Abstract
Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a family of lipid transfer proteins conserved in eukaryotes. ORP1 transports cholesterol at the interface between the late endosomes/lysosomes (LELs) and the endoplasmic reticulum (ER). ORP1 is targeted to the endosomal membranes by forming a tripartite complex with the LE GTPase Rab7 and its effector RILP (Rab7-interacting lysosomal protein). Here, we determined the crystal structure of human ORP1 ANK domain in complex with the GTP-bound form of Rab7. ORP1 ANK binds to the helix α3 of Rab7 located away from the switching regions, which makes the interaction independent of the nucleotide-binding state of Rab7. Thus, the effector-interacting switch regions of Rab7 are accessible for RILP binding, allowing formation of the ORP1-Rab7-RILP complex. ORP1 ANK binds to Rab7 and the Rab7-RILP complex with similar micro-molar affinities, which is consistent with the independence binding of ORP1 and RILP to Rab7. The structural model of the ORP1-Rab7-RILP complex correlates with the recruitment of ORP1 at the LEL-ER interface and the role in lipid transport and regulation.
Highlights
The proper intracellular distribution of sterols and other lipids are crucial for the membrane identity and function
This study provides a framework for understanding the molecular basis of the ORP1– Rab7 interaction and how Rab7 GTPase recruits Rab7-interacting lysosomal protein (RILP) and ORP1 simultaneously to the late endosomes/lysosomes (LELs) membrane for lipid transport and regulation
Escherichia coli BL21(DE3) cells transformed with the plasmid encoding the ORP1 ANK, Rab7, or RILP were grown to an OD600 of 0.8 at 37 ̊C
Summary
The proper intracellular distribution of sterols and other lipids are crucial for the membrane identity and function. Lipid transport mediated by the oxysterol-binding protein (OSBP)-related protein (ORPs) has been proposed as a major route of transporting of sterols and other phospholipids between intracellular membranes [4]. ORP1L, a long ORP1 variant (hereafter referred to as ORP1), is localized to the contact sites between the late endosomes/lysosomes (LELs) and the ER and implicated in nonvesicular sterol transport [3,17,18]. The N-terminal ANK domain targets ORP1 to the LELs via interaction with the Rab anchored to the endosomal membrane. This study provides a framework for understanding the molecular basis of the ORP1– Rab interaction and how Rab GTPase recruits RILP and ORP1 simultaneously to the LEL membrane for lipid transport and regulation
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