The proton channel, CF0, in thylakoid membranes

Abstract

We investigated the conductance of pea thylakoid membranes and their capacity for photophosphorylation as function of the extraction of chloroplast coupling factor CF₁. The degree of extraction was varied via the incubation time in EDTA‐containing hypo‐osmolar medium and was measured by rocket electroimmunodiffusion. The conductance of thylakoid membranes was measured by flash kinetic spectrophotometry. The time course of extraction followed the time course of thylakoid swelling. Contrary to expectation increasing loss of CF₁ did not primarily increase the velocity of proton efflux from each vesicle. Instead proton‐tight vesicles were converted to leaky ones, which lost phosphorylating activity. Two subpopulations occurred, although both types of vesicles, leaky and proton‐tight ones, were CF₁‐depleted to a similar degree. This implied that only a small fraction of CF₁‐lacking CF₀ was functional as a proton channel. Tight vesicles had no functional channels while leaky ones had at least one. We determined the proportion of tight vesicles in three independent ways: via the residual phosphorylation activity, via measurements of proton efflux and via measurements of the electric relaxation across the membrane. The results obtained were identical. A statistical evaluation of the data led us to the following conclusions. (a) EDTA treatment produced vesicles containing approximately 10⁵ chlorophyll molecules, equivalent to a total of approximately 100 CF₀CF₁ per vesicle. (b) Even at the highest degree of extraction (75% of total CF₁ extracted) only 2.5 out of 75 exposed CF₀ per vesicle were proton‐conducting. (c) The unit conductance of one open CF₀ channel was 169 ± 18 fS at pH 7.5 and room temperature. At an electrical driving force of 100 mV this was equivalent to the passage of approximately 10⁵ protons/s. The most important consequence of this relatively high unit conductance was that a single open CF₀ channel was capable of dissipating the protonmotive force of one vesicle, thereby deactivating the whole remaining catalytic capacity of this vesicle.