Figure 4.

ESV formation can be reconstituted in a cell-free assay. (A and B) Cytosol dependence of ESV formation in vitro. CHO/GLUT4myc cells were incubated with either ¹²⁵I-9E10 (A) or ¹²⁵I-Tf (B), respectively, for 80 min at 15°C. Labeled crude membranes (1 mg/ml) were incubated for 10 min at 37°C with or without 2 mg/ml rat brain cytosol in the presence of an ATP-regenerating system. The entire reaction mixture was fractionated on glycerol gradients as described above. (C and D) Absence of detectable ESVs after in vitro budding from membranes labeled by incubation with ¹²⁵I-9E10 (C) or ¹²⁵I-Tf (D) for 2 h at 0°C. (E) Efficiency of ESV formation increases using donor membranes enriched in endosomal membranes. The formation of ESVs from different preparations of donor membranes was compared. P1, a pellet centrifuged at 500 × g (enriched in plasma membranes); P2, a pellet from the supernatant obtained after a 500 × g spin centrifuged at 27,000 × g (enriched in endosomal membranes). Values shown indicate the amount of specific radioactivity associated with de novo ESVs assayed on glycerol gradients normalized relative to the amount of specific radioactivity associated with the starting material in each reaction. The total amount of radioactivity recovered was the same for plus- and minus-cytosol conditions.