Optimal Conditions for Formation and Separation

Plasma membrane preparations of high purity (about 95%) are easily obtained by partitioning in aqueous polymer two-phase systems. These preparations, however, mainly contain sealed right-side-out (apoplastic side out) vesicles. Part of these vesicles have been turned inside-out by freezing and thawing, and sealed inside-out and right-side-out vesicles subsequently separated by repeating the phase partition step. Increasing the KCI concentration in the freeze/thaw medium as well as increasing the number of freeze/thaw cycles significantly increased the yield of insideout vesicles. At optimal conditions, 15 to 25% of total plasma membrane protein was recovered as inside-out vesicles, corresponding to 5 to 10 milligrams of protein from 500 grams of sugar beet (Beta vulgaris L.) leaves. Based on enzyme latency, trypsin inhibition of NADH-cytochrome c reductase, and H+ pumping capacity, a cross-contamination of about 20% between the two fractions of oppositely oriented vesicles was estimated. Thus, preparations containing about 80% inside-out and 80% rightside-out vesicles, respectively, were obtained. ATPase activity and H+ pumping were both completely inhibited by vanadate (K, ; 10 micromolar), indicating that the fractions were completely free from nonplasma membrane ATPases. Furthermore, the polypeptide patterns of the two fractions were close to identical, which shows that the vesicles differed in sidedness only. Thus, preparations of both inside-out and right-side-out plasma membrane vesicles are now available. This permits studies on transport, signal transduction mechanisms, enzyme topology, etc., using plasma membrane vesicles of either orientation. A typical feature ofbiological membranes is the asymmetric arrangement of constituents across the lipid bilayer. This asymmetry is absolute for proteins: integral proteins, which span the membrane, expose different regions on either side of This paper is dedicated to Professor Per-Ake Albertsson, the pioneer of aqueous polymer two-phase partitioning, on the occasion of his 60th birthday. Supported by grants from the Swedish Natural Science Research Council (C. L., M. S., S. W.), the Danish Agricultural and Veterinary Research Council (M. G. P.), the Danish Natural Science Research Council (M. G. P.), the Danish Research Academy (M. G. P.), and the Carl Tesdorpf Foundation (C. L.). the membrane, whereas peripheral proteins are bound to either surface of the membrane. For lipids, the asymmetry is rather relative than absolute, such that each lipid species usually only shows some enrichment to either half of the bilayer. This asymmetric, transverse organization of membrane constituents forms the basis for all the vectorial activities exerted by biological membranes, and is created through the asymmetric assembly of membranes (review, 22). The most useful approach for characterizing the asymmetric properties ofa membrane, including its vectorial activities, is to prepare sealed membrane vesicles of either orientation. With such preparations, each membrane surface can be probed selectively using impermeable agents, and transport in either direction can be measured as uptake into vesicles. The formation and subsequent separation of vesicles of opposite orientation was first achieved with the erythrocyte membrane through the pioneering work ofSteck et al. (review, 30), and later with the mitochondrial inner membrane (review, 8) and the chloroplast thylakoid membrane (review, 2). The access to sealed membrane vesicles of either orientation made extensive studies on the topology of these membranes possible. With both the erythrocyte and the thylakoid membrane, aqueous two-phase partitioning was used to separate the oppositely oriented vesicles (2, 30). Indeed, two-phase partitioning should be a very suitable method in such cases, since it separates particles according to their surface properties (1) and vesicles of opposite orientation are expected to differ in this respect but not in size or density. For the plant plasma membrane, the separation of insideout (cytoplasmic side out) and right-side-out (apoplastic side out) vesicles was only recently achieved using either free-flow electrophoresis (7) or two-phase partitioning (21). We now report a number of essential improvements on the phase partition procedure, as well as a thorough characterization of the membrane fractions obtained. MATERIALS AND METHODS