CALCIUM-DEPENDENT INTERACTION OF CHLORPROMAZINE WITH THE CHLOROPLAST 8-KILODALTON CF(0) PROTEIN AND CALCIUM GATING OF H+ FLUXES BETWEEN THYLAKOID MEMBRANE DOMAINS AND THE LUMEN

Earlier work suggested that Ca2+ ions in the chloroplast thylakoid lumen interact with thylakoid membrane proteins to produce a proton flux gating structure which functions to regulate the expression of the energy-coupling H+ gradient between localized and delocalized modes [Chiang, G., & Dilley, R. A. (1987) Biochemistry 26, 4911-4916]. In this work, one of the phenothiazine Ca2+ antagonists, chlorpromazine, was used as a photoaffinity probe to test for Ca2+-dependent binding of the probe to thylakoid proteins. [H-3]Chlorpromazine photoaffinity-labels thylakoid polypeptides of M(r) 8K and 6K, with generally much less label occurring in other proteins (some experiments showed labeled proteins at M(r) 13K-15K). More label was incorporated in circumstances where it is expected that Ca2+ occupies the putative H+ flux gating site, compared to when the gating site is not occupied by calcium. The photoaffinity labeling of the 8-kDa protein was also influenced by the energization level of the thylakoids (less labeling under H+ uptake energization). The 8-kDa protein was identified by partial amino acid sequence data as subunit III of the thylakoid CF0 H+ channel complex. The partial amino acid sequence of the 6-kDa protein (19 residues were determined with some uncertainties) was compared to data in the GCG sequence analysis data base, and no clear identity to a known sequence was revealed. Neither the exact site of putative Ca2+ binding to the CF0 proteolipid nor the site of covalent attachment of the chlorpromazine to the CF0 component has been identified. Evidence for gating of energy-linked H+ fluxes by the hypothesized Ca2+-CF0 gating site came from the correlation between Ca2+-dependent binding of chlorpromazine to the CF0 8-kDa protein with inhibition of light-driven H+ uptake into the lumen but no inhibition of H+ uptake into sequestered membrane domains. When conditions favored a delocalized DELTA-mu(H+) coupling mode, less chlorpromazine was bound to the CF0 structure, and much larger amounts of H+ ions were accumulated in the lumen. The data support the hypothesis that Ca2+ ions act in concert with the 8-kDa CF0 protein (and perhaps another protein, the 6-kDa polypeptide?) in a gating mechanism for regulating the expression of the energy-coupling H+ gradient between localized or delocalized coupling modes.