Inside-out membrane vesicles isolated from spinach thylakoids

doi:10.1016/0005-2728(78)90145-7

Biochimica et Biophysica Acta (BBA) - Bioenergetics

Volume 503, Issue 3, 7 September 1978, Pages 462-472

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Abstract

Inside-out thylakoid vesicles have been separated from right-side-out material after press disruption of chloroplast lamellae. The separation was obtained by partition in an aqueous dextran-polyethylene glycol two-phase system, a method which utilizes differences in surface properties for separation of membrane particles. The isolated thylakoid vesicles showed the following inside-out properties: (1) light-induced reversible proton extrusion into the surrounding medium when supplied with the Photosystem II electron acceptor phenyl-p-benzoquinone; (2) a pH rise in the internal phase accompanying the external proton release, (3) sensitivity to trypsin treatment different from that of thylakoid membranes of normal orientation; (4) concave EF and convex PF freeze-fracture faces.

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Cited by (111)

Fragmentation and separation analysis of the photosynthetic membrane from spinach
2009, Biochimica et Biophysica Acta - Bioenergetics
Citation Excerpt :
The separation of grana vesicles from the stroma/end membrane vesicles, Fig. 3A, is due to the different sidedness of the two types of vesicles. The grana vesicles are inside-out and prefer the lower, dextran rich, phase while the stroma/end membrane vesicles are right-side out and prefer the upper, PEG rich, phase [43,44]. The heterogeneity observed within the grana vesicles, grana core vesicles, margin vesicles, and the stroma lamellae/end membrane vesicles (Fig. 3B–E) respectively is not due to different sidedness but probably to differences in the relative amount of different thylakoid proteins exposed to the surface.
Membrane vesicles, originating from grana, grana core (appressed grana regions), grana margins and stroma lamellae/end membranes, were analysed by counter current distribution (CCD) using aqueous dextran-polyethylene glycol two-phase systems. Each vesicle population gave rise to distinct peaks in the CCD diagram representing different vesicle subpopulations. The grana vesicles and grana core vesicles each separated into 3 different subpopulations having different chlorophyll a/b ratios and PSI/PSII ratios. Two of the grana core subpopulations had a chlorophyll a/b ratio of 2.0 and PSI/PSII ratio of 0.10 and are among the most PSII enriched thylakoid vesicle preparation obtained so far by a non detergent method. The margin vesicles separated into 3 different populations, with about the same chlorophyll a/b ratios, but different fluorescence emission spectra. The stroma lamellae/end membrane vesicles separated into 4 subpopulations. Plastoglobules, connected to membrane vesicles, were highly enriched in 2 of these subpopulations and it is proposed that these 2 subpopulations originate from stroma lamellae while the 2 others originate from end membranes. Fragmentation and separation analysis shows that the margins of grana constitute a distinct domain of the thylakoid and also allows the estimation of the chlorophyll antenna sizes of PSI and PSII in different thylakoid domains.
The topology of phosphatidylglycerol populations is essential for sustaining photosynthetic electron flow activities in thylakoid membranes
2000, Biochimica et Biophysica Acta - Biomembranes
Citation Excerpt :
This has been verified in thylakoids by measuring the osmotic responsiveness towards sorbitol, a molecule having a much smaller size than phospholipase A2[5]; (e) each type of structure (thylakoids, thylakoids rightside-out and inside-out vesicles) must have, within the same population, an identical sidedness; and (4) the inside-out and rightside-out vesicle fractions should contain the lowest amount of the other category of vesicles. Andersson and Akerlund [24] found that the inside-out fraction contains a contamination of 26% of rightside-out vesicles. The cross-contamination between these two fractions might explain the low chlorophyll a/b ratio value in rightside-out vesicles as well as the lower uptake of protons in rightside-out vesicles than in intact thylakoids.
The transmembrane distribution of phosphatidylglycerol (PG) was determined in rightside-out (RO) and inside-out vesicles (IO) obtained by fragmentation of spinach thylakoids in a Yeda press, followed by partition in an aqueous dextran-polyethyleneglycol two-phase system. Using the phospholipase A₂ from porcine pancreas to digest selectively PG molecules in the outer monolayer (exposed to the incubation medium) of the membrane, we found the molar outside/inside distribution to be 70/30±5 in RO and 40/60±3 in IO. The transmembrane distribution of PG in IO was the opposite of that in intact thylakoids (molar ratio 58/42±3). The phospholipid population which sustained most of the uncoupled photosystem II electron flow activity was localized in the inner monolayer (exposed to the thylakoid lumen) of both thylakoid and RO membranes. In contrast, the activity in IO membranes was highly dependent on the PG population located in the outer monolayer. This finding brings the first direct demonstration of the dependence of the photosynthetic electron flow activity on the integrity of the inner topological pool of PG in the thylakoid membrane.
The thylakoid lumen of chloroplasts. Isolation and characterization
1998, Journal of Biological Chemistry
The chloroplast compartment enclosed by the thylakoid membrane, the “lumen,” is poorly characterized. The major aims of this work were to design a procedure for the isolation of the thylakoid lumen which could be generally used to characterize lumenal proteins. The preparation was a stepwise procedure in which thylakoid membranes were isolated from intact chloroplasts. Loosely associated thylakoid surface proteins were removed, and following Yeda press fragmentation the lumenal content was recovered in the supernatant following centrifugation. The purity and yield of lumenal proteins were determined using appropriate marker proteins specific for the different chloroplast compartments. Quantitative immunoblot analyses showed that the recovery of soluble lumenal proteins was 60–65% (as judged by the presence of plastocyanin), whereas contamination with stromal enzymes was less than 1% (ribulose-bisphosphate carboxylase) and negligible for thylakoid integral membrane proteins (D1 protein). Approximately 25 polypeptides were recovered in the lumenal fraction, of which several were identified for the first time. Enzymatic measurements and/or amino-terminal sequencing revealed the presence of proteolytic activities, violaxanthin de-epoxidase, polyphenol oxidase, peroxidase, as well as a novel prolyl cis/trans-isomerase.
Antenna protein composition of PS I and PS II in thylakoid sub-domains
1997, Biochimica et Biophysica Acta - Bioenergetics
Spinach thylakoids were separated into grana core, grana margin, and two different stromal lamella fractions in the absence of detergents. The levels of all light-harvesting chlorophyll a/b-binding (LHC) proteins were determined in all fractions, and were normalised to the amount of Photosystem I (PS I) and Photosystem II (PS II) centres. PS Iβ in the stroma lamellae was found to have a full complement of Lhca polypeptides and, probably, one attached LHC II trimer. PS Iα binds additional LHC II trimers, but PS I centres located in the inner parts of the grana stack lack Lhca1 and are depleted in Lhca4. PS IIβ, found in grana margins and stroma lamellae, seems to associate one monomer each of Lhcb4, Lhcb5 and Lhcb6 (CP29, CP26 and CP24, respectively) and one LHC II trimer consisting of two Lhcb1 and one Lhcb3 subunit. PS IIα has additional LHC II trimers (consisting of Lhcb1 and Lhcb2) attached. We also find evidence for the existence of both PS I and PS II centres in the extreme stroma (probably centres being synthesised or repaired), that lack all LHC proteins.
The rate constant of photoinhibition in vitro is independent of the antenna size of Photosystem II but depends on temperature
1994, BBA - Bioenergetics
Photoinhibition of Photosystem (PS) II was studied in thylakoid membranes, inside-out and rightside-out thylakoid vesicles derived from appressed and non-appressed membrane regions, respectively, in detergent fractionated PS II membranes and in oxygen evolving PS II core complexes. The preparations were illuminated without added electron acceptors, and care was taken to keep the oxidizing side of PS II in a functional condition during the experiments. The first-order rate constant of photoinhibition, measured under given photon flux density, was similar in all preparations derived from appressed thylakoid regions and independent of the antenna size. This antenna size independence indicates that under photoinhibitory conditions in vitro, when most PS II traps are closed, the probability of finding an exciton in the reaction centre is not much larger than the probability of finding it in the antenna. Spillover of excitation energy from PS II to PS I may be an important factor protecting the PS IIβ of stroma thylakoids from photoinhibition. Photoinhibition in vitro is a first-order reaction even at low temperatures where the degradation of the D1 protein is slow, which demonstrates that the photoinhibited PS II centres do not protect the remaining active ones from photoinhibition, at least not in vitro. The activation energy of photoinhibition in pumpkin thylakoids, as measured between 6 and 25°C, was 15 kJ/mol; the rate constant of photoinhibition in pumpkin thylakoids increased both below 6 and above 25°C.
Supports for liquid-liquid partition chromatography in aqueous two-phase systems: A comparison of LiChrospher and LiParGel
1994, Journal of Chromatography A
Polyacrylated particles of silica (LiChrospher Diol 4000) or hydrophilic polyvinyl polymers (LiParGel 650) were used as supports for liquid-liquid partition chromatography in an aqueous polyethylene glycol-dextran two-phase system. High packing pressures gave high and reproducible values of the stationary mobile phase volume ratio (V_S/V_M = 1.5) on both supports. The volume of one theoretical plate was only 6 μl on LiChrospher and 20 μl on LiParGel. The performance of a 3-ml LiChrospher column was comparable to that of 15-ml LiParGel columns. In addition, the resolution of immunoglobulins was higher on LiChrospher. The influence of the supports on the retention of proteins is discussed.

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Inside-out membrane vesicles isolated from spinach thylakoids

Abstract

FEBS Lett.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

FEBS Lett.

Biochim. Biophys. Acta

J. Biol. Chem.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Science