Selective MOSPD2-STARD3 interaction at ER contact sites governs late endosome/lysosome dynamics and cholesterol homeostasis

Membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and other organelles or the plasma membrane play essential roles in lipid exchange and organelle homeostasis; however, how the molecular composition of these sites is functionally specified remains poorly understood. Here, we identify the ER-resident protein MOSPD2 as a novel regulator of late endosome/lysosome (LE/Lys) homeostasis and show that MOSPD2 selectively recruits the cholesterol transporter STARD3 at ER-LE/Lys contacts. Loss of either MOSPD2 or STARD3 leads to an expansion of the LE/Lys compartment, accumulation of free cholesterol at LE/Lys, and impaired LE/Lys fusion dynamics, indicating that both proteins act within a common pathway at ER-LE/Lys contact sites. STARD3 interacts with three members of the MSP-domain protein family, VAP-A, VAP-B and MOSPD2, yet, these tethers are not functionally equivalent at ER-LE/Lys MCSs. Using a series of quantitative binding assays, we show that STARD3 preferentially associates with MOSPD2 rather than with VAP-A or VAP-B, and that neither VAP-A nor VAP-B can compensate for the loss of MOSPD2 to restore LE/Lys biology and cholesterol distribution. This work establishes the MOSPD2-STARD3 complex as a unique functional unit that regulates LE/Lys homeostasis. Collectively, these findings reveal that selective pairing between MSP-domain proteins and their FFAT-containing partners exist and shape the interaction landscape of MCSs to define their functions.