Blue-Native Page Analysis Validates Heterogeneity in the Thylakoids of Synechocystis 6803

Abstract

Thylakoids from mature chloroplasts of higher plants show distinct ultra structural differentiation into grana and stroma lamellae where the photosystems, PSII and PSI, are differentially distributed. The thylakoids from cyanobacteria do not show such differentiation, even if they have both PSII and PSI as components of electron transport like higher plants. Recently we have demonstrated that the thylakoids from cell free extracts of Synechocystis 6803 can be fractionated using differential ultra-centrifugation at 40,000 × g (40 k), 90,000 × g (90 k) and 150,000 × g (150 k). These native thylakoid fractions showed biochemical as well as proteomic heterogeneity. We have analyzed these fractions using Blue-Native (BN) PAGE followed by ESI-MS/MS or MALDI-MS. The 1-D BN gels showed protein super-complexes bands ranging from ∼200∼900 kDa. The protein super-complexes from 1-D gels were further separated into individual subunits in second dimension on the denaturing tricine SDS-PAGE. 44 differing bands from 1-D gels and 38 differing protein spots form 2-D gels were selected for identification. Our results showed that there were qualitative and quantitative differences in the pigment-protein complexes among the three fractions validating the hypothesis of heterogeneity in thylakoids of Synechocystis 6803.