STARD3 coordinates Endoplasmic Reticulum-late endosome/lysosome contacts and organelle positioning through a GSK3-regulated phosphorylation switch

Eichler, J.; Wendling, C.; Huver, S.; Zouiouich, M.; Hanss, V.; Cardinal, A.; Fimbel, V.; Birck, C.; McEwen, A. G.; Knorr, C.; Fromental-Ramain, C.; Boutry, M.; Chenard, M.-P.; DRIN, G.; Tomasetto, C.; ALPY, F.

doi:10.1101/2025.05.29.656564

bioRxiv · 10.1101/2025.05.29.656564

STARD3 coordinates Endoplasmic Reticulum-late endosome/lysosome contacts and organelle positioning through a GSK3-regulated phosphorylation switch

Eichler, J.·Wendling, C.·Huver, S.·Zouiouich, M.·Hanss, V.·Cardinal, A.·Fimbel, V.·Birck, C.·McEwen, A. G.·Knorr, C.·Fromental-Ramain, C.·Boutry, M.·Chenard, M.-P.·DRIN, G.·Tomasetto, C.·ALPY, F.

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Abstract

Membrane contact sites (MCS) are dynamic regions where the membranes of two organelles come into close apposition. MCSs play many roles in cellular homeostasis by facilitating inter-organelle lipid and ion exchange as well as organelle positioning. The late endosome/lysosome (LE/Lys) cholesterol transfer protein STARD3 (StAR-related lipid transfer (START) domain containing protein 3) forms reversible contacts between the LE/Lys and the endoplasmic reticulum (ER). This tether protein contains a Phospho-FFAT motif (two phenylalanines (FF) in an acidic tract (AT)) whose interaction with ER-resident VAP proteins is phosphorylation-dependent. In this study, we identify Glycogen Synthase Kinase 3 (GSK3 and GSK3{beta}) as the kinases responsible for phosphorylating serine 209 within the Phospho-FFAT motif of STARD3. This phosphorylation event is both necessary and sufficient to activate STARD3s tethering activity, thereby promoting ER-LE/Lys contacts. Furthermore, we show that the ER-LE/Lys tethering activity made by STARD3 regulates endosome positioning, revealing an additional function for STARD3 on endosome biology. Our findings establish a direct and critical role for GSK3 in regulating MCS dynamics via STARD3 phosphorylation. This novel insight into GSK3-mediated phosphorylation expands our understanding of the molecular mechanisms governing inter-organelle communication.

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Eichler, J., Wendling, C., Huver, S., Zouiouich, M., Hanss, V., Cardinal, A., Fimbel, V., Birck, C., McEwen, A. G., Knorr, C., Fromental-Ramain, C., Boutry, M., Chenard, M.-P., DRIN, G., Tomasetto, C., ALPY, F.. 2025-05-30. STARD3 coordinates Endoplasmic Reticulum-late endosome/lysosome contacts and organelle positioning through a GSK3-regulated phosphorylation switch. https://doi.org/10.1101/2025.05.29.656564

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