biostudies-literatureChung WYHHS | NIH | National Institute of Dental and Craniofacial Researche2301410120https://www.ebi.ac.uk/biostudies/studies/S-EPMC10469337biostudies-literatureUnknown120(35)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 tProceedings of the National Academy of Sciences of the United States of AmericaPtdSer 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.PMC10469337DE000735-13Ohman HKEChung WYMcNally BALeibow SRMovahed Abtahi AMuallem SAhuja MfalsePtdSer 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 t2023-01-01T00:00:00Z2023 Aug2025-04-26T08:06:25.368Z2025-04-06T12:32:20.87ZS-EPMC104693373760723010.1073/pnas.2301410120