Chloroplast thylakoid proteins associated with sequestered proton-buffering domains. Plastocyanin contributes...

Thylakoid membrane proteins are organized so as to shield 30-50 nmol H+ (mg Chl)-1 from freely equilibrating with either the external or the lumen aqueous phases. Amine groups provide binding sites for this metastable buffering array and can be quantitatively measured by acetylation using [3H]acetic anhydride. The principle of the assay is that a metastable acidic domain will have relatively less of the reactive neutral form of the amine compared to the amount present after addition of an uncoupler. The extent of the acetylation reaction is strongly influenced by whether the lumen pH comes to complete equilibrium with the external pH prior to adding the acetic anhydride. Determination of the lumen pH by [14C]methylamine distribution after the standard 3 or 5 min equilibration in pH 8.6 buffer indicated that the lumen may have been 0.2 to 0.3 pH more acidic than the external phase. This effect was taken into account by determining the pH dependence, in the pH 8.2-8.6 range, of acetylation of the membrane proteins studied, and the labeling data were conservatively corrected for this possible contribution. Experiments were carried out to identify the thylakoid proteins that contribute such metastable domain amine groups, using the above conservative correction. Surprisingly, plastocyanin contributes buried amine groups, but cytochrome f did not give evidence for such a contribution, if the conservative correction in the labeling was applied. If the correction was too conservative, cytochrome f may contribute amines to the sequestered domains. The new methodology verified earlier results suggesting that three Tris-releasable photosystem II-associated proteins also contribute significantly to the sequestered amine-buffering array.