Calcium-dependent interaction of chlorpromazine with the chloroplast 8-kilodalton CF0 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 Ca(2+)-dependent binding of the probe to thylakoid proteins. [3H]Chlorpromazine photoaffinity-labels thylakoid polypeptides of Mr 8K and 6K, with generally much less label occurring in other proteins (some experiments showed labeled proteins at Mr 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 Ca(2+)-CF0 gating site came from the correlation between Ca(2+)-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.
Chiang
G G
GG
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
Wooten
D C
DC
Dilley
R A
RA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels; D000595:Amino Acid Sequence; D002118:Calcium / Q000494:pharmacology*; D002736:Chloroplasts / Q000378:metabolism*; D002746:Chlorpromazine / Q000378:metabolism* / Q000494:pharmacology; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism; D004734:Energy Metabolism; D007887:Fabaceae; D007425:Intracellular Membranes / Q000378:metabolism*; D015640:Ion Channel Gating / Q000187:drug effects*; D007473:Ion Channels / Q000502:physiology*; D008027:Light; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010640:Phenothiazines / Q000494:pharmacology; D010766:Phosphorylation; D010777:Photochemistry; D010946:Plants, Medicinal; D011522:Protons
D000345:Affinity Labels; D007473:Ion Channels; D008565:Membrane Proteins; D010640:Phenothiazines; D011522:Protons; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D002118:Calcium; D002746:Chlorpromazine