Isolation of the Ochromanas danica plasma membrane and identification of several membrane enzymes

Ochromonas danica cell homogenate can be fractionated by differential centrifugation into chloroplast, mitochondrial, ribosome, lysosomal, plasma membrane and soluble fractions. The plasma membrane fraction was further purified by discontinuous sucrose density gradient centrifugation and was found to be enriched 4–16-fold in the following enzymes: β-galactosidase, acid phosphatase, alkaline phosphatase, 5′-nucleotidase, and (Na⁺, K⁺)-ATPase. The role of plasma membrane phosphatase in the phosphate metabolism of plants is discussed.     

Cytochrome b of the Dictyostelium discoideum plasma membrane

Cytochrome b of the plasma membrane of Dictyostelium discoideum was investigated in purified plasma membranes and in solubilized form. The membrane-bound cytochrome b can be reduced by NADH. This reduction is inhibited by p-hydroxymercuribenzoate. The reduced cytochrome b does not react with carbon monoxide. Its apparent molecular weight lies between 13000 and 16000. Tryptic digestion yields a large, heme-containing peptide with an apparent molecular weight between 12000 and 15000. After solubilization with cholate, cytochrome b can be enriched by reversed-phase HPLC, indicating that it contains also a hydrophobic component. With these properties, cytochrome b of the D. discoideum plasma membrane resembles microsomal cytochrome b₅.     

Detergent-insoluble glycosphingolipid/cholesterol microdomains of the myelin membrane

Glycosphingolipids and cholesterol form lateral assemblies, or lipid 'rafts', within biological membranes. Lipid rafts are routinely studied biochemically as low-density, detergent-insoluble complexes (in non-ionic detergents at 4 degrees C; DIGs, detergent-insoluble glycosphingolipid/cholesterol microdomains). Recent discrepancies recommended a re-evaluation of the conditions used for the biochemical analysis of lipid rafts. We have investigated the detergent insolubility of several known proteins present in the glycosphingolipid/cholesterol-rich myelin membrane, using four detergents representing different chemical classes (TX-100, CHAPS, Brij 96 and TX-102), under four conditions: detergent extraction of myelin either at (i) 4 degrees C or (ii) 37 degrees C, or at 4 degrees C after pre-extraction with (iii) saponin or (iv) methyl-beta-cyclodextrin (MbetaCD). Each detergent was different in its ability to solubilize myelin proteins and in the density of the DIGs produced. Brij 96 DIGs floated to a lower density than other detergents tested, possibly representing a subpopulation of DIGs in myelin. DIGs pre-extracted with saponin were denser than DIGs pre-extracted with MbetaCD. Furthermore, pre-extraction with MbetaCD solubilized proteolipid protein (known to associate with cholesterol), whereas pre-extraction with saponin did not, suggesting that saponin is less effective as a cholesterol-perturbing agent than is MbetaCD. These results demonstrate that DIGs isolated by different detergents are not necessarily comparable, and that these detergent-specific DIGs may represent distinct biochemical, and possibly physiological, entities based on the solubilities of specific lipids/proteins in each type of detergent.     

Retrograde trafficking from the vacuole/lysosome membrane

Membrane protein recycling is a fundamental process from yeast to humans. The lysosome (or vacuole in yeast) receives membrane proteins from the secretory, endocytic, and macroautophagy/autophagy pathways. Although some of these membrane proteins appear to be recycled, the molecular mechanisms underlying this retrograde trafficking are poorly understood. Our recent study revealed that the transmembrane autophagy protein Atg27 is recycled from the vacuole membrane using a 2-step recycling process. First, the Snx4 complex recycles Atg27 from the vacuole to the endosome. Then, the retromer complex mediates endosome-to-Golgi retrograde transport. Thus, 2 distinct protein complexes facilitate the sequential retrograde trafficking for Atg27. As far as we know, Atg27 is the first physiological substrate for the vacuole-to-endosome retrograde trafficking pathway.     

Influence of membrane lipids on the photochemistry of bacteriorhodopsin in the purple membrane of Halobacterium halobium

Purple membrane fragments from Halobacterium halobium were reconstituted with the native lipids replaced by dipalmitoyl phosphatidylcholine and by egg lecithin. In parallel studies the temperature dependence of bacteriorhodopsin phototransient lifetime and absorption dichroism and of in situ lipid microviscosity were determined; the former two by, respectively, conventional and polarization flash photometry, and the latter by observation of emission depolarization of an embedded fluorescent dye, 1,6-diphenyl-1,3,5-hexatriene. Discontinuities in lipid microviscosity profiles in native and egg lecithin purple membrane were reflected in both the photochemical cycle frequency and bacteriorhodopsin chromophore rotational mobility. The influence exerted by membrane-lipid viscosity appears to be a secondary effect, and points to the bacteriorhodopsin chromophoric group being situated in the protein interior.     

Relation between the gradient of the ATP/ADP ratio and the membrane potential across the mitochondrial membrane.

The relation between the intramitochondrial and extramitochondrial ratio ATP/ADP, the transmembrane potential and pH gradient is investigated in the present communication. For this purpose mitochondria are equilibrated with added [14C]ATP in the presence of substrate and oligomycin for eliminating phosphate transfer by ATPase. The membrane potential was measured by the distribution of 86Rb+ in the presence of valinomycin, the deltapH by the distribution of [14C]acetate. In the energized state by varying deltapsi between 60 and 160 mV, the internal (ATP/ADP)i is decreased 30-fold, the external (ATP/ADP)e remains largely constant. As a result, the deltalog (ATP/ADP)e/(ATP/ADP)i = deltalogphi is increased linerly with deltapsi according to the following relation: deltalogphi = 0.85 deltapsi - 0.35. The deltapH was changed between 0.1 and 0.8 by increasing the Pi concentration causing only a minor decrease of deltalogphi would be expected if the ATP-ADP exchange has a significant electroneutral portion. Also in the uncoupled and respiration-inhibited state the same function between deltalogphi and deltapsi is found as in the energized states. It is concluded that under these conditions the ATP-ADP exchange is largely electrical.     

Assembly of the semliki forest virus membrane glycoproteins in the membrane of the endoplasmic reticulum in vitro

A cell-free system has been constructed to study the mechanism by which a single messenger RNA directs the synthesis of proteins destined for two different cellular locations. The Semliki Forest virus (SFV) 26 S mRNA codes for the viral capsid protein (C protein) and the membrane proteins p62 and E1. The three virus proteins are read in this order from the messenger RNA using one initiation site. The C protein is left on the cytoplasmic side and the p62 and the El proteins are inserted into the endoplasmic reticulum membrane. Translation of 26 S mRNA in a HeLa cell-free system in the presence of microsomes from dog pancreas reproduced the segregation, and proteolytic processing and glycosylation observed in infected cells. The signal for membrane binding was in the amino-terminal end of p62. The results indicate that the membrane proteins become inserted in the nascent state. The cleavage between p62 and El was coupled to membrane insertion. If the membranes were added after a period corresponding to the synthesis of about 100 amino acids of the p62 protein, segregation, glycosylation and cleavage between p62 and E1 failed to occur.     

Characterization of the membrane beta-lactamase in Bacillus cereus 569/H/9

The membrane-bound beta-lactamase from Bacillus cereus, strain 569/H/9, has been purified to apparent homogeneity. Nonionic detergent (0.5% Triton X-100) is required to keep the enzyme (traditionally called gamma-penicillinase and now called beta-lactamase III) in solution. Antibodies to beta-lactamase III have been prepared, and the membrane-bound enzyme is immunochemically distinct from the extracellular enzymes. beta-Lactamase III has a molecular weight of 31 500, in contrast to the extracellular enzymes beta-lactamase I and beta-lactamase II which have molecular weights of 30 000 and 22 000, respectively. The isoelectric point of beta-lactamase III is pH 6.8, whereas beta-lactamase I and beta-lactamase II have isoelectric points about 8.6 and 8.3. The amino acid composition of beta-lactamase III differs from those of beta-lactamase I and beta-lactamase II; however, the difference index between the compositions of beta-lactamase I and beta-lactamase III (52%) suggests relatedness. beta-Lactamase III is inactivated by 6 beta-bromopenicillanic acid and by the sulfone of 6 alpha-chloropenicillanic acid, and cephalosporins are poorer substrates than penicillins. beta-Lactamase III may be a membrane-bound class A beta-lactamase.     

The water permeability of the monoolein/triolein bilayer membrane

The water permeability of the lipid bilayer can be used as a probe of membrane structure. A simple model of the bilayer, the liquid hydrocarbon model, views the membrane as a thin slice of bulk hydrocarbon liquid. A previous study (Petersen, D. (1980) Biochim. Biophys. Acta 600, 666–677) showed that this model does not accurately predict the water permeability of the monoolein/n-hexadecane bilayer: the measured activation energy for water permeation is 50% above the predicted value. From this it was inferred that the hydrocarbon chains in the lipid bilayer are more ordered than in the bulk hydrocarbon liquid. The present study tests the liquid hydrocarbon model for the monoolein/triolein bilayer, which has been shown to contain very little triolein in the plane of the membrane (Waldbillig, R.C. and Szabo, G. (1979) Biochim. Biophys. Acta 557, 295–305). Measurements of the water permeability coefficient of the bilayer are compared with predictions of the liquid hydrocarbon model based on measurements of the water permeability coefficient of bulk 8-heptadecene. The predicted and measured values agree quite closely over the temperature range studied (15–35°C): the predicted activation energy is 11.1±0.2 kcal/mol, whereas the measured activation energy for the bilayer is 9.8±0.7 kcal/mol. This close agreement is in contrast with the monoolein/n-hexadecane results and suggests that, insofar as water permeation is concerned, the liquid hydrocarbon model quite closely represents the monoolein/triolein bilayer.     

Changes in the membrane surface charge density and/or membrane potential of the porcine intestinal brush-border membrane vesicles induced by treatment with neuraminidase

The effects of neuraminidase treatment on the membrane surface charge density and/or membrane potential of the porcine intestinal brush-border membrane vesicles were studied by using three fluorescent dyes, 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-anilino-8-naphthalene sulfonate (ANS), and 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide (DiS-C3(5]. The results of quenching studies of DPH-labeled membranes using cationic (T1+) and anionic (I-) quenchers suggested an increase of negative charge on the membrane surface by desialylation upon neuraminidase treatment. This interpretation was further supported by a decrease of ANS-binding affinity of the membranes after treatment with the enzyme. In addition, the degree of valinomycin-induced fluorescence change of DiS-C3(5)-probed membranes in the presence of various concentrations of KCl was reduced by treatment of the membranes with neuraminidase. This suggests that penetration of the dye molecules into the vesicle interior is facilitated by the treatment. The membrane potentials estimated from the null point of valinomycin-induced changes in the DiS-C3(5) fluorescence of the control and neuraminidase-treated membranes were -25 to -29.7 and -40 to -48.8 mV, respectively. From these results, it is suggested that the membrane surface charge density and/or membrane potential of the intestinal brush-border membranes are susceptible to modification of carbohydrate moieties on the membrane surface by neuraminidase treatment.     

Outer membrane of Salmonella typhimurium: Reconstitution of sucrose-permeable membrane vesicles

Outer membrane vesicles were reconstituted from phospholipids, lipopolysaccharide, and outer membrane proteins isolated from Salmonella typhimurium. The vesicles appeared to be permeable to sucrose and other small oligosaccharides only when membrane proteins were added to the reconstitution system. The size of saccharides that could pass through the vesicle membranes was found to be close to the size of saccharides that penetrate through the intact outer membrane of S. typhimurium.     

Arrangement of the light-harvesting chlorophyll a/b protein complex in the thylakoid membrane

The transverse orientation of the light-harvesting chlorophyll a/b protein complex of Photosystem II (LHC II) in the thylakoid membrane of pea was investigated using surface radioiodination with Iodo-Gen^TM. The labelling effects on LHC II of four different membrane preparations were compared. One preparation was oriented right-side-out (intact thylakoids); two of them had an inside-out orientation exposing the lumenal surface (inside-out vesicles; PS II particles) and one had both sides of the membrane exposed (mechanically damaged thylakoids). It was found that LHC II could be iodinated only in membrane preparations with an exposed lumenal surface. Isolated apoproteins were chemically cleaved. Fragments analysis revealed a tyrosine residue located eight amino acids from the C-terminus as the single iodination site. It is concluded that the C-terminus of LHC II points towards the lumental side of the thylakoid. Differences in the labelling behaviour of the LHC apoproteins could be assigned to a heterogeneity in the C-terminal region in which the tyrosine residue is replaced by phenylalanine.     

Interactions of Plasmodium falciparum erythrocyte membrane protein 3 with the red blood cell membrane skeleton

Plasmodium falciparum parasites express and traffick numerous proteins into the red blood cell (RBC), where some associate specifically with the membrane skeleton. Importantly, these interactions underlie the major alterations to the modified structural and functional properties of the parasite-infected RBC. P. falciparum Erythrocyte Membrane Protein 3 (PfEMP3) is one such parasite protein that is found in association with the membrane skeleton. Using recombinant PfEMP3 proteins in vitro, we have identified the region of PfEMP3 that binds to the RBC membrane skeleton, specifically to spectrin and actin. Kinetic studies revealed that residues 38-97 of PfEMP3 bound to purified spectrin with moderately high affinity (K_D(kin) = 8.5 × 10^− 8 M). Subsequent deletion mapping analysis further defined the binding domain to a 14-residue sequence (IFEIRLKRSLAQVL; K_D(kin) = 3.8 × 10^− 7 M). Interestingly, this same domain also bound to F-actin in a specific and saturable manner. These interactions are of physiological relevance as evidenced by the binding of this region to the membrane skeleton of inside-out RBCs and when introduced into resealed RBCs. Identification of a 14-residue region of PfEMP3 that binds to both spectrin and actin provides insight into the potential function of PfEMP3 in P. falciparum-infected RBCs.     

Morphological evidence for the association of plasma membrane glycoprotein IIb/IIIa with the membrane skeleton in human platelets

Summary We examined the association between glycoprotein (GP) IIb/IIIa, a receptor for fibrinogen, and membrane skeletons in both unstimulated and thrombin-activated human platelets. After a treatment with dithiobis succinimidyl propionate (DTSP), a cross-linker, unstimulated and activated platelets were simultaneously extracted and fixed with a fixing solution containing Triton X-100. Also, the localization of GPIIb/IIIa on the plasma membrane was observed by a preembedding staining method of unextracted platelets. In unstimulated platelets, 20–40% of the whole plasma membrane remained in the detergent-extracted samples. Amorphous structures with 10–70 nm in diameters are distributed at 20 to 100-nm intervals on the surface of plasma membrane. Similar structures also were identified in the intact platelets by the immunocytochemical method. By careful inspection, we found that most of the amorphous structures that contained gold particles were connected to the submembrane zone just beneath the plasma membrane. The submembrane zone was identified as the membrane skeleton because actin was detected in the zone. After activation, detergent-insoluble granules were surrounded by dense networks of microfilaments in the central part of platelets. The filaments were identified as actin and became associated with myosin. These results demonstrate that GPIIb/IIIa on the plasma membrane is connected to the membrane skeleton and suggest that, during activation, actin filaments which extend into the cytoplasm from the membrane skeleton increase and form dense networks around Triton-insoluble granules.     

Morphological evidence for the association of plasma membrane glycoprotein IIb/IIIa with the membrane skeleton in human platelets

We examined the association between glycoprotein (GP) IIb/IIIa, a receptor for fibrinogen, and membrane skeletons in both unstimulated and thrombin-activated human platelets. After a treatment with dithiobis succinimidyl propionate (DTSP), a cross-linker, unstimulated and activated platelets were simultaneously extracted and fixed with a fixing solution containing Triton X-100. Also, the localization of GPIIb/IIIa on the plasma membrane was observed by a preembedding staining method of unextracted platelets. In unstimulated platelets, 20-40% of the whole plasma membrane remained in the detergent-extracted samples. Amorphous structures with 10-70 nm in diameters are distributed at 20 to 100-nm intervals on the surface of plasma membrane. Similar structures also were identified in the intact platelets by the immunocytochemical method. By careful inspection, we found that most of the amorphous structures that contained gold particles were connected to the submembrane zone just beneath the plasma membrane. The submembrane zone was identified as the membrane skeleton because actin was detected in the zone. After activation, detergent-insoluble granules were surrounded by dense networks of microfilaments in the central part of platelets. The filaments were identified as actin and became associated with myosin. These results demonstrate that GPIIb/IIIa on the plasma membrane is connected to the membrane skeleton and suggest that, during activation, actin filaments which extend into the cytoplasm from the membrane skeleton increase and form dense networks around Triton-insoluble granules.     

Effect of temperature on membrane fluidity and calcium conductance of the excitable ciliary membrane from Paramecium

Fluorescence anisotropy and average fluorescence lifetime of diphenylhexatriene were measured in artificial lipid membrane vesicles. Within the temperature range investigated (15–52°C) both parameters correlate and can be used interchangeably to measure membrane fluidity. Fluorescence anisotropy of DPH in membrane vesicles of cilia from the protozoan Paramecium tetraurelia decreased slightly from 5 to 37°C, yet, no phase transition was observed. An estimated flow activation energy of approx. 2 kcal/mol indicated that the ciliary membrane is very rigid and not readily susceptible to environmental stimuli. The ciliary membrane contains two domains of different membrane fluidity as indicated by two distinct fluorescence lifetimes of diphenylhexatriene of 7.9 and 12.4 ns, respectively. Ca²⁺ flux into ciliary membrane vesicles of Paramecium as measured with the Ca²⁺ indicator dye arsenazo III showed a nonlinear temperature dependency from 5 to 35°C with a minimum around 15°C and increasing flux rates at higher and lower temperatures. The fraction of vesicles permeable for Ca²⁺ remained unaffected by temperature. The differences in temperature dependency of Ca²⁺ conductance and membrane fluidity indicate that the Ca²⁺ permeability of the ciliary membrane is a membrane property which is not directly affected by the fluidity of its lipid environment.     

The involvement of the plasma membrane in the development of Dictyostelium discoideum. I. Purification of the plasma membrane

A method for the isolation and purification of plasma membranes of Dictyostelium discoideum by equilibrium centrifugation on sucrose followed by Renografin continuous density gradients has been developed and monitored both with electron microscopy and a number of enzyme assays. On the basis of electron microscopy, the final plasma membrane fractions are judged to be free of nuclei, rough endoplasmic reticulum, lysosomes and peroxisomes. Some profiles of the mitochondrial inner membranes are found within the plasma membrane fractions, but this contamination has been estimated to be only 5%. On the basis on enzyme assays, the plasma membrane fractions contain all the 5′-nucleotidase activity in the final gradients and are free of catalase, acid phosphatase and malate dehydrogenase activity (markers for peroxisomes, lysosomes, soluble enzymes and the matrix of mitochondria). Their content of glucose-6-phosphatase is reduced by more than 70%. The large majority of RNA and DNA have been removed from the preparation.