On the lateral organisation of the thylakoid membrane

Abstract: In this work the lateral heterogeneity of the thylakoid membrane was investigated by quantifying the amount of photosystem I (PSI), photosystem II (PSII) and some PSII complexes and PSII proteins in different domains of the thylakoid. Grana, grana core, grana margins and stroma lamellae + end fractions were obtained by sonication and separation in a non-detergent, aqueous polymer two-phase system. Y-100 was obtained by Yeda press and centrifugation. EPR spectroscopy was used to quantify PSI (P700+) and PSII (YD·) in the different fractions. These measurements showed a successively increasing amount of PSI from grana core, grana, grana margins and stroma lamellae + end membranes to Y-100. On the contrary the amount of PSII is decreased successively from grana core to Y-100. The ratio PSI/PSII increases from 0,25 in grana core to 13 in Y-100. For the entire thylakoid the PSI/PSII ratio is 1,13. Gel electrophoresis was performed to trace five PSII complexes (supercomplexes, dimers, monomers with CP43 and monomers without CP43, and core reaction centres) in the different fractions of the thylakoid. These were then quantified by western blotting. The supercomplexes are most common in grana core and the fragments of the complexes are most common in Y-100, in a gradient through the fractions. This gives strong support to the notion of a lateral transport of PSII centres from grana core to Y-100 and back again, in a repair process. Thirteen PSII proteins were also analysed and quantified in the different fractions. PsbS, PsbW, PsbZ were overrepresented in grana or grana core and absent or nearly absent in Y-100. It agrees with their assumed function of preserving and stabilising the fully developed PSII complexes in grana core. Using counter-current distribution, a diagram was obtained showing heterogeneity within all the sub-fractions according to their surface properties.