EVIDENCE THAT THE INTRINSIC MEMBRANE PROTEIN-LHCII IN THYLAKOIDS IS NECESSARY FOR MAINTAINING LOCALIZED DELTA-MU-H+ ENERGY COUPLING

This work tested the hypothesis that thylakoid localized proton-binding domains, suggested to be involved in localized DELTAmu(H+)-driven ATP formation, are maintained with the involvement of several membrane proteins, including the LHCII (Laszlo, J. A., Baker, G. M., and Dilley, R. A. (1984) Biochim. Biophys. Acta 764, 160-169), which comprises about 50% of the total thylakoid protein. The concept we have in mind is that several membrane proteins cooperate to shield a localized proton diffusion pathway from direct contact with the lumen, thus providing a physical barrier to H+ equilibration between the sequestered domains and the lumen. A barely mutant, chlorina f2, that lacks Chl b and does not accumulate some of the LHCII proteins, was tested for its capacity to carry out localized-proton gradient-dependent ATP formation. Two previously developed assays permit clear discrimination between localized and delocalized DELTAmu(H+) gradient-driven ATP formation. Those assays include the effect of a permeable buffer, pyridine, on the number of single-turnover flashes needed to reach the energetic threshold for ATP formation and the more recently developed assay for lumen pH using 8-hydroxy-1,3,6-pyrene trisulfonic acid as a lumenally loaded pH-sensitive fluorescent probe. By those two criteria, the wild-type barley thylakoids revealed either a localized or a delocalized energy coupling mode under low- or high-salt storage conditions, respectively. Addition of Ca++ to the high-salt storage medium caused those thylakoids to maintain a localized energy-coupling response, as previously observed for pea thylakoids. In contrast, the chlorina f2 mutant thylakoids had an active delocalized energy coupling activity but did not show localized DELTAmu(H+) energy coupling under any conditions, and added Ca++ to the thylakoid storage medium did not alter the delocalized energy coupling mode. One interpretation of the results is that the absence of the LHCII polypeptides produces a ''leaky'' pathway for protons which allows the DELTAmu(H+) gradient to equilibrate with the lumen under all conditions. Another interpretation is possible but seems less likely, that being that the absence of the LHCII polypeptides in some way causes the proposed Ca2++-gated H+ flux site on the membrane sector (CF0) of the energy coupling complex to lose its gating function.