Localization of membrane proteins in membrane vesicles of Bacillus subtilis.

Electrons can be transferred to the respiratory chain in whole cells and in membrane vesicles of Bacillus subtilis W 23 by the membrane impermeable electron donor reduced 5-N-methyl-phenazonium-3-sulfonate as efficiently as by the membrane permeable electron donor reduced 5-N-methyl-phenazonium methyl-sulfate, indicating that the respiratory chain is accessible from the outside of the membrane.Succinate is oxidized by whole cells and membrane vesicles at a low rate and does not energize transport of l-glutamate. In the presence of 5-N-methyl-phenazonium-3-sulfonate or 5-N-methyl-phenazonium methyl-sulfate, the oxidation rate and the rate of l-glutamate transport are increased considerably. The electrons are transferred directly from succinic dehydrogenase to these acceptors. Succinic dehydrogenase must therefore be exposed to the outside surface of the membrane in both membrane vesicles and whole cells. The exposure of succinic dehydrogenase to the outside is also indicated by the observations that only a 5% increase in the oxidation rates of succinate-5-N-methyl-phenazonium methylsulfate and succinate-5-N-methyl-phenazonium-3-sulfonate is observed upon solubilization of the membrane with the nonionic detergent Brij-58. Furthermore, treatment of membrane vesicles with trypsin decreases by more than 95% these oxidation rates.NADH is oxidized at a high rate and energizes transport of l-glutamate in whole cells and membrane vesicles effectively. The NADH-oxidation is not effected by trypsin treatment of the vesicles indicating that the oxidation occurs at the inside-surface of the membrane. Trypsin treatment of the vesicles, however, significantly decreases the rate of l-glutamate transport driven by NADH. Therefore component(s) of the transport system for l-glutamate must be effected by trypsin treatment. No apparent differences could be observed in the localization of membrane-bound functions between membrane vesicles and whole cells. This strongly supports the contention that the vesicle membrane of B. subtilis has the same orientation as the cytoplasmic membrane of whole cells.     

Inhibition of membrane fusion by suppression of lateral movement of membrane proteins

Chicken erythrocytes were fused either by Sendai virus or by the combination of Ca²⁺ and ionophore A23187.Intramembrane particles and external anionic sites of cells undergoing fusion were found to acquire the ability to undergo a process of cold-induced clustering (thermotropic separation).Cationized ferritin (200 μg/ml 5% (v/v) cell suspension) inhibited both the fusion process and the thermotropic separation of intramembrane particles and external anionic sites. The correlation between the mobility of membrane proteins and the fusion process is discussed. It is suggested that an increase in the lateral mobility of membrane proteins is a prerequisite for initiation of membrane fusion.     

Junctional membrane permeability

Summary Substitution of extracellular Na⁺ by Li⁺ causes depression of junctional membrane permeability inChironomus salivary gland cells; within 3 hr, permeability falls to so low a level that neither fluorescein nor the smaller inorganic ions any longer traverse the junctional membrane in detectable amounts (uncoupling). The effect is Li-specific: if choline⁺ is the Na⁺ substitute, coupling is unchanged. The Li-produced uncoupling is not reversed by restitution of Na⁺. Long-term exposure (>1 hr) of the cells to Ca, Mg-free medium leads also to uncoupling. This uncoupling is fully reversible by early restitution of Ca⁺⁺ or Mg⁺⁺. Coupling is maintained in the presence of either Ca⁺⁺ or Mg⁺⁺, so long as the total divalent concentration is about 12mm. The uncoupling in Ca, Mg-free medium ensues regardless of whether the main monovalent cation is Na, Li or choline.The uncouplings are accompanied by cell depolarization. Repolarization of the cells by inward current causes restoration of coupling; the junctional conductance rises again to its normal level. The effect was shown for Li-produced uncoupling, for uncoupling by prolonged absence of external Ca⁺⁺ and Mg⁺⁺, and for uncoupling produced by dinitrophenol. In all cases, the recoupling has the same features: (1) it develops rapidly upon application of the polarizing current; (2) it is cumulative; (3) it is transient, but outlasts the current; and (4) it appears not to depend on the particular ions carrying the current from the electrodes to the cell. The recoupling is due to repolarization of nonjunctional cell membrane; recoupling can be produced at zero net currernt through the junctional membrane. Recoupling takes place also as a result of chemically produced repolarization; restoration of theK gradients in uncoupled cells causes partial recoupling during the repolarization phase.An explanation of the results on coupling is proposed in terms of known mechanisms of regulation of Ca⁺⁺ flux in cells. The uncouplings are explained by actions raising the Ca⁺⁺ level in the cytoplasmic environment of the junctional membranes; the recoupling is explained by actions lowering this Ca⁺⁺ level.     

Viscosity changes of erythrochyte membrane and membrane lipids at transition temperature

A non-linearity in the changes of viscosity with temperature was found in sonicated human erythrocyte membranes at 18–19 °C. At the same temperature, a break was observed in the viscosity of the extracted membrane lipids, the cholesterol content of which was varied by means of Sephadex LH 20 column chromatography. It is inferred that the break observed in the membranes corresponds to the transition temperature of the erythrocyte membrane lipids. The applied method of direct viscosimetry is relatively simple and cheap in comparison to the well known methods of ESR spectroscopy or differential scanning calorimetry, which have been hitherto widely used in determining thermal transition points in different systems.Viscosity measurements may be compared to light scattering or fluorescence measurements, introduced recently for the determination of phase transitions (Träuble, H. (1971) Naturwissenschaften 58, 277–284, and Lussan, C. and Faucon, J.F. (1971) FEBS Lett. 19, 186–188).     

Erythrocyte membrane proteins and membrane skeleton

Considerable advances in the research field of erythrocyte membrane were achieved in the recent two decades. New findings in the structure-function correlation and interactions of erythrocyte membrane proteins have attracted extensive attention. Interesting progress was also made in the molecular pathogenesis of erythrocyte membrane disorders. Advances in the composition, function and interaction of erythrocyte membrane proteins, erythrocyte membrane skeleton, and relevant diseases are briefly described and summarized here on the basis of domestic and world literatures. Translated from Life Science Research, 2005, 9(4): 283–291 [译自: 生命科学研究]     

Modulation of membrane receptor endocytosis by chemical effectors of membrane fluidity

Several chemical effectors were used to induce changes in spleen B cell membrane fluidity. Membrane fluidity was monitored by fluorescence polarization analysis of the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene (DPH) and cell viability was checked not to be affected by the treatments. Membrane immunoglobulin (Ig) endocytosis by the living B cells with modified or unmodified membranes was quantitatively measured by flow cytometry, using a previously described method (Métézeau et al., 1982, 1984). The kinetics of endocytosis of membrane Ig was not affected by chemical effectors increasing membrane fluidity. On the contrary, increasing membrane microviscosity resulted in the slowing down and eventually the blocking of membrane Ig endocytosis. It is suggested that a step depending on membrane microviscosity is involved in the process of endocytosis; this step may become rate limiting when membranes are artificially rendered or naturally become (i.e. for pathological or particularly differentiated cells) more viscous.     

Dynamic imaging of mitochondrial membrane proteins in specific sub-organelle membrane locations

Mitochondria are cellular organelles with multifaceted tasks and thus composed of different sub-compartments. The inner mitochondrial membrane especially has a complex nano-architecture with cristae protruding into the matrix. Related to their function, the localization of mitochondrial membrane proteins is more or less restricted to specific sub-compartments. In contrast, it can be assumed that membrane proteins per se diffuse unimpeded through continuous membranes. Fluorescence recovery after photobleaching is a versatile technology used in mobility analyses to determine the mobile fraction of proteins, but it cannot provide data on subpopulations or on confined diffusion behavior. Fluorescence correlation spectroscopy is used to analyze single molecule diffusion, but no trajectory maps are obtained. Single particle tracking (SPT) technologies in live cells, such as tracking and localization microscopy (TALM), do provide nanotopic localization and mobility maps of mitochondrial proteins in situ. Molecules can be localized with a precision of between 10 and 20 nm, and single trajectories can be recorded and analyzed; this is sufficient to reveal significant differences in the spatio-temporal behavior of diverse mitochondrial proteins. Here, we compare diffusion coefficients obtained by these different technologies and discuss trajectory maps of diverse mitochondrial membrane proteins obtained by SPT/TALM. We show that membrane proteins in the outer membrane generally display unhindered diffusion, while the mobility of inner membrane proteins is restricted by the inner membrane architecture, resulting in significantly lower diffusion coefficients. Moreover, tracking analysis could discern proteins in the inner boundary membrane from proteins preferentially diffusing in cristae membranes, two sub-compartments of the inner mitochondrial membrane. Thus, by evaluating trajectory maps it is possible to assign proteins to different sub-compartments of the same membrane.     

Studies on membrane topology, N-glycosylation and functionality of SARS-CoV membrane protein

Background

Dengue is the most important arbovirus disease in tropical and subtropical countries. The viral envelope (E) protein is responsible for cell receptor binding and is the main target of neutralizing antibodies. The aim of this study was to analyze the diversity of the E protein gene of DENV-3. E protein gene sequences of 20 new viruses isolated in Ribeirao Preto, Brazil, and 427 sequences retrieved from GenBank were aligned for diversity and phylogenetic analysis.

Results

Comparison of the E protein gene sequences revealed the presence of 47 variable sites distributed in the protein; most of those amino acids changes are located on the viral surface. The phylogenetic analysis showed the distribution of DENV-3 in four genotypes. Genotypes I, II and III revealed internal groups that we have called lineages and sub-lineages. All amino acids that characterize a group (genotype, lineage, or sub-lineage) are located in the 47 variable sites of the E protein.

Conclusion

Our results provide information about the most frequent amino acid changes and diversity of the E protein of DENV-3.     

Studies of asymmetric membrane assembly

The major capsid protein of M13 bacteriophage is incorporated at each stage of infection into the host plasma membrane with its amino terminus exposed on the outer surface. Purified M13 coat protein is incorporated with the same asymmetry into synthetic phosphatidylcholine vesicles formed near the T_m of the lipid by a cholate dilution technique. We now report that the lipid in the pre-dilution mixture exists as mixed micelles of uniform size. Prior to dilution, the coat protein is present in at least two states of aggregation, both of which behave similarly in the model membrane assembly reaction. No detectable lipid-protein interaction occurs prior to dilution. Upon dilution there is rapid production of small closed vesicles and coat protein is converted to a chymotrypsin-resistant form, presumably reflecting its incorporation into these vesicle bilayers. Formation of large ($\text{>6000}\text{A}\text{?}$ diameter) vesicles occurs slowly with preservation of coat protein asymmetry and internal volume. A model for this assembly reaction is proposed.     

Non-covalent binding of membrane lipids to membrane proteins

Polar lipids and membrane proteins are major components of biological membranes, both cell membranes and membranes of enveloped viruses. How these two classes of membrane components interact with each other to influence the function of biological membranes is a fundamental question that has attracted intense interest since the origins of the field of membrane studies. One of the most powerful ideas that driven the field is the likelihood that lipids bind to membrane proteins at specific sites, modulating protein structure and function. However only relatively recently has high resolution structure determination of membrane proteins progressed to the point of providing atomic level structure of lipid binding sites on membrane proteins. Analysis of X-ray diffraction, electron crystallography and NMR data over 100 specific lipid binding sites on membrane proteins. These data demonstrate tight lipid binding of both phospholipids and cholesterol to membrane proteins. Membrane lipids bind to membrane proteins by their headgroups, or by their acyl chains, or binding is mediated by the entire lipid molecule. When headgroups bind, binding is stabilized by polar interactions between lipid headgroups and the protein. When acyl chains bind, van der Waals effects dominate as the acyl chains adopt conformations that complement particular sites on the rough protein surface. No generally applicable motifs for binding have yet emerged. Previously published biochemical and biophysical data link this binding with function. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

Analog circuit of theAcetabularia membrane

Summary The high membrane potential ofAcetabularia (E _m=–170 mV) is due to an electrogenic pump in parallel with the passive diffusion system (E _d=–80 mV) which could be studied separately in the cold, when the pump is blocked. Electrical measurements under normal conditions show that the pump pathway consists of its electromotive forceE _p with two elementsP ₁ andP ₂ in series;P ₂ is shunted by a large capacitance (C _p=3 mF cm^–2). The nonlinear current-voltage relationship ofP ₁ (light- and temperature-sensitive) could be determined separately; it reflects the properties of a carrier-mediated electrogenic pump. The value ofE _p (–190 mV) indicates a stoichiometry of 21 between electrogenically transported charges and ATP. The electrical energy, normally stored inC _p, compares well with the metabolic energy, stored in the ATP pool. The nonlinear current-voltage relationship ofP ₂ (attributed to phosphorylating reactions) is also sensitive to light and temperature and is responsible for the region of negative conductance of the overall current-voltage relationship. The power of the pump (1 W cm^–2) amounts to some percent of the total energy turnover. The high Cl^– fluxes (1 nmol cm^–2 sec^–1) and the electrical properties of the plasmalemma are not as closely related as assumed previously. For kinetic reasons, a direct and specific Cl^– pathway between the vacuole and outside is postulated to exist.     

Thylakoid membrane protein phosphorylation in correlation with photosynthetic membrane activation

The phosphorylation of five $\text{E.}\text{gracilis}$ thylakoid membrane polypeptides was studied, in isolated chloroplasts. Using [³²P] labelling, in the light, we found that phosphorylation was inhibited by ethanol and DCMU. Inhibition curves were characteristic of photosynthetic inhibition. [γ-³²P] ATP labelling was used to distinguish between two groups of phosphoproteins: the first one, includes protein I, II, V which require only ATP for phosphorylation while the second one includes protein III and IV whose phosphorylation is light-requiring. Phosphorylation of protein III and IV was inhibited by CCCP, NH₄Cl and DCMU, and was reversible in the dark.     

Accessibility of plasma membrane antigens

Serological techniques applied to intact cells register only those antigens of the plasma membrane that are exposed at the cell surface and are therefore accessible to antibody. Solubilization of the plasma membrane by detergent, used in the conventional surface-iodination immunoprecipitation technique, renders other plasma membrane antigens accessible. We have shown this by using a modified version of the technique in which lysis with detergent is postponed until after the cells have been reacted with antibody. Comparison of the conventional and modified methods confirms that the plasma membrane glycoprotein gp70 has antigen that is not exposed on the intact cells as well as accessible antigen, for example, G_IX. The modified surface-iodination immunoprecipitation method is useful for distinguishing cell-surface antigens from plasma membrane antigens that normally are not accessible. This is exemplified by the fact that standard anti-TL and anti-X.1 sera identify gp70 antigen in the plasma membrane that is registered by the conventional, but not by the modified method.Abbreviations used in this paper are anti - BALB BALB/c - gp70 MuLV envelope glycoprotein of molecular weight about 70,000 daltons, sometimes referred to as gp69/71 - gs group-specific - ¹²⁵I-imm-pptn surface labeling of viable cells with¹²⁵I followed by immunoprecipitation analysis - Ig immunoglobulin - MuLV murine leukemia virus - NMS normal mouse serum - PAGE polyacrylamide gel electrophoresis - PBS Dulbecco's phosphate-buffered saline, Ca⁺⁺- and Mg⁺⁺-free - SDS sodium dodecyl sulfate - TL thymus leukemia antigen     

Fusion of photoreceptor membrane vesicles

Summary Then-alkyl bromides with 6 to 10 carbons induce formation of vesicles of 5 to 100 m diameter from the small vesicles (0.1 m average diameter) produced by disruption of the discs from frog rod photoreceptors. Then-alkanes,n-alkyl iodides andn-alkyl chlorides are relatively ineffective. The formation of large vesicles is independent of calcium concentration and is distinguished from fusion processes previously reported by the large number of vesicles involved. The results reported here together with others suggest the occurrence of multiple fusion (and/or rupture-resealing) events between vesicles, induced by then-alkyl bromides.     

A novel complete reconstitution system for membrane integration of the simplest membrane protein

A complete reconstitution system for membrane integration of the simplest protein was developed by means of defined factors. A mutant version of Pf3 coat protein, 3L-Pf3 coat, requires neither signal recognition particle/Sec factors nor a membrane potential for its integration into the cytoplasmic membrane of Escherichia coli. Although 3L-Pf3 coat is spontaneously integrated into liposomes composed of phospholipids, diacylglycerol completely blocks such spontaneous integrations at a physiological level. Under the conditions where spontaneous integration does not occur, 3L-Pf3 coat integration was absolutely dependent on a novel integration-stimulating factor. Combination of the PURE system, an in vitro translation system composed of the purified factors involved in translation in E. coli, with liposomes containing the highly purified integration-stimulating factor revealed multiple cycles of 3L-Pf3 coat integration, achieving the complete reconstitution of membrane integration. Based on the function of the factor, we propose that the factor is named MPIase (Membrane Protein Integrase).     

Properties of membrane stationary states

Summary The flux of permeant species through a membrane is examined using discrete state stochastic models for the transport process within the membrane. While a membrane flux is maintained due to a concentration gradient between bathing solutions, the distribution of species within the membrane evolves to a time invariant configuration which can differ significantly from the equilibrium configuration. Some special properties of these stationary states are examined using linear, microcanonical models for the membrane transport process. Analysis of these models reveals properties which are masked by the phenomenological analysis of irreversible thermodynamics. For example, the models can be used to study the nature of multi-state relaxation within the membrane by observation of the time dependence of the net membrane flux when the membrane is perturbed from its stationary state distribution. Under some conditions, multi-state models will produce relaxation similar to that observed for single-state processes. The symmetry within the membrane is a critical factor for monitoring relaxation processes within the membrane. Because of the stationary nature of the membrane configuration, statistical thermodynamic variables can be defined for the membrane configuration. The total system is not in equilibrium since the baths must still be described by dissipation functions. In the stationary state, the configurational entropy of the membrane is lowered relative to equilibrium and is shown to depend quadratically on the time independent parameter (j/p) wherej is the membrane flux andp is a characteristic transition probability for intra-membrane transitions. The basic membrane system serves as a quantitative example of the entropy reduction possible in a stationary state system. An allosteric transition mediated by the stationary state configuration is examined as a means of utilizing this negentropy production.     

Modulation of membrane dynamics and cell motility by membrane tension

The plasma membrane of most cells is drawn tightly over the cytoskeleton of the cell, resulting in a significant tension being developed in the membrane. The tension in the membrane can be calculated from the force required to separate it from the cytoskeleton; and the force itself can be measured rapidly by using laser tweezers. Recent observations indicate that decreasing membrane tension stimulates endocytosis and increasing tension stimulates secretion. Thus, membrane tension provides a simple physical mechanism to control the area of the plasma membrane. Here, we speculate that tension is a global parameter that the cell uses to control physically plasma membrane dynamics, cell shape and cell motility.     

Open-vacuole method for measuring membrane potential and membrane resistance of Characeae cells

A simple method called the open-vacuole (o.v.) method was developedto measure the vacuolar potential (E_vo) and membrane resistance(R_m) of Characeae cells without inserting a microelectrode intothe vacuole. Values of E_vo and R_m measured by this method arehigher than those measured by the microelectrode method. Usingthe o.v. method we can measure E_vo and R_m exactly in cells withinternal media of extremely low ionic concentrations. In respect to E_vo and R_m, the tonoplast is less sensitive thanthe plasmalemma to a change in ionic concentration. The existenceof a significant amount of E_vo (–40 mV), even when boththe internal and external media are isotonic artificial pondwater with a high Ca^2$content, may be accounted for by the differencein sensitivity to ionic species between these two membranes. Irrespective of the presence or absence of Ca^2$in the vacuole,practically the same values of E_vo and R_m were measured withthe open-vacuole method, when measurements were carried outwithin 20 min after the end of perfusion. The discrepanciesbetween the present and previous results (16) may be accountedfor by the difference in methods. ¹Present address: Sanki Engineering Ltd., Nagaokakyo, Kyoto. (Received January 31, 1975; )     

Effect of sodium on potassium fluxes at the cell membrane and vacuole membrane of red beet

Slices of red beet (Beta vulgaris) washed for 5 to 6 days are known to accumulate Na⁺ in preference to K⁺ from solutions containing both ions. The present work, using ion concentrations of 1.0 mm or less, with Ca²⁺ added in some cases, shows that Na⁺ strongly inhibits K⁺ influx at the cell membrane (plasmalemma) while K⁺ efflux is increased to a lesser extent. This result from compartmental analysis is confirmed by short (15-minute) influx experiments, which indicate an immediate inhibitory effect of Na⁺ on K⁺ influx at the cell membrane. It is concluded that cation selectivity, even when Na⁺ is favored for uptake, is primarily determined at the cell membrane. Nevertheless, a high level of K⁺ in the cytoplasm is maintained during Na⁺ influx, by an inhibition of K⁺ transfer to the vacuole.