This chapter describes solubilization, isolation, and reconstitution of the clathrin-coated vesicle proton-translocating complex. By sucrose density gradient centrifugation determination, the molecular weight of the proton pump is 530K. In addition, the purification of a 116K ATPase from clathrin-coated vesicles is described. An ATP-driven proton pump activity is present in clathrin-coated vesicles, which are distinguished from the well-characterized proton pump of mitochondria by its insensitivity to oligomycin, azide, and efrapeptin. The activity of the pump is expressed in arbitrary optical density units, either in terms of the initial slope or the extent of ATP-induced decrease in absorbance, under the assay conditions described. Coated vesicles contain a transporter through which chloride or bromide pass—thereby, balancing the electrogenic proton translocation. Chloride transport can be functionally dissociated from the proton pump. It is facilitated either by ATP-driven proton movement, or by an inside positive membrane potential generated by K+moving with valinomycin from the outside to the inside of the vesicles.
ASJC Scopus subject areas
- Molecular Biology