STARD3 regulates lysosome positioning and contacts via a GSK3-controlled phosphorylation switch

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 exchange and organelle positioning. The late endosome/lysosome (LE/Lys) cholesterol transfer protein STARD3 forms reversible contacts between LE/Lys and the endoplasmic reticulum (ER). This tether protein contains a Phospho-FFAT motif (two phenylalanines in an acidic tract) whose interaction with ER-resident VAPs (vesicle-associated membrane protein-associated proteins) is phosphorylation-dependent. In this study, we identify GSK3α and GSK3β 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 STARD3's tethering activity, thereby promoting ER-LE/Lys contacts. Furthermore, we show that when ER-LE/Lys tethering is prevented, STARD3 triggers LE/Lys homotypic interactions, revealing an additional function for STARD3 on endosome biology. Our findings establish a direct and critical role for GSK3 in regulating MCS via STARD3 phosphorylation, and expand our understanding of the molecular basis of inter-organelle communication.