Edited by Melanie Cobb, The University of Texas Southwestern Medical Center, Dallas, TX; received January 25, 2023; accepted July 11, 2023
August 22, 2023
120 (35) e2301410120
Significance
Membrane proteins are surrounded by lipids that regulate their functions. Lipids are concentrated at membrane contact sites, including the ER/PM junctions that are enriched with phosphatidylserine (PtdSer). The ER/PM junctions are also a hub for Ca2+ signaling that mediates most cellular functions. Understanding how lipids regulate Ca2+ signaling has multiple physiological implications. The present findings manipulated the junctional lipid transfer proteins (LTP) ORP5 and ORP8 and show that they function as a rheostat that sets the junctional PtdSer/PI(4)P ratio. The PtdSer/PI(4)P ratio controls Ca2+ influx by determining the clustering of the ER Ca2+ sensor STIM1 and thus all Ca2+-dependent cell functions. These findings should have implications for the function of other LTP and the physiological and pathological roles of PtdSer.
Abstract
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.
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Data, Materials, and Software Availability
Acknowledgments
We thank Dr. Pietro De Camilli (Yale University) for generously providing the plasmids coding for ORP5, ORP8, FKBP-ΔPHD-ORP5, and FKBP-ΔPHD-ORP8, Dr. Jen Liou (UT Southwestern Medical Center) for MAPPER-s, Dr. Sergio Grinstein (University of Toronto) for the LactC2 plasmid, Dr. Tamas Balla (NICHD/NIH) for the GFP-evt2-2XPH and ORP8(PLCδPHD) plasmids, and Dr. Mohamed Trebak (University of Pittsburg) for the STIM1−/− cells. We also thank Dr. Andrew Doyle and the NIDCR imaging core for the help with the Airy Scan imaging. This work was funded by an intramural NIH grant NIH/NIDCR DE000735-13.
Author contributions
S.M. designed research; W.Y.C., M.A., B.A.M., S.R.L., H.K.E.O., and A.M.A. performed research; W.Y.C., M.A., B.A.M., S.R.L., H.K.E.O., A.M.A., and S.M. analyzed data; and W.Y.C., M.A., B.A.M., S.R.L., H.K.E.O., A.M.A., and S.M. wrote the paper.
Competing interests
The authors declare no competing interest.
Supporting Information
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Information & Authors
Information
Published in
Proceedings of the National Academy of Sciences
Vol. 120 | No. 35
August 29, 2023
Classifications
Copyright
Data, Materials, and Software Availability
Submission history
Received: January 25, 2023
Accepted: July 11, 2023
Published online: August 22, 2023
Published in issue: August 29, 2023
Keywords
- ER/PM junctions
- phosphatidylserine
- STIM1
- Weather1
- Ca2+ signaling
Acknowledgments
We thank Dr. Pietro De Camilli (Yale University) for generously providing the plasmids coding for ORP5, ORP8, FKBP-ΔPHD-ORP5, and FKBP-ΔPHD-ORP8, Dr. Jen Liou (UT Southwestern Medical Center) for MAPPER-s, Dr. Sergio Grinstein (University of Toronto) for the LactC2 plasmid, Dr. Tamas Balla (NICHD/NIH) for the GFP-evt2-2XPH and ORP8(PLCδPHD) plasmids, and Dr. Mohamed Trebak (University of Pittsburg) for the STIM1−/− cells. We also thank Dr. Andrew Doyle and the NIDCR imaging core for the help with the Airy Scan imaging. This work was funded by an intramural NIH grant NIH/NIDCR DE000735-13.
Author Contributions
S.M. designed research; W.Y.C., M.A., B.A.M., S.R.L., H.K.E.O., and A.M.A. performed research; W.Y.C., M.A., B.A.M., S.R.L., H.K.E.O., A.M.A., and S.M. analyzed data; and W.Y.C., M.A., B.A.M., S.R.L., H.K.E.O., A.M.A., and S.M. wrote the paper.
Competing Interests
The authors declare no competing interest.
Notes
This article is a PNAS Direct Submission.
Authors
Affiliations
Woo Young Chung1
Epithelial Signaling and Transport Section, National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD 20892
Malini Ahuja1
Epithelial Signaling and Transport Section, National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD 20892
Beth A. McNally
Epithelial Signaling and Transport Section, National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD 20892
Spencer R. Leibow
Epithelial Signaling and Transport Section, National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD 20892
Henry K. E. Ohman
Epithelial Signaling and Transport Section, National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD 20892
Ava Movahed Abtahi
Epithelial Signaling and Transport Section, National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD 20892
Epithelial Signaling and Transport Section, National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD 20892
Notes
1
W.Y.C. and M.A. contributed equally to this work.
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