Isolation of Inside-Out and Right-Side-Out Thylakoid Membrane Vesicles from the Cyanobacterium Synechocystis 6803

Abstract

Cyanobacteria are prokaryotic organisms that perform oxygenic photosynthesis. The functional organization of the photosynthetic thylakoid membranes in cyanobacteria is similar to that in the chloroplasts of green plants. Synechocystis sp. PCC 6803, a unicellular cyanobacterium, offers an attractive system to study the biogenesis and functions of protein complexes such as photosystems I and II in the thylakoid membrane. Synechocystis 6803 is the first photosynthetic organism for which the complete genome sequence has been determined. More than twenty years ago, a method was developed to isolate vesicles of higher plant thylakoid membranes with different surface orientations (1). Inside-out thylakoid vesicles have also been isolated from the filamentous cyanobacterium Phormidium laminosum (2). However, the yield of such vesicles is low. In this communication, we describe a method for the isolation of right-side-out and inside-out thylalcoid vesicles from Synechocystis 6803. To isolate inside-out vesicles, cells were broken by French-press treatment, whereas for right-side-out vesicles, cells were disrupted using glass beads. In both cases, membrane vesicles were obtained after partitioning in an aqueous two-phase polymer system. The orientations of the membranes were determined by treatments with various salts, protease and sonication. Using such isolated vesicles, we have determined that BtpA, a newly identified protein involved in the biogenesis of the PSI protein complex (3), is associated with the outer surface of the thylakoid membrane.