Isolation of Inside-Out Thylakoid Vesicles

Abstract

To understand the function of a biological membrane like that of chloroplast thylakoids, it is important to understand the arrangement of its different protein and lipid components. Preparations that have proven to be particularly suited for such studies are those consisting of membrane vesicles that are turned inside-out. Inside-out vesicles from the thylakoid membrane were first obtained from spinach chloroplasts by a combination of mechanical fragmentation and separation by aqueous two-phase partition (1,2). By the same or very similar procedures, inside-out thylakoid vesicles have now also been obtained from other plant sources such as pea (3), barley (4), mangrove (Avicennia marina) (5), lettuce (6), Euglena gracilis (7), cyanobacteria (8,9) and the photosynthetic bacteria Rhodopseudomonas viridis (10). Because the isolation procedure does not involve the use of detergents, the inside-out thylakoids have a preserved membrane structure and are ideally suited for structure-function studies. They have been used extensively in studies on thylakoid membrane topography (11-14) and for the identification of proteins associated with oxygen evolution (15,16). An important finding was that the inside-out vesicles are only formed from appressed (not exposed to stroma) thylakoid membranes, whereas right side-out vesicles derive from nonappressed (stroma exposed) membranes (17). As a result, the usefulness of inside-out vesicles could be extended to include studies on the lateral organization of the thylakoid membrane. The use of inside-out thylakoids in studies on structure and function of the thylakoid membrane has been reviewed by Andersson et al. (18).