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. 2018 Sep 7;293(36):14155-14164.
doi: 10.1074/jbc.RA118.001854. Epub 2018 Jul 16.

A non-canonical GTPase interaction enables ORP1L-Rab7-RILP complex formation and late endosome positioning

Xinli Ma  1   2 , Kai Liu  2   3   4   5 , Jian Li  1 , Huanhuan Li  1   2 , Jun Li  1 , Yingfang Liu  1   6 , Chonglin Yang  7   4   5 , Huanhuan Liang  8   9
Affiliations

A non-canonical GTPase interaction enables ORP1L-Rab7-RILP complex formation and late endosome positioning

Xinli Ma et al. J Biol Chem. .

Abstract

Endosomal transport represents the primary mode for intracellular trafficking and signaling transduction and thus has to be tightly controlled. The molecular processes controlling the endosomal positioning utilize several large protein complexes, one of which contains the small GTPase Rab7, Rab-interacting lysosomal protein (RILP), and oxysterol-binding protein-related protein 1 (ORP1L). Rab7 is known to interact with RILP through a canonical binding site termed the effector-interacting switch region, but it is not clear how Rab7 interacts with ORP1L, limiting our understanding of the overall process. Here, we report structural and biochemical investigation of the Rab7-ORP1L interaction. We found that, contrary to prior studies, the interaction between Rab7 and the N-terminal ankyrin repeat domain (ARDN) of ORP1L is independent of Rab7's GTP- or GDP-binding state. Moreover, we show that Rab7 interacts with ORP1L ARDN via a unique region consisting of helix3 (α3) and 310-helix 2 (η2). This architecture leaves the canonical effector-interacting switch regions available for RILP binding and thus allows formation of the ORP1L-Rab7-RILP tripartite complex. Mutational disruption of the interacting interface between ORP1L and Rab7 compromised the ability of ORP1L-Rab7-RILP to regulate the late endosome positioning. Collectively, our results again manifested the versatility in the interaction between GTPase and its effector.

Keywords: GTPase; ORP1L; RILP; Rab7; cholesterol; crystal structure; endosome; protein complex; protein-protein interaction; trafficking.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
The interaction between ORP1L ARDN and Rab7 is GTP-independent. A, the cellular interaction between full-length hORP1L and WT or mutated Rab7 (Q67L and T22N) was tested using a coimmunoprecipitation assay in HEK 293 cells. The GFP-Rab7 proteins were immobilized using GFP-Trap beads, and hORP1L, the precipitated protein, was detected using anti-FLAG antibody. B, pulldown of Rab7-Q67L by GST-tagged ORP1L proteins. A truncated form of GST-tagged ORP1L was incubated with purified His-tagged Rab7-Q67L protein in the presence of GTP and then immobilized on GSH-Sepharose beads. After extensive washing, Rab7-Q67L bound to ORP1L was detected using anti-His antibody. C, pulldown of ARDN by WT or mutated Rab7 (Q67L or T22N). The purified GST-tagged Rab7 proteins were incubated with His-tagged ORP1L ARDN (residues 1–136) and GTP or GDP as indicated, and then immobilized on GSH-Sepharose beads. After extensive washing, the ORP1L ARDN protein was detected using anti-His antibody. Data shown in A–C are representative of two independent experiments. D, the dissociation constants for ARDN binding to GTP- or GDP-bound Rab7 are identical as determined by ITC. IP, immunoprecipitation.
Figure 2.
Figure 2.
Structures of mORP1L ARDN and ARDN-Rab7 complex. A, gel filtration profiles of ARDN, Rab7-Q67L, and ARDN-Rab7 complex. B, overall structure of ARDN (ORP1L(1–136)), composed of four ANKs and three fingers. C, overall structure of the ARDN-Rab7 complex. ARDN is shown in green, and Rab7 is shown in light blue. The switch I, interswitch, and switch II regions of Rab7 are shown in purple, cyan, and orange, respectively. GTP is shown as red sticks, and magnesium is shown as a yellow sphere. The secondary structure elements are shown. D, ARDN, Rab7, and RILP associate into a ternary complex in solution. GST-tagged ARDN was incubated with His-tagged RILP and His-tagged Rab7 and then immobilized on GSH-Sepharose beads. After extensive washing, RILP and Rab7 were detected using anti-His antibody. E, structure model of a ternary ORP1L-Rab7-RILP complex shows that Rab7 binds ORP1L and RILP simultaneously using two distinct regions. The RILP dimer is shown in pink. ARDN and Rab7 are shown in colors identical to those used in C.
Figure 3.
Figure 3.
The structural details of ARDN-Rab7 interaction. A, the important residues on the ARDN-Rab7 interface. The hydrogen bonds are shown in dashed gray lines. The salt bridges are shown in dashed black lines. B, detailed hydrophobic interactions on the interface. C, sequence alignment of human Rab7, Rab3, Rab4, Rab5, and Rab34 around the α3-η2 region. Residues involved in ORP1L interaction are highlighted with blue squares. D and E, pulldown of Rab7-Q67L by GST-tagged ARDN (ORP1L(1–136)) mutants. F and G, pulldown of ARDN (ORP1L(1–136)) by GST-tagged Rab7 mutants. Data shown are representative of two independent experiments.
Figure 4.
Figure 4.
Effects of mutations on ORP1L-Rab7 interaction and colocalization in cells. A, left panel, coimmunoprecipitation between full-length GFP-tagged WT or mutant ORP1L and FLAG-tagged Rab7. Immunoprecipitations (IP) were performed with anti-FLAG M2-agarose beads, and precipitated proteins were detected with anti-GFP antibody. Right panel, coimmunoprecipitation between GFP-tagged WT or mutant Rab7 and FLAG-tagged ORP1L. Immunoprecipitations were performed with anti-FLAG M2-agarose beads, and precipitated proteins were detected with anti-GFP antibody. B, confocal images of the colocalization of full-length GFP-ORP1L-WT or -Y57A/F58A mutant with mCherry-Rab7 in HeLa cells transfected with these plasmids. Scale bars, 5 μm. The graph shows quantification of protein colocalization. Pearson's correlation coefficient is plotted on the y axis. ≥50 cells were scored. Error bars represent S.E. Significance was tested by unpaired Student's t test: ***, p < 0.001. C, confocal images of the colocalization of full-length GFP-ORP1L-WT or -Y57A/F58A mutant with mCherry-CD63 in HeLa cells transfected with these plasmids. Scale bars, 5 μm. D, colocalization between full-length ORP1L and Rab7-WT or -S111A mutant. HeLa cells were treated with control siRNA or Rab7 siRNA to knock down (KD) endogenous Rab7 expression. Then GFP-ORP1L and mCherry-Rab7-WT or -S111A mutant plasmids were cotransfected. Scale bars, 5 μm. E, immunoblotting (IB) of Rab7 in HeLa cells treated with control (Ctrl) siRNA or Rab7 siRNA.
Figure 5.
Figure 5.
ORP1L-Rab7 interaction affects ER-LE contact and LE positioning. A, effects of Rab7 interaction on ORP1L(1–514)-induced ER-LE membrane contact site. HeLa cells were transfected with the indicated expression vectors and stained with Rab7 and HA antibodies 12 h post-transfection. Quantifications are Pearson's coefficients between Rab7 and HA-VAP in ≥50 cells. Scale bars, 5 μm. B, effects of Rab7 interaction on ORP1L(1–514)-mediated LE positioning in the presence of RILP. HeLa cells were transfected with the indicated expression vectors and observed 12 h post-transfection. Quantifications are Pearson's coefficients between GFP-ORP1L and mCherry-RILP in ≥50 cells. Scale bars, 5 μm. Error bars represent S.E. Significance in this figure was tested by one-way analysis of variance: ***, p < 0.001; NS, not significant. BFP, blue fluorescent protein.
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