Hans Erik Akerlund 1 Introduction

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 thyla-koids 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).

The isolation of inside-out thylakoid vesicles can be divided principally into two steps; fragmentation and subsequent separation. The ionic conditions imposed during fragmentation have a profound influence on both the proper-

From: Methods in Biotechnology, Vol. 11: Aqueous Two-Phase Systems: Methods and Protocols Edited by: R. Hatti-Kaul © Humana Press Inc., Totowa, NJ

ties and relative proportions of inside-out thylakoids and right side-out thyla-koids (17). Basically, three different fragmentation procedures have been described. The original procedure (1,2; see Note 1) gives rise to inside-out vesicles originating from the appressed thylakoid region, highly enriched in photosystem II, but with relatively low yield. The procedure that will be described here (19) gives inside-out vesicles in a high yield and somewhat lower enrichment in photosystem II. The inside-out vesicles obtained by these two procedures are particularly suited for studies on the lateral distribution of thylakoid components and for studies on the organization and function of photosystem II and its oxygen evolving system. The third procedure (17, see Note 1) gives inside-out vesicles with the same relative amounts of photosystem I and II as whole thylakoids and is therefore especially suited for studies on transverse arrangements of all components of the thylakoid membrane.

The key step for the isolation of inside-out vesicles from right side-out vesicles is aqueous polymer two-phase partition. This method separates membrane particles according to differences in surface properties such as charge and hydrophobicity. It is, therefore, ideally suited for separation of membrane vesicles that only differ in sidedness. This separation step is essentially the same for all three preparation procedures. The procedures described here were developed for thylakoids from spinach, but should be suitable for most other species too.

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