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.
Highlights
Endosomal transport represents the primary mode for intracellular trafficking and signaling transduction and has to be tightly controlled
We found that full-length Human ORP1L (hORP1L) coprecipitated with both Q67L and T22N in HEK 293 cells (Fig. 1A), the amount of the hORP1L protein precipitated by Q67L was higher than that precipitated by T22N
We found that ankyrin repeat domain (ARDN) (ORP1L(1–136)) is sufficient for ORP1L to interact with Rab7
Summary
Endosomal transport represents the primary mode for intracellular trafficking and signaling transduction and has to be tightly controlled. The molecular processes controlling the endosomal positioning utilize several large protein complexes, one of which contains the small GTPase Rab, Rab-interacting lysosomal protein (RILP), and oxysterol-binding protein– related protein 1 (ORP1L). When cholesterol molecules in the late endosomes are present at high levels, ORP1L binds to cholesterol through the ORD and adopts a conformation that enables the interaction of ARD in ORP1L with the Rab7-RILP complex. Analysis of the Rab7-RILP structure at high resolution showed that GTP-bound Rab interacts with RILP using its “effector-interacting switch region” and Rab subfamily motifs 1 and 4 [10] These effectorinteracting switch regions represent canonical binding sites recognized by most Rab effectors [11,12,13]. Our structural data clearly show that ORP1L ARDN binds to a noncanonical region of Rab, which is positioned away from the effector-interacting switch region, enabling Rab to bind both ORP1L and RILP in a simultaneous manner
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
