significant differences were assessed using the unpaired Student's t-test. For surface biotinylation, living confluent HeLa cells or living dissociated hippocampal neurons were washed three times with ice-cold PBS and biotinylated for 10 min on ice using membrane impermeable EZ-link Sulfo-NHS-SS-biotin (HeLa: 0.1 mg/ml in PBS, neurons: 0.15 mg/ml in PBS; Thermo Scientific). To quench the remaining unbound biotin, cells were washed two times with ice-cold PBS supplemented with 50 mM NH4Cl and once with ice-cold PBS alone. Cells were harvested, lysed by sonification, and crude membrane fractions were isolated by ultracentrifugation at 125,000× g for 20 min. Membrane protein complexes were solubilized in ComplexioLyte buffer 91 (LOGOPHARM GmbH) for 30 min at 4°C followed by ultracentrifugation (15 min at 125,000× g). Solubilisates were incubated with streptavidin agarose resin (Thermo Scientific) for 1 hour at 4°C to separate biotinylated proteins. After washing with PBS, biotinylated proteins were eluted by incubation in Laemmli buffer for 10 min at 37°C. Protein samples were finally resolved by SDS-PAGE and identified by Western blotting.
