Homotypic SCOTIN assemblies form ER‐endosome membrane contacts and regulate endosome dynamics

The ER regulates the spatiotemporal organization of endolysosomal systems by membrane contact. In addition to tethering via heterotypic interactions on both organelles, we present a novel ER‐endosome tethering mechanism mediated by homotypic interactions. The single‐pass transmembrane protein SCOTIN is detected in the membrane of the ER and endosomes. In SCOTIN‐knockout (KO) cells, the ER‐late endosome contacts are reduced, and the perinuclear positioning of endosomes is disturbed. The cytosolic proline‐rich domain (PRD) of SCOTIN forms homotypic assemblies in vitro and is necessary for ER‐endosome membrane tethering in cells. A region of 28 amino acids spanning 150–177 within the SCOTIN PRD is essential to elicit membrane tethering and endosomal dynamics, as verified by reconstitution in SCOTIN‐KO cells. The assembly of SCOTIN (PRD) is sufficient to mediate membrane tethering, as purified SCOTIN (PRD), but not SCOTIN (PRDΔ150‐177), brings two different liposomes closer in vitro. Using organelle‐specific targeting of a chimeric PRD domain shows that only the presence on both organellar membranes enables the ER‐endosome membrane contact, indicating that the assembly of SCOTIN on heterologous membranes mediates organelle tethering.