The reversibility of freeze/thaw injury to spinach thylakoids; restoration of light-induced proton pumping, membrane-conformational changes and proton gradient formation

Abstract

Spinach thylakoids have been frozen under a variety of salt / sucrose concentrations to remove varying amounts of peripheral membrane proteins, including the water-soluble part of the coupling factor complex (CF 1). This leads to a defined degree of uncoupling by exposing the CF 0 proton channel. The ability of thylakoids, subjected to this treatment, to reconstitute light-induced proton pumping, membrane-conformational changes and proton gradient formation when treated with DCCD, an energy transfer inhibitor which interacts with the CF 0 proton channel, thus reducing the proton permeability of the membrane, has been investigated. Full reconstitution of proton pumping and ΔpH formation could be obtained in thylakoids in which up to 75% of the coupling factor complex had been removed by the freezing regime. Even under the most severe conditions employed, in which over 80% of the CF 1 was removed from the membrane, there was still between 25 and 50% reconstitution of proton pumping. Reconstitution of membrane conformational changes as monitored by 90° scattering changes showed a strong positive correlation to the restoration of proton pumping. Reconstitution of slower, light-induced transmittance changes, in contrast, exhibited a more variable response. Little reconstitution of the slow transmittance changes was found under conditions which removed more than 60–70% of the coupling factor complex.