The proton gradient across the vacuo-lysosomal membrane of lutoids from the latex ofHevea brasiliensis. I. Further evidence for a proton-translocating ATPase on the vacuo-lysosomal membrane of intact lutoids

Abstract

Lutoids (vacuo-lysosomal particles) were isolated from the latex ofHevea brasiliensis. Using flow dialysis with¹⁴C-methylamine uptake as a ΔpH probe and⁸⁶Rb rubidium+valinomycin distribution for estimations of transmembrane electrical potential, intact lutoids exhibited a ΔpH of 1 unit (interior more acid) and a ΔΨ of −70 mV (interior negative), when suspended in an isotonic medium at physiological concentration of potassium (30mm) and pH 7.0, in the absence of ATP. In most cases, the Donnan potential was shown to fully account for ΔpH in nonenergized lutoids. The addition of Mg-ATP (5mm) resulted in a marked acidification of the lutoidic internal space (0.7 to 1 pH unit) depending on the composition of the medium, and in a membrane depolarization by 60 mV (interior becoming less negative). The resulting electrochemical potential of protons ( $\Delta \tilde \mu H$ ) increased by a hundred millivolts when lutoids were energized by ATP. These data strongly support an inward electrogenic proton translocating function for the ATPase of the vacuo-lysosomal membrane of lutoids. Results are discussed in terms of thein vivo maintenance of large “lutoids/cytoplasm” proton gradients, and of the rôle of these vacuo-lysosomes in the homeostasis of the cytoplasmic metabolism.