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-bindin g domains, suggested to be involved in localized DELTAmu(H+)-driven AT P formation, are maintained with the involvement of several membrane p roteins, including the LHCII (Laszlo, J. A., Baker, G. M., and Dilley, R. A. (1984) Biochim. Biophys. Acta 764, 160-169), which comprises ab out 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 doe s not accumulate some of the LHCII proteins, was tested for its capaci ty to carry out localized-proton gradient-dependent ATP formation. Two previously developed assays permit clear discrimination between local ized and delocalized DELTAmu(H+) gradient-driven ATP formation. Those assays include the effect of a permeable buffer, pyridine, on the numb er of single-turnover flashes needed to reach the energetic threshold for ATP formation and the more recently developed assay for lumen pH u sing 8-hydroxy-1,3,6-pyrene trisulfonic acid as a lumenally loaded pH- sensitive fluorescent probe. By those two criteria, the wild-type barl ey thylakoids revealed either a localized or a delocalized energy coup ling mode under low- or high-salt storage conditions, respectively. Ad dition of Ca++ to the high-salt storage medium caused those thylakoids to maintain a localized energy-coupling response, as previously obser ved for pea thylakoids. In contrast, the chlorina f2 mutant thylakoids had an active delocalized energy coupling activity but did not show l ocalized DELTAmu(H+) energy coupling under any conditions, and added C a++ to the thylakoid storage medium did not alter the delocalized ener gy coupling mode. One interpretation of the results is that the absenc e of the LHCII polypeptides produces a ''leaky'' pathway for protons w hich allows the DELTAmu(H+) gradient to equilibrate with the lumen und er all conditions. Another interpretation is possible but seems less l ikely, that being that the absence of the LHCII polypeptides in some w ay causes the proposed Ca2++-gated H+ flux site on the membrane sector (CF0) of the energy coupling complex to lose its gating function.