Phosphatidylserine transporters ORP5 and ORP8 control cholesterol trafficking from the plasma membrane to the endoplasmic reticulum

Phosphatidylserine (PS), the most abundant negatively charged phospholipid in mammalian cells, is made in the endoplasmic reticulum (ER) but concentrated in the plasma membrane (PM). Similarly, cellular cholesterol is synthesized in the ER, yet enriched in the PM. Recently, PS has been shown to govern the transport of low-density lipoprotein-derived cholesterol from the PM to the ER. Here, we investigated how cholesterol regulates the delivery of PS from the ER to PM by the lipid-transfer proteins, ORP5 and ORP8. Adding exogenous cholesterol markedly increased the level of PI(4,5)P₂ on the PM, which recruited ORP5/8 to promote the delivery of PS to the PM from the ER. Similar results were also obtained when the level of PM cholesterol was increased upon sphingomyelinase treatment. The increased delivery of PS to the PM helps recruit GRAMD1b, a cholesterol carrier transporting cholesterol from the PM to the ER. Importantly, we show ORP5 interacts with GRAMD1b, and this interaction further facilitates the recruitment of GRAMD1b to the PM. Our results thus unveil a new mechanism by which excess PM cholesterol promotes its own trafficking to the ER via PI(4,5)P₂ and ORP5/8. Our results also provide fundamental new insights into how two major lipid species, PS and cholesterol, can impact each other's cellular homeostasis.