Delineation between sequestered domain and lumen buffering in the chloroplast thylakoid membrane

Robert Glen Ewy, Purdue University

Abstract

The dual energy coupling hypothesis posits that thylakoids can use either a membrane-localized or a delocalized $\Delta\tilde\mu\ \sb{\rm H}+$ to drive ATP formation, but in either scenario, redox-generated H$\sp+$ are presumed to be initially deposited directly into the sequestered domains, not the thylakoid lumen. Lumen acidification is thought to be dependent on proton translocation through the sequestered domains and passage through a Ca$\sp{2+}$-regulated "H$\sp+$ gate" comprised of the CF$\sb0$ subunit III. The main work in this thesis was directed toward defining more clearly proton uptake into sequestered domains as distinguished from H+ uptake into the lumen space. Low pK$\sb{\rm a}$ lysines (pK$\sb{\rm a}$ ca. 7.8) have been shown to comprise part of the buffering array of the sequestered domains. Acetic anhydride selectively acetylates these low pK$\sb{\rm a}$ lysines. It was reasoned that acetic anhydride might be a useful tool to delineate better H$\sp+$ in the sequestered domains from those in the lumen. The effect on lumen acidification by redox-generated protons in thylakoids treated with acetic anhydride was determined by three criteria. Violaxanthin deepoxidation was used to determine lumen acidification because the violaxanthin deepoxidase enzyme (VDE) requires a lumen pH near 6 or below for activation. Anhydride treatment prevented lumen acidification as judged by the inhibition of light-driven violaxanthin deepoxidation in assays done at pH of 7.6. The anhydride treatment did not inhibit the VDE as violaxanthin was converted to zeaxanthin in the dark at pH 5.6. The anhydride treatment also inhibited the permeable amine dependent stimulation of proton uptake as well as fluorescence quenching of permeable amines, both diagnostic indicators for lumen acidification. The calcium channel blocker verapamil prevented a localized to delocalized $\Delta\tilde\mu\ \sb{\rm H}+$ switch as determined by the lack of a pyridine-dependent increase in the onset of photophosphorylation. Verapamil also prevented large HEM-induced stimulations of proton uptake compared to the controls. The effect of verapamil to prevent this switch in energy coupling was not due to uncoupling of electron transport. The results are consistent with there being a H$\sp+$ uptake-dependent Ca$\sp{2+}$ efflux which accompanies the switch from localized to delocalized energy coupling.

Degree

Ph.D.

Advisors

Dilley, Purdue University.

Subject Area

Botany

COinS