first arrow). The ligand was allowed to bind for 100 s and then DMSO (1 % final) or CID 1067700 (10 μM final) was added at 150 s (second arrow). While the addition of a competitive guanine nucleotide-binding inhibitor (CID 1067700) causes dissociation of BODIPY-GTP, addition of DMSO has no effect on BODIPY-GTP-binding kinetics

Fig. 6

GSH bead-based flow cytometry assay establishes that a competitive guanine nucleotide-binding inhibitor (CID 1067700) retains Rab7 in an inactive conformation. (a) Flow cytometry-based measurement of Rab7 binding to RILP by detecting fluorescent GFP-Rab7 in the presence of CID 1067700. GFP-Rab7 increasingly binds to RILP at increasing concentrations of nonhydrolyzable GTP-γ-S but fails to bind to RILP with increasing concentrations of GDP or CID 1067700. Rab7 does not adopt ‘active’ like conformation in the presence of CID 1067700 alone. (b) Graphical display of the two distinct possible scenarios that can result from competitive inhibitor (CID 1067700) binding to Rab7. In scenario 1, binding of the competitive inhibitor dissociates GTP from the nucleotide-binding pocket, but keeps Rab7 in the active conformation, which would still allow binding to the RILP effector. In scenario 2, binding of the competitive inhibitor to Rab7 causes the GTPase to assume or remain in the inactive conformation, which does not favor interaction with RILP. The data we have presented support scenario 2 and suggest that the guanine nucleotide-binding inhibitor should functionally inhibit Rab7 in cell-based assays