ATPase assay

The ATPase assay was conducted with a commercially available ATPase assay Kit (#601–0120, Innova Biosciences Ltd, Babraham, Cambridge, UK) with minor modifications. Briefly, isolated melanosomes were incubated at + 37°C for 30 min with or without 1.6 μM bafilomycin A1 (Sigma-Aldrich) in MES buffer. Then, an equal volume of ATP-containing buffer (1 mM ATP, Innova Biosciences) was added and melanosomes were incubated at room temperature for 30 min. After incubation, melanosomes were removed by centrifugation (20 000 g 5 min) and the supernatant was recovered. Color reagent and stabilizer were added to the supernatant. After 30 min, free phosphate (P_i) concentrations were measured with a spectrophotometer at A595 nm.