Inside-out membrane vesicles isolated from spinach thylakoids

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.     

Symport of proton and sucrose in plasma membrane vesicles isolated from spinach leaves

The mechanism of sucrose transport was investigated in plasma membrane (PM) vesicles isolated from spinach (Spinacia oleracea L.) leaves. PM vesicles were isolated by aqueous two-phase partitioning and were equilibrated in pH 7.8 buffer containing K⁺. The vesicles rapidly accumulated sucrose in the presence of a transmembrane pH gradient (ΔpH) with external pH set at 5.8. The uptake rate was slow at pH 7.8. The K⁺-selective ionophore, valinomycin, stimulated uptake in the presence of a ΔpH, and the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), greatly inhibited ΔpH-dependent sucrose uptake. Addition of sucrose to the vesicles resulted in immediate alkalization of the medium. Alkalization was stimulated by valinomycin, was abolished by CCCP, and was sucrose-specific. These results demonstrate the presence of a tightly coupled H⁺/sucrose symporter in PM vesicles isolated from spinach leaves.     

Inside-out basolateral plasma membrane vesicles from rat kidney proximal tubular cells

A method for preparation of highly purified basolateral plasma membranes from rat kidney proximal tubular cells is reported. These membranes were assayed for the presence of vesicles as well as for their orientation. (Na+ + K+)-ATPase activity and [3H]ouabain binding studies with membranes treated with or without SDS revealed that the preparation consisted of almost 100% vesicles. The percentage of inside-out vesicles was found to be approx. 70%. This percentage was determined measuring the (Na+ + K+)-ATPase activity in K+-loaded vesicles and in membranes treated with or without trypsin and SDS. These membranes represent a very efficient tool to assay the correlation between active transport and ATPase activities in basolateral plasma membranes from rat kidney proximal tubular cells.     

Protein identification of purified particles isolated from spinach thylakoids by deoxycholate electrophoresis

Three thylakoid complexes were isolated by deoxycholate preparative electrophoresis. The protein composition of each fraction was analyzed by SDS analytical electrophoresis. No protein of the PS 1 enriched fraction (fraction 1) was found in the PS 2 enriched fraction (fraction 2) and inversely. The antenna complex (fraction 3) did not have any contamination by proteins of fraction 1 or fraction 2. Fraction 1 was mainly composed of the CP1, the reaction center complex of the PS1, and by low molecular weight proteins, previously found in other PS 1 preparations. Tentative assignments of these proteins are presented; among them are iron sulfur proteins. After analytical SDS electrophoresis of fraction 2, the reaction center complex was dissociated. Nevertheless three proteins of 50 kD, 42 kD and 35 kD were assigned to this complex. Fraction 2 contained also the three cytochromes of the thylakoid membranes: cyt f, cyt b6, cyt b559. Fraction 3 was exclusively composed of one protein pigment complex, CP2.Abbreviations SDS sodium dodecyl sulfate - PS 1 photosystem 1 - PS 2 photosystem 2 - CP1, CP2 protein pigment complexes isolated by SDS electrophoresis - cyt cytochromes - P700 primary electron donor of PS 1 - P680 primary electron donor of PS 2 - DOC deoxycholate - Q primary plastoquinone electron acceptor - CF coupling factor     

Salt treatment induces frost hardiness in leaves and isolated thylakoids from spinach

Frost hardiness of spinach (Spinacia oleracea L.) leaves was increased by high concentrations of NaCl in the hydroponic culture medium. Freezing damage was determined by measurement of slow chlorophyll fluorescence quenching after freezing of leaves. Both the osmolality of the leaf sap and forst hardiness of the leaves were linearly correlated with the salt concentration in the hydroponic culture medium. Freezing damage occurred, irrespective of the extent of frost hardening, when dehydration of cells during extracellular ice formation decreased cellular volume to approximately 14% of the volume of unfrozen cells. The resistance of isolated, washed thylakoids against mechanical and chemical damage by freezing was investigated. Chemical damage by freezing caused by salt accumulation was measured as release of chloroplast coupling factor (CF1; EC 3.6.1.3), and mechanical damage was measured as release of the lumenal protein plastocyanin from the membranes during an in-vitro freeze-thaw cycle. Isolated thylakoids from salt-treated frost-hardy spinach and those from plants hardened under natural conditions did not exhibit improved tolerance against chemical freezing stress exerted by high salt concentrations. They were, however, more hardy than thylakoids from unhardened control leaves against mechanical damage by freezing.Abbreviation CF1 peripheral part of chloroplast coupling factor ATPase     

Quantitative separation of spinach thylakoids into Photosystem II-enriched inside-out vesicles and Photosystem I-enriched right-side-out vesicles

Yeda press disruption of thylakoids in the presence of magnesium followed by aqueous polymer two-phase partitioning fractionated the total thylakoid membrane material into two distinctly different fractions. One fraction comprised approx. 60% of the material on a chlorophyll basis and contained inside-out vesicles while the other fraction (40%) contained right-side-out vesicles. The sidedness of the vesicles was determined from the direction of their light-induced proton translocation. The inside-out vesicles showed a pronounced Photosystem (PS) II enrichment as judged by their high PS II and low PS I activities. Moreover, they showed a high ratio between the PS II reaction centre chlorophyll-protein complex and the PS I reaction centre chlorophyll-protein complex (CP I). The chlorophyll $\text{a}\text{b}$ ratio was as low as 2.3 compared to 3.2 for the starting material. In contrast, the right-side-out vesicles showed a pronounced PS I enrichment. Their chlorophyll $\text{a}\text{b}$ ratio was 4.3–4.9. The tight stacking induced by Mg²⁺ allows a quantitative formation of inside-out vesicles from the appressed thylakoid regions while mainly non-appressed thylakoids turn right-side-out. The possibility of fractionating all of the thylakoid material into two sub-populations with markedly different composition with respect to PS I and PS II argues against a close physical association between the two photosystems and in favour of their spatial separation in the plane of the membrane. This fractionation procedure, which can be completed within 1 h and gives high yields of both PS II inside-out thylakoids and PS I right-side-out thylakoids, should be very useful for facilitating and improving studies on both the transverse and lateral organization of the thylakoid membrane.     

Composition of photosynthetic pigments in thylakoid membrane vesicles from spinach

Thylakoid membranes from spinach were fragmented mechanically and separated into vesicles originating from grana and stroma-exposed lamellae (Andreasson et al. (1988) Biochim Biophys Acta 936: 339–350). The grana vesicles were further fragmented and separated into smaller vesicles originating from different parts of the grana (Svensson and Albertsson (1989) Photosynth Res 20: 249–259). All vesicles so obtained were analyzed with respect to chlorophyll and carotenoid composition by reverse phase HPLC. For all fractions the following relations (mole/mole) were found: 1 carotenoid per 4 chlorophyll (a+b), 2 lutein per 5 chlorophyll b and 5 violaxanthin per 100 chlorophyll (a + b). The contents of lutein and neoxanthin were each linearly related to chlorophyll b and -carotene was linearly related to chlorophyll a.     

Rotavirus interaction with isolated membrane vesicles.

To gain information about the mechanism of epithelial cell infection by rotavirus, we studied the interaction of bovine rotavirus, RF strain, with isolated membrane vesicles from apical membrane of pig enterocytes. Vesicles were charged with high (quenching) concentrations of either carboxyfluorescein or calcein, and the rate of fluorophore release (dequenching) was monitored as a function of time after mixing with purified virus particles. Purified single-shelled particles and untrypsinized double-shelled ones had no effect. Trypsinized double-shelled virions induced carboxyfluorescein release according to sigmoid curves whose lag period and amplitude were a function of virus concentration and depended on both temperature and pH. The presence of 100 mM salts (Tris Cl, NaCl, or KCl) was required, since there was no reaction in isoosmotic salt-free sorbitol media. Other membrane vesicle preparations such as apical membranes of piglet enterocyte and rat placenta syncytiotrophoblasts, basolateral membranes of pig enterocytes, and the undifferentiated plasma membrane of cultured MA104 cells all gave qualitatively similar responses. Inhibition by a specific monoclonal antibody suggests that the active species causing carboxyfluorescein release is VP5*. Ca2+ (1 mM), but not Mg2+, inhibited the reaction. In situ solubilization of the outer capsid of trypsinized double-shelled particles changed release kinetics from sigmoidal to hyperbolic and was not inhibited by Ca2+. Our results indicate that membrane destabilization caused by trypsinized outer capsid proteins of rotavirus leads to fluorophore release. From the data presented here, a hypothetical model of the interaction of the various states of the viral particles with the membrane lipid phase is proposed. Membrane permeabilization induced by rotavirus may be related to the mechanism of entry of the virus into the host cell.     

Plasma membrane vesicles isolated from epimastigote forms of Trypanosoma cruzi.

Membrane vesicles can be obtained from epimastigote forms of Trypansoma cruzi by incubating cells with either cross-linking reagents or acid pH. Acetate, phtalate or citrate, at pH 4.0, but not at higher pH values, were able to induce plasma membrane vesiculation. Vesicles have been purified by sucrose density centrifugation and their membrane origin was demonstrated by the following criteria: (a) Vesicles are 5--10 times richer in protein-bound iodine when they are prepared from cells previously labeled with 131I by the lactoperoxidase catalyzed reaction. (b) Electron microscopy of vesiculating cells shows physical continuity between cell plasma membrane and vesicle membrane. (c) Antibodies prepared against purified vesicles are able to agglutinate epimastigote forms of T. cruzi with sera dilutions up to 1 : 256 to 1 : 512. (d) Freeze-fracture studies of the purified vesicles have shown images of faces P and E compatible with known images of the intact cell plasma membrane. Typical preparations of acetate vesicles present the following characteristics: total carbohydrate : protein=1.5--2.0; orcinol : protein-0.07 and absence of diphenylamine reaction. Vesicles contain 0.2--0.5% and 0.3--1.0% of the total homogenate protein and carbohydrate, respectively. The presence of 10 major protein bands and 30--50-fold enrichment of the four sugar-containing macromolecules present in epimastigote forms of T. cruzi have been demonstrated in these preparations.     

Chloride permeability of membrane vesicles isolated from Torpedo californica electroplax.

The Cl- permeability of membrane vesicles prepared from the electric organ of the marine ray Torpedo californica was studied by means of radioactive tracer exchange and by measuring the changes in the scattered-light intensity caused by osmotically induced volume changes. Both types of experiments indicate that a substantial fraction of the vesicles is extremely permeable of Cl-. Furthermore, this permeability pathway is inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, a well-known inhibitory of anion transport in a variety of systems. The properties of this permeability pathway are consistent with its identification as the voltage-aged Cl- channel studied in planar bilayers.     

Isolation of chestnut chloroplasts: membrane potentials of chestnut and spinach thylakoids

Typical chestnut thylakoid extracts isolated by mechanical disruption of leaf tissues had an equivalent of 0.28 kg m⁻³ chlorophyll (Chl) which is six times less than in thylakoids obtained from spinach, although Chl content in leaves was only half as small. According to optical microscopy, the vesicles showed a good integrity, exhibiting at 21 °C a high capacity of photon-induced potential membrane generation, which was demonstrated by the almost full 9-amino-6-chloro-2-methoxyacridine fluorescence quenching in a hyper-saline medium containing 150 mM KCl and having osmotic potential of −1.5 MPa. The half-time of the thylakoid potential generation was 11.7 s with the time of dissipation around 8.9 s. In such conditions, spinach thylakoids showed an increased swelling and also differences in the half-time generation which was almost four times faster than was observed in chestnut. However, when spinach thylakoids were incubated in a typical hypo-saline medium without KCl with osmotic potential −0.8 MPa, no additional swelling was observed. Consequently the half-time of potential dissipation was 35 s. Studies with nigericin suggested a chestnut thylakoid ΔpH significantly smaller than that observed in spinach, which was confirmed by the measurements of the ATP driven pumping activity.     

Distribution of calmodulin-stimulated ca transport into membrane vesicles from green spinach leaves

A microsomal fraction isolated from green spinach leaves exhibited a Mg(2+) and ATP-dependent (45)Ca(2+) uptake. Addition of 10 micromolar carbonyl cyanide m-chlorophenylhydrazine had no effect. The cationophore A23187 (10 micromolar) induced the release of (45)Ca(2+) accumulated by membrane vesicles. Membranes prepared from lower epidermis showed the highest Ca(2+) accumulation activity. Microsomal fractions from petiole, lamina, and midrib were less active. The stimulation by bovine brain calmodulin was about 30% for the lower epidermis, 23.5% for midrib, and below 20% for petiole and lamina.     

Interaction of 1,4-benzoquinones with Photosystem II in thylakoids and Photosystem II membrane fragments from spinach

The interaction of exogenous quinones with the Photosystem II (PS II) acceptor side has been analyzed by measurements of flash-induced 320 nm absorption changes, transient flash-induced variable fluorescence changes, thermoluminescence emission and oxygen yield in dark-adapted thylakoids and PS II membrane fragments. Two classes of 1,4-benzoquinones were shown to give rise to remarkably different reaction patterns. (A) Phenyl-p-benzoquinone (Ph-p-BQ) -type compounds give rise to a marked binary oscillation of the initial amplitudes of 320 nm absorption changes induced by a flash train in dark-adapted PS II membrane fragments and a retardation of the decay kinetics of the flash-induced variable fluorescence. The electron transfer reactions to these type of quinones are severely inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). (B) In the presence of tribromotoluquinone (TBTQ) a different oscillation pattern of the 320 nm absorption changes is observed characterized by a marked relaxation after the first flash in the 5 ms domain. This relaxation is insensitive to 10 μM DCMU. Likewise the decay of the flash-induced variable fluorescence in TBTQ-treated samples is much less sensitive to DCMU than in control. The thermoluminescence emission exhibits an oscillation in samples incubated for 5 min with TBTQ before addition of 30 μM DCMU. Under the same conditions a significant flash-induced oxygen evolution is observed only after the third and fourth flash, respectively, whereas in the presence of TBTQ alone a normal oscillation pattern is observed. The different functional patterns of PS II caused by the two types of classes of exogenous quinones are interpreted by their binding properties: a noncovalent association with the Q_B-site of Ph-p-BQ-type quinones versus a tight (covalent?) binding in the vicinity of Q_A (possibly also at the Q_B-site) in the case of halogenated 1,4-benzoquinones. The mechanistic implications of these findings are discussed.     

Phosphate transport into brush-border membrane vesicles isolated from rat small intestine.

Uptake of Pi into brush-border membrane vesicles isolated from rat small intestine was investigated by a rapid filtration technique. The following results were obtained. 1. At pH 7.4 in the presence of a NaCl gradient across the membrane (sodium concentration in the medium higher than sodium concentration in the vesicles), phosphate was taken up by a saturable transport system, which was competitively inhibited by arsenate. Phosphate entered the same osmotically reactive space as D-glucose, which indicates that transport into the vesicles rather than binding to the membranes was determined. 2. The amount of phosphate taken up initially was increased about fourfold by lowering the pH from 7.4 to 6.0.3. When Na+ was replaced by K+, Rb+ or Cs+, the initial rate of uptake decreased at pH 7.4 but was not altered at pH 6.0.4. Experiments with different anions (SCN-,Cl-, SO42-) and with ionophores (valinomycin, monactin) showed that at pH 7.4 phosphate transport in the presence of a Na+ gradient is almost independent of the electrical potential across the vesicle membrane, whereas at pH 6.0 phosphate transport involves the transfer of negative charge. It is concluded that intestinal brush-border membranes contain a Na+/phosphate co-transport system, which catalyses under physiological conditions an electroneutral entry of Pi and Na+ into the intestinal epithelial cell. In contrast with the kidney, probably univalent phosphate and one Na+ ion instead of bivalent phosphate and two Na+ ions are transported together.     

Tyrosine transport by membrane vesicles isolated from rat brain

Tyrosine uptake by membrane vesicles derived from rat brain has been investigated. The uptake is dependent on an Na⁺ gradient ([$\text{Na}{}^{\mathrm{+}}\text{]}\text{outside}\text{> [}\text{Na}{}^{\mathrm{+}}\text{]}\text{inside}$). The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. The process is stimulated by a membrane potential (negative inside) as demonstrated by the effect of the ionophores valinomycin and carbonyl cyanide $\text{m-}\text{chlorophenylhydrazone}$ and anions with different permeabilities. Kinetic data show that tyrosine is accumulated by two systems with different affinities. Tyrosine uptake is inhibited by the presence of phenylalanine and tryptophan.     

Inside-out: from endosomes to extracellular vesicles in fungal RNA transport

Membrane-coupled RNA transport is an emerging theme in fungal biology. This review focuses on the RNA cargo and mechanistic details of transport via two inter-related sets of organelles: endosomes and extracellular vesicles for intra- and intercellular RNA transfer. Simultaneous transport and translation of messenger RNAs (mRNAs) on the surface of shuttling endosomes is a conserved process pertinent to highly polarised eukaryotic cells, such as hyphae or neurons. Here we detail the endosomal mRNA transport machinery components and mRNA targets of the core RNA-binding protein Rrm4. Extracellular vesicles (EVs) are newly garnering interest as mediators of intercellular communication, especially between pathogenic fungi and their hosts. Landmark studies in plant–fungus interactions indicate EVs as a means of delivering various cargos, most notably small RNAs (sRNAs), for cross-kingdom RNA interference. Recent advances and implications of the nascent field of fungal EVs are discussed and potential links between endosomal and EV-mediated RNA transport are proposed.