PtdSer as a signaling lipid determined by privileged localization of ORP5 and ORP8 at ER/PM junctional foci to determine PM and ER PtdSer/PI(4)P ratio and cell function

The membrane contact site ER/PM junctions are hubs for signaling pathways, including Ca2+ signaling. Phosphatidylserine (PtdSer) mediates various physiological functions; however, junctional PtdSer composition and the role of PtdSer in Ca2+ signaling and Ca2+- dependent gene regulation are not understood. Here, we show that STIM1-formed junctions are required for PI(4)P/PtdSer exchange by ORP5 and ORP8, which have reciprocal lipid exchange modes and function as a rheostat that sets the junctional PtdSer/PI(4)P ratio. Targeting the ORP5 and ORP8 and their lipid transfer ORD domains to PM subdomains revealed that ORP5 sets low and ORP8 high junctional PI(4)P/PtdSer ratio that controls STIM1- STIM1 and STIM1- Orai1 interaction and the activity of the SERCA pump to determine the pattern of receptor-evoked Ca2+ oscillations, and consequently translocation of NFAT to the nucleus. Significantly, targeting the ORP5 and ORP8 ORDs to the STIM1 ER subdomain reversed their function. Notably, changing PI(4)P/PtdSer ratio by hydrolysis of PM or ER PtdSer with targeted PtdSer-specific PLA1a1 reproduced the ORPs function. The function of the ORPs is determined both by their differential lipid exchange modes and by privileged localization at the ER/PM subdomains. These findings reveal a role of PtdSer as a signaling lipid that controls the available PM PI(4)P, the unappreciated role of ER PtdSer in cell function, and the diversity of the ER/PM junctions. The effect of PtdSer on the junctional PI(4)P level should have multiple implications in cellular signaling and functions.

Automated summary

Membrane contact site ER/PM junctions play a crucial role in cellular signaling, including Ca2+ signaling. Phosphatidylserine (PtdSer) regulates the composition of these junctions and is involved in Ca2+ signaling, Ca2+-dependent gene regulation, and cellular functions. ORP5 and ORP8 mediate the exchange of PtdSer and PI(4)P at the ER/PM junctions, and their differential lipid exchange modes determine the PtdSer/PI(4)P ratio, which controls the interaction between STIM1-STIM1 and STIM1-Orai1, the activity of the SERCA pump, Ca2+ oscillations, and the translocation of NFAT. The localization of ORP5 and ORP8 at specific subdomains further regulates their function, and the hydrolysis of PtdSer alters the PI(4)P/PtdSer ratio and reproduces the function of ORPs.