Calcium ion-gated proton fluxes in choroplast thylakoid membranes

Gisela Guanhsin Chiang, Purdue University

Abstract

Replacement of sucrose by 100 mM KCl in the choroplast thylakoid storage media, shifts proton gradients in thylakoids from a localized to a delocalized mode of energy coupling. This conversion appears to be regulated by Ca$\sp{2+}$ ions. Addition of divalent cations to the KCl containing medium maintained thylakoids in a localized coupling response. Inclusion of lipid soluble Ca$\sp{2+}$ chelators to sucrose stored thylakoids normally displaying localized coupling behavior, resulted in a conversion to a delocalized pattern typical of KCl stored thylakoids. These results suggest that Ca$\sp{2+}$ ions associated with the lumenal side of thylakoid membranes or with the interior of the membrane, regulate a gating function that can switch proton diffusion pathways from localized to delocalized energy coupling gradients. The intact choroplast system was analogous to isolated thylakoids, in regard to the effects of KCl and Ca$\sp{2+}$ on the energy coupling mode. Suspending thylakoids in stromal material previously isolated from intact chloroplast preparations showed that the stromal milieau has sufficient Ca$\sp{2+}$ to cause a localized coupling response even in the presence of 80 mM endogenous K$\sp +$ concentration. Extrapolating the intact choroplast data to the whole leaf level suggests that proton energy coupling is normally of the localized mode, but under certain conditions it could either be localized or delocalized, depending on factors that affect the putative Ca$\sp{2+}$ regulated gate. The Ca$\sp{2+}$ antagonists directed towards calmodulin, chorpromazine and trifluoperazine, perturb the H$\sp+$ gating function in a way consistent with the Ca$\sp{2+}$ gating action, involving a membrane protein analogous to a calmodulin-Ca$\sp{2+}$ complex functioning as the closed gate structure. Chlorpromazine bound at the Ca$\sp{2+}$ occupied sites causes partial blockage of the proton flux between the membrane domains and the lumen. ($\sp3$H) Chlorpromazine used as a photoaffinity probe, identified four membrane proteins of Mr 7-14 kDa, as possibly being part of the gating function. One of the proteins was identified as the 8 kDa CF$\sb0$ subunit by its N-terminus amino acid sequence analysis and co-labeling with ($\sp{14}$C) DCCD. A 6 kDa protein also bound ($\sp3$H) CPZ similarly, from which a 10 amino acid peptide sequence was determined. This polypeptide may be part of a yet unidentified protein involved in the gating mechanism.

Degree

Ph.D.

Advisors

Dilley, Purdue University.

Subject Area

Biology

COinS