ORP5 and ORP8 bind phosphatidylinositol-4, 5-biphosphate (PtdIns(4,5)P2) and regulate its level at the plasma membrane

Rajesh Ghai,Robert G Parton,Ximing Du,Andrew J Brown,Huan Wang,Jia-Wei Wu,Hongyuan Yang,Charles Ferguson,Jiangqing Dong

doi:10.1038/s41467-017-00861-5

Abstract

ORP5 and ORP8, members of the oxysterol-binding protein (OSBP)-related proteins (ORP) family, are endoplasmic reticulum membrane proteins implicated in lipid trafficking. ORP5 and ORP8 are reported to localize to endoplasmic reticulum–plasma membrane junctions via binding to phosphatidylinositol-4-phosphate (PtdIns(4)P), and act as a PtdIns(4)P/phosphatidylserine counter exchanger between the endoplasmic reticulum and plasma membrane. Here we provide evidence that the pleckstrin homology domain of ORP5/8 via PtdIns(4,5)P2, and not PtdIns(4)P binding mediates the recruitment of ORP5/8 to endoplasmic reticulum–plasma membrane contact sites. The OSBP-related domain of ORP8 can extract and transport multiple phosphoinositides in vitro, and knocking down both ORP5 and ORP8 in cells increases the plasma membrane level of PtdIns(4,5)P2 with little effect on PtdIns(4)P. Overall, our data show, for the first time, that phosphoinositides other than PtdIns(4)P can also serve as co-exchangers for the transport of cargo lipids by ORPs.

Highlights

ORP5 and ORP8, members of the oxysterol-binding protein (OSBP)-related proteins (ORP) family, are endoplasmic reticulum membrane proteins implicated in lipid trafficking
Our results confirm the critical importance of ORP5 and ORP8 in endoplasmic reticulum (ER)–plasma membrane (PM) lipid homeostasis, but show, for the first time, that phosphoinositides other than PtdIns(4)P serve as co-exchangers for the transport of cargo lipids by ORPs
We have previously shown endogenous ORP5A associates predominantly with ER membranes and the carboxyl terminal transmembrane domain is responsible for ER anchoring[17]

Summary

Results

ORP5 accumulates at the ER–PM junctions. We have previously shown endogenous ORP5A (isoform A) associates predominantly with ER membranes and the carboxyl terminal transmembrane domain is responsible for ER anchoring[17]. Overexpressed mCherry–ORP5A labels the cell periphery with punctate structures (Fig. 1b), which almost completely co-localized with the ER–PM junction marker MAPPER Both mCherry-tagged isoforms of ORP8 demonstrated only reticular distribution (Fig. 1b). A single amino acid change (L99A) abolished the targeting of ORP5A to ER–PM contact site These data establish that while the transmembrane domain (TM) of ORP5 maintains anchoring to the ER, the N-terminal coiled coil region together with the PH domain mediates ORP5 tethering to the PM. Close inspection of the distinct positively charged cavity of ORP8 PH domain shows the amino acids forming the basic patch are lined on the β1–β2 and β5–β6 loops (non-canonical mode) (Fig. 2c and Supplementary Fig. 5A, B). Both the point mutations in ORP5 and ORP8 PH domains β5–β6

C Extended loop β6–β7 d β1

Discussion

Methods