Use of inside-out chloroplast thylakoid membrane vesicles for studying electron transport and membrane structure

Emmanuel Atta-Asafo-Adjei, Purdue University

Abstract

Inside-out and right-side-out thylakoid vesicles were isolated from spinach chloroplasts by aqueous-polymer two-phase (dextran/polyethylene glycol) partitioning following mechanical fragmentation of thylakoid membranes by Yeda press treatment. Externally added plastocyanin stimulated the whole-chain and PSI electron transport rates in the inside-out thylakoid vesicles by about 500 and 350%, respectively, compared to about 50% stimulation for both assays in the fraction enriched in right-side-out vesicles. The electron transport between PSII and PSI in inside-out thylakoid vesicles appears to be interrupted due to plastocyanin release from the thylakoids by the Yeda press treatment, but it was restored by externally added plastocyanin. The P700 contents of the inside-out and right-side-out vesicles were 1 P700 per 1100 to 1500 chlorophylls and 1 P700 per 500 chlorophylls respectively. Photosystem 1 is not totally excluded from the appressed grana regions (the hypothesized origin of inside-out thylakoid vesicles) as suggested by the concept of extreme lateral heterogeneity of PS I and II in the thylakoid membranes. Although PSI is somewhat depleted in the appressed grana membrane region, there is adequate photochemically active P700, when sufficient plastocyanin is available, to effectively couple PSI electron transfer with the preponderant PSII in linear electron transport. Acetic anhydride chemical modification and uncoupler-induced proton release from dark-adapted membranes are probes for detecting the sequested proton domains in thylakoid membranes. Both assays were used to find out if inside-out membranes retain metastable, localized proton binding domains. Treatment of dark-maintained inside-out thylakoid membrane vesicles with ($\sp3$H) acetic anhydride showed no uncoupler-induced increase in acetylation of the 33, 24, and 18 kDa polypeptides of the oxygen-evolving-complex, indicating complete loss of the implicated proton domains in these polypeptides. The various steps in the inside-out preparation were studied to discern which step(s) leads to the loss of the metastable domain proton pool. It appears that the Yeda press treatment disrupts the proton domains probably due to the damaging effects of the sonication-like events that occur as particles traverse the Yeda press orifice thus making inside-out thylakoid vesicles unsuitable for localized proton domain studies.

Degree

Ph.D.

Advisors

Dilley, Purdue University.

Subject Area

Biology

COinS