Abstract
Thylakoid membranes contain a number of proteins involved in photosynthetic electron transport and ATP synthase. Some proteins present in the thylakoid membranes form multi-protein complexes such as photosystem I and II (PSI and PSII, respectively), cytochrome b6f (Cytb6f), light-harvesting complex (LHC) as well as ATP synthase. Recently Blue-Native-PAGE (BN-PAGE) is extensively used to separate multi-protein complexes with high resolution. Here we used 2-dimensional (2D)-PAGE combining BN-PAGE and SDS-PAGE to carry out stoichiometric estimation of the multi-protein complexes in the thylakoid membranes. First we searched for optimal conditions for the 2D-PAGE to separate multi-protein complexes and their constituent proteins. Then we prepared thylakoid membranes uniformly labeled with 14C from which separated multi-protein complexes and their constituent proteins. Quantitative analyses of the radiolabeled proteins revealed that PSI, PSII, Cytb6f and ATP synthase accumulate at nearly equal amounts. In contrast the accumulation of LHCII was about 30 times higher than that of PSII. We also report the stoichiometry of constituent proteins of ATP synthase and LHCII complexes.
