Background

Thylakoids are specialized membranes of algal and plant chloroplasts and cyanobacteria. The thylakoid lipid bilayer shares characteristic features with prokaryotic bacterial membranes but it is enriched in glycolipids, namely galactolipids, while the level of acidic lipids is relatively low and does not exceed 20% of the overall lipid content. Thylakoids contain a large number of proteins and their complexes and over 40% of them are involved in light-dependent reactions of photosynthesis. The next largest functional group includes proteins involved in protein targeting, processing and folding, oxidative stress response and translation. The most integral membrane proteins of thylakoids form large protein complexes which play an important role in the harvesting of light and its photochemical conversion. The key complexes performing this conversion are PSI and PSII. They both perform the light-driven primary charge separation resulting in the thylakoid-associated flow of electrons reducing NADP⁺ and generating a pH gradient used for the synthesis of ATP. Both complexes are subject to intensive research focused mainly on the elucidation of their function and underlying structural features as well as the mechanism of their assembly. For this research the isolation of native membranes and their detailed analysis as concerns the content, assembly and oligomerization state of both photosystems is essential. The existing and frequently used protocols for the isolation of cyanobacterial thylakoids either do not sufficiently preserve the native structure of fragile membrane complexes like Photosystem II dimers (e.g., Komenda and Barber, 1995), or the obtained preparations contain high concentrations of compounds (for instance 1 M glycine betaine) which are not compatible with the clear native gel electrophoresis (e.g., Gombos et al., 1994). The described protocol for thylakoid preparation is optimized from both points of view. The analysis of cyanobacterial membranes by clear native PAGE (CN PAGE) using a protocol modified from Wittig et al. (2007) gives direct information about the quantity and oligomeric (or assembly) state of multisubunit pigmented photosystem complexes with unprecedented speed and resolution. The isolation, solubilization, and analysis of membranes does not disrupt weak non-covalent interactions between pigments and proteins and therefore the gel with separated individual complexes can be directly scanned. Unlike denaturing SDS gels, no fixation, staining and destaining, or, blotting and immunodetection is needed for distinguishing between PSII and PSI (see Figure 2). Green PSI complexes do not show red fluorescence at room temperature and are present as dominant green bands in the gel while PSII complexes fluoresce and their green bands are hard to distinguish on the simple transparency scan. Thus, PSI quantification is done using the simple transparency scan of green non-fluorescent bands while PSII quantification is done using the red fluorescence scan of fluorescent bands.