Aqueous Polymer Two-phase Partitioning Research Articles

A mechanism for the formation of inside-out membrane vesicles. Preparation of inside-out vesicles from membrane-paired randomized chloroplast lamellae

Inside-out thylakoid membrane vesicles can be isolated by aqueous polymer two-phase partition of Yeda press-fragmented spinach chloroplasts (Andersson, B. and Åkerlund, H.-E. (1978) Biochim. Biophys. Acta 503, 462–472). The mechanism for their formation has been investigated by studying the yield of inside-out vesicles after various treatments of the chloroplasts prior to fragmentation. No inside-out vesicles were isolated during phase partitioning if the chloroplasts had been destacked in a low-salt medium prior to the fragmentation. Only in those cases where the chloroplast lamellae had been stacked by cations or membrane-paired by acidic treatment did we get any yield of inside-out vesicles. Thus, the intrinsic properties of chloroplast thylakoids seem to be such that they seal into right-side out vesicles after disruption unless they are in an appressed state. This favours the following mechanism for the formation of inside-out thylakoids. After press treatment, a ruptured membrane still remains appressed with an adjacent membrane. Resealing of such an appressed membrane pair would result in an inside-out vesicle. If the compartmentation of chloroplast lamellae into appressed grana and unappressed stroma lamellae is preserved by cations before fragmentation, the inside-out vesicles are highly enriched in photosystem II. This indicates a granal origin which is consistent with the proposed model outlined. Inside-out vesicles possessing photosystem I and II properties in approximately equal proportions could be obtained by acid-induced membrane-pairing of chloroplasts which had been destacked and randomized prior to fragmentation. Since this new preparation of inside-out thylakoid vesicles also exposes components derived from the stroma lamellae it complements the previous preparation. It is suggested that fragmentation of paired membranes followed by phase partitioning should be a general method of obtaining inside-out vesicles from membranes of various biological sources.

Scanning electron microscopy of different populations of chloroplasts isolated by phase partition

Scanning electron microscopy has been used to get information about purity and three-dimensional structure of the chloroplast populations that can be isolated by aqueous polymer two-phase partition. The critical point technique was used for drying the chloroplast preparations to avoid destructive surface tension and freeze artifacts. The three populations (intact chloroplasts, unstacked chloroplast lamellae and multiorganelle particles containing intact chloroplasts) showed essentially no cross contamination. Micrographs of intact chloroplasts show lens-shaped particles, regular in size and with a relatively smooth surface. In contrast the unstacked chloroplast lamellae have a very rough surface and a very flat appearance. Traditionally prepared stacked lamellar systems also look very flat but have a pattern of structures, which probably correspond to grana end membranes. The membrane-bound multiorganelle complexes containing chloroplasts, mitochondria, peroxisomes and cytoplasm have varying sizes and forms.