Endoplasmic reticulum (ER)–lysosome membrane contact sites (MCSs) are crucial for intracellular lipid transport, but owing to their dynamic and transient nature, identifying the proteins involved in their function and regulation can be difficult. The abundant lysosomal membrane protein LIMP-2 transports cholesterol from the lysosomal lumen into the lysosomal membrane, from which it can be delivered to the ER and lipid droplets via MCSs, but whether LIMP-2 itself is a component of ER–lysosome contacts has not been established. Here (Rudnik et al., 2024), Paul Saftig and colleagues investigate the association of LIMP-2 with MCSs using complementary biochemical and imaging-based approaches. Using proximity proteomics, co-immunoprecipitation and bimolecular fluorescence complementation, they identify interactions between LIMP-2, the ER contact site tether protein VAPB and another lysosomal membrane protein involved in cholesterol transfer, STARD3. Binding of STARD3 to LIMP-2 at the lysosome is necessary for their interaction with VAPB at the ER, suggesting that a trimeric complex of these proteins could contribute to ER–lysosome contacts. However, LIMP-2-mediated cholesterol transport function is not required for formation of the trimeric complex, indicating that these proteins play a primarily structural role at MCSs. Overall, these findings contribute to the further elucidation of the complex machinery present at ER–lysosome contacts, which are emerging as significant regulators of cholesterol homeostasis.
