Role of the Cytosolic Loop C2 and the C Terminus of YidC in Ribosome Binding and Insertion Activity

Members of the YidC/Oxa1/Alb3 protein family mediate membrane protein insertion, and this process is initiated by the assembly of YidC·ribosome nascent chain complexes at the inner leaflet of the lipid bilayer. The positively charged C terminus of Escherichia coli YidC plays a significant role in ribosome binding but is not the sole determinant because deletion does not completely abrogate ribosome binding. The positively charged cytosolic loops C1 and C2 of YidC may provide additional docking sites. We performed systematic sequential deletions within these cytosolic domains and studied their effect on the YidC insertase activity and interaction with translation-stalled (programmed) ribosome. Deletions within loop C1 strongly affected the activity of YidC in vivo but did not influence ribosome binding or substrate insertion, whereas loop C2 appeared to be involved in ribosome binding. Combining the latter deletion with the removal of the C terminus of YidC abolished YidC-mediated insertion. We propose that these two regions play an crucial role in the formation and stabilization of an active YidC·ribosome nascent chain complex, allowing for co-translational membrane insertion, whereas loop C1 may be involved in the downstream chaperone activity of YidC or in other protein-protein interactions.     

From mosaic to patchwork: Matching lipids and proteins in membrane organization

Abstract

Biological membranes encompass and compartmentalize cells and organelles and are a prerequisite to life as we know it. One defining feature of membranes is an astonishing diversity of building blocks. The mechanisms and principles organizing the thousands of proteins and lipids that make up membrane bilayers in cells are still under debate. Many terms and mechanisms have been introduced over the years to account for certain phenomena and aspects of membrane organization and function. Recently, the different viewpoints – focusing on lipids vs. proteins or physical vs. molecular driving forces for membrane organization – are increasingly converging. Here we review the basic properties of biological membranes and the most common theories for lateral segregation of membrane components before discussing an emerging model of a self-organized, multi-domain membrane or ‘patchwork membrane'.     

Tamoxifen Increases Membrane Fluidity at High Concentrations

There are contradictory results in the literature relating to the effect oftamoxifen on membrane fluidity. The present work investigates the effect oftamoxifen on membrane dynamics to find out whether the concentration oftamoxifen can be one of the factors in this discrepancy. Turbidity(absorbance at 440 nm) and Fourier transform infrared spectroscopicstudies reveal that tamoxifen causes opposite effects on membranefluidity at low (1 mol.%) and high (30 mol.%) tamoxifen concentrations. Lowtamoxifen concentrations increase the absorbance in the gel and liquidcrystalline phase, whereas high tamoxifen concentrations decrease theabsorbance in gel and liquid crystalline phase, whereas tamoxifenconcentrations decrease the absorbance. Observations on both phasesshow that the bandwidth of the CH2 stretching bands decreases with1 mol.% tamoxifen and increases with 30 mol.% tamoxifen present, indicatinga decrease in membrane fluidity at low tamoxifen concentrations and anincrease in fluidity at high tamoxifen concentrations. It is seen that theapparent discrepancy in the literature on the effect of tamoxifen onmembrane fluidity mainly arises from the tamoxifen concentration used andthe confusion on the concept of lipid fluidity and lipid order.     

Association of spectrin with its membrane attachment site restricts lateral mobility of human erythrocyte integral membrane proteins

Interactions between spectrin and the inner surface of the human erythrocyte membrane have been implicated in the control of lateral mobility of the integral membrane proteins. We report here that incubation of “leaky” erythrocytes with a water-soluble proteolytic fragment containing the membrane attachment site for spectrin achieves a selective and controlled dissociation of spectrin from the membrane, and increases the rate of lateral mobility of fluorescein isothiocyanate-labeled integral membrane proteins (> 70% of label in band 3 and PAS-1). Mobility of membrane proteins is measured as an increase in the percentage of uniformly fluorescent cells with time after fusion of fluorescent with nonfluorescent erythrocytes by Sendai virus. The cells are permeable to macromolecules since virus-fused erythrocytes lose most of their hemoglobin. The membrane attachment site for spectrin has been solubilized by limited proteolysis of inside-out erythrocyte vesicles and has been purified (V). Bennett, J Biol Chem 253:2292 (1978). This 72,000-dalton fragment binds to spectrin in solution, competitively inhibits association of ³²P-spectrin with inside-out vesicles with a K_i of 10^?7M, and causes rapid dissociation of ³²P-spectrin from vesicles. Both acid-treated 72,000-dalton fragment and the 45,000 dalton-cytoplasmic portion of band 3, which also was isolated from the proteolytic digest, have no effect on spectrin binding, release, or membrane protein mobility. The enhancement of membrane protein lateral mobility by the same polypeptide that inhibits binding of spectrin to inverted vesicles and displaces spectrin from these vesicles provides direct evidence that the interaction of spectrin with protein components in the membrane restricts the lateral mobility of integral membrane proteins in the erythrocyte.     

Single-channel analysis of the conductance fluctuations induced in lipid bilayer membranes by complement proteins C5b-9

Summary Single-channel analysis of electrical fluctuations induced in planar bilayer membranes by the purified human complement proteins C5b6, C7, C8, and C9 have been analyzed. Reconstitution experiments with lipid bilayer membranes showed that the C5b-9 proteins formed pores only if all proteins were present at one side of the membrane. The complement pores had an average single-channel conductance of 3.1 nS at 0.15m KCl. The histogram of the complement pores suggested a substantial variation of the size of the single channel. The linear relationship between single-channel conductance at fixed ionic strength and the aqueous mobility of the ions in the bulk aqueous phase indicated that the ions move inside the complement pore in a manner similar to the way they move in the aqueous phase. The minimum diameter of the pores as judged from the conductance data is approximately 3 nm. The complement channels showed no apparent voltage control or regulation up to transmembrane potentials of 100 mV. At neutral pH the pore is three to four times more permeable for alkali ions than for chloride, which may be explained by the existence of fixed negatively charged groups in or near the pore. The significance of these observations to current molecular models of the membrane lesion formed by these cytolytic serum proteins is considered.     

Involvement of carboxyl groups in the divalent cation permeability of rat parotid gland basolateral plasma membrane

Divalent cation permeability of rat parotid gland basolateral plasma membranes was examined in dispersed parotid acini (by Ca²⁺ or Mn²⁺ entry) and in isolated basolateral plasma membrane vesicles (BLMV, by⁴⁵Ca²⁺ influx). Mn²⁺ entry (fura2 quenching) was about 1.6 fold higher in internal Ca²⁺ pool-depleted acini (Ca²⁺-depl acini) than in unstimulated cells. Mn²⁺ entry into Ca²⁺-depl acini was increased at external pH>7.4 and decreased at pH<7.4. Pretreatment of Ca²⁺-depl acini with the relatively hydrophobic carboxylic group reagent, N,N-dicyclohexylcarbodiimide (DCCD, 50 M for 30 min) resulted in the inhibition of Mn²⁺ entry into Ca²⁺-depl acini to unstimulated levels. Another hydrophobic carboxyl group reagent, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) and the relatively hydrophilic carboxyl group reagents, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMCD) did not affect Mn²⁺ entry.Similar to the effects in intact acini, Ca²⁺ influx into BLMV was decreased when the external pH was lowered below 7.4. Also DCCD (5 mM, 30 min), but not EEDQ, decreased (40%) Ca²⁺ influx in BLMV. However, unlike in acini, the hydrophilic reagents, EDC, EAC, and CMCD decreased Ca²⁺ permeability in BLMV and the effects were nonadditive with the decrease induced by DCCD. The aggregate effects of carboxyl group reagents on the Ca²⁺ and Mn²⁺ permeability in BLMV and intact acini, respectively, suggest that a critical carboxyl group (most likely accessible from the cytoplasmic side of the plasma membrane) is involved in divalent cation flux in rat parotid acinar cells.     

Immunological approach to the characterization of the outer acrosomal membrane of boar spermatozoa

Antiserum to purified boar spermatozoan outer acrosomal membrane (OAM) was raised in rabbits and adsorbed with boar liver and serum glutaraldehyde cross-linked immunoadsorbents. The IgG fraction of the antiserum was purified by (NH₄)₂SO₄ precipitation followed by ion-exchange chromatography. Indirect immunofluorescence showed bright fluorescent staining of the acrosomal cap of boar spermatozoa and to a lesser extent of the acrosomes of bull and goat spermatozoa after incubation with anti-OAM-IgG. Immuno-electron microscopy further confirmed the specificity of the antibody for the OAM. Preincubation of the anti-OAM-IgG with isolated OAM, completely abolished its reactivity. When tested by ELISA, anti-OAM-IgG reacted with boar, bull, goat, and human spermatozoa; however, its binding activity to boar spermatozoa was significantly greater as compared to spermatozoa from the other species tested. In an effort to identify OAM antigens recognized by this antiserum, the isolated boar OAM was labeled either with ³H or with ¹²⁵I and solubilized by mild detergent treatment. The extracted components were immunoprecipitated with anti-OAM-IgG and protein A-bearing S. aureus and the thus isolated antigens were analysed on SDS-PAGE. The results suggest that anti-OAM-IgG recognized one high molecular ³H-labeled glycoprotein (270 kd), and four ¹²⁵I-labeled polypeptides of lower molecular weight of the boar OAM.     

Developing an antibacterial super-hydrophilic barrier between bacteria and membranes to mitigate the severe impacts of biofouling

Biofouling produces concentrated microbial populations with highly resistive biofilms and is considered to be a serious obstacle for a wide range of membrane technology applications. An antibacterial super-hydrophilic barrier could help to reduce biofouling by preventing direct contact between membranes and bacteria. In this study, an antibacterial super-hydrophilic barrier consisting of a layer of TiO₂ nanoparticles (NPs) was developed on polyvinylidene fluoride (PVDF)-based membrane via a facile technique. The results demonstrated that the presence of TiO₂ NPs eliminated the first step of biofouling, ie bacterial adhesion to the membrane. In addition, after bacterial deposition onto the membrane during ultrafiltration (UF), the TiO₂ NPs significantly retarded bacterial growth and reproduction (the second step of biofouling). During UF, the membrane flux decreased due to bacterial deposition, but 85% of the flux was recovered through physical cleaning using water. This study sheds light on the potential advantages of antibacterial super-hydrophilic membranes for biofouling mitigation.     

The influence of electrochemical gradients of Na+ and K+ upon the membrane binding and pore forming activity of the terminal complement proteins

Summary The hemolytic activity of the terminal complement proteins (C5b-9) towards erythrocytes containing high potassium concentration has been reported to be dramatically increased when extracellular Na⁺ is substituted isotonically by K⁺ (Dalmasso, A.P., et al., 1975,J. Immunol. 115:63–68). This phenomenon was now further investigated using resealed human erythrocyte ghosts (ghosts), which can be maintained at a nonlytic osmotic steady state subsequent to C5b-9 binding: (1) The functional state of C5b-9-treated ghosts was studied from their ability to retain trapped [¹⁴C]-sucrose or [³H]-inulin when suspended either in the presence of Na⁺ or K⁺. A dramatic increase in the permeability of the ghost membrane to both nonelectrolytes-in the absence of significant hemoglobin release-was observed for C5b-9 assembly in the presence of external K⁺. (2) The physical binding of the individual¹²⁵I-labeled terminal complement proteins to ghost membranes was directly measured as a function of intra- and extracellular K⁺ and Na⁺. The uptake of¹²⁵I-C7,¹²⁵I-C8, and¹²⁵I-C9 into membrane C5b-9 was unaltered by substitution of Na⁺ by K⁺. (3) The binding of the terminal complement proteins to ghosts subjected to a transient membrane potential generated by the K⁺-ionophore valinomycin (in the presence of K⁺ concentration gradients) was measured. No significant change in membrane binding of any of the C5b-9 proteins was detected under the influence of both depolarizing and hyperpolarizing membrane potentials. It can be concluded that the differential effect of Na⁺ versus K⁺ upon the erythrocyte membrane isnot due to an effect upon the binding of the complement proteins to the membraneper se, but upon the functional properties of the assembled C5b-9 pore site.     

The protein coat in membrane fusion: lessons from fission

Multiple cell biological processes involve two opposite rearrangements of membrane configuration, referred to as fusion and fission. While membrane intermediates in protein-mediated fusion have been studied in some detail, the global force which drives sequential stages of the fusion reaction from early local intermediates to an expanding fusion pore remains unknown. Fusion proceeds via stages, which are analogous but in the opposite direction to that of membrane budding-off and fission driven by protein coats. On the basis of this analogy, we propose that an interconnected coat formed by membrane-bound activated fusion proteins surrounding the membrane contact zone generates the driving force for fusion. This fusion protein coat has a strongly curved intrinsic shape opposite to that of the protein coat driving fission. To relieve internal stresses, the fusion protein coat spontaneously bends out of the initial shape of the membrane surface. This bending produces elastic stresses in the underlying lipid bilayer and drives its fusion with the apposing membrane. The hypothesis that 'bystander' proteins (i.e. fusion proteins outside the contact zone) generate the driving force for fusion offers a new interpretation for a number of known features of the fusion reaction mediated by the prototype fusion protein, influenza hemagglutinin, and might bring new insights into mechanisms of other fusion reactions.     

动物细胞膜的分离纯化及其在病毒受体研究中的应用

细胞膜是动物细胞与胞外环境之间的屏障。病毒只有与细胞膜上的病毒受体特异性结合 ,才能进入细胞 ,进而启动其增殖周期。因此 ,病毒受体是病毒学研究的重要组成部分。分离纯化病毒受体所在的细胞膜作为病毒受体研究的实验材料 ,已经在许多病毒的研究中得到应用 ,并取得了很好的效果。现就动物细胞膜的分离纯化及其在病毒受体研究中的应用作一综述。     

Hypotonically induced changes in the plasma membrane of cultured mammalian cells

Summary Cells from three cell lines were electrorotated in media of osmotic strengths from 330 mOsm to 60 mOsm. From the field-frequency dependence of the rotation speed, the passive electrical properties of the surfaces were deduced. In all cases, the area-specific membrane capacitance (C _m) decreased with osmolality. At 280 mOsm (iso-osmotic), SP2 (mouse myeloma) and G8 (hybridoma) cells had C _mvalues of 1.01 ± 0.04 F/cm² and 1.09 ± 0.03 F/cm², respectively, whereas dispase-treated L-cells (sarcoma fibroblasts) exhibited C _m=2.18±0.10/F/cm². As the osmolality was reduced, the C _mreached a well-defined minimum at 150 mOsm (SP2) or 180 mOsm (G8). Further reduction in osmolality gave a 7% increase in C _m, after which a plateau close to 0.80F/cm2²was reached. However, the whole-cell capacities increased about twofold from 200 mOsm to 60 mOsm. L-cells showed very little change in C _mbetween 280 mOsm and 150 mOsm, but below 150 mOsm the C _mdecreased rapidly. The changes in C _mcorrelate well with the swelling of the cells assessed by means of van't Hoff plots. The apparent membrane conductance (including the effect of surface conductance) decreased with C _m, but then increased again instead of exhibiting a plateau. The rotation speed of the cells increased as the osmolality was lowered, and eventually attained almost the theoretical value. All measurements indicate that hypo-osmotically stressed cells obtain the necessary membrane area by using material from microvilli. However, below about 200 mOsm the whole-cell capacities indicate the progressive incorporation of extra membrane into the cell surface.We thank Mr. B.G. Klarmann for his help with the measurements. This work was supported by grants of the DFG (SFB 176 B5 to U.Z. and W.M.A.) and of the BMFT (DARA 50 WB 9212 to U.Z.). We also thank the Umweltbundesamt, Berlin, for support enabling the construction of some of the rotation generators used in this work.     

Contamination of an anion-exchange membrane by glutathione

Electrodialysis, which can separate electrolytes under mild conditions by using ion-exchange membranes, is a strong candidate for separation of GSH from yeast extracts, because GSH is unstable and easily oxidized forming a disulfide bond especially under alkali conditions. In this paper, sorption behavior of GSH on an anion-exchange membrane, in the pH 3–6 region that is expected to be the most preferable for its electrodialytic separation, was examined. Sorption of GSH on a Selemion-AMV anion-exchange membrane was accelerated as the pH of the membrane-contact solution increased, and there was a good correlation between the sorbed amounts and the molar fraction of monovalent anionic species of GSH. However, the amounts of GSH desorbed from the membrane by a NaCl desorbing solution were much lower than the initial sorbed amounts, and the difference between them was enlarged with increasing pH. The GSH which was lost could be recovered by the addition of DTT in the membrane-contact and desorbing solutions. Similar results were also obtained with Cys. We thus concluded that an anion-exchange membrane would be contaminated by thiol compounds, such as GSH and Cys, through oxidative binding of the thiol group with the membrane, the local OH^- concentration in which was enhanced due to attraction by the positively charged anion-exchange membrane.     

Epigallocatechin-3-gallate location and interaction with late endosomal and plasma membrane model membranes by molecular dynamics

Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol in green tea and it has been reported to have many beneficial properties against many different types of illnesses and infections. However, the exact mechanism/s underlying its biological effects are unknown. It has been previously shown that EGCG is capable of binding to and disrupting the membrane, so that some of its effects on biological systems could be ascribed to its capacity to incorporate into the biological membrane and modulate its structure. In this work, we have used atomistic molecular dynamics (MD) to discern the location and orientation of EGCG in model membranes and the possible existence of specific interactions with membrane lipids. For that goal, we have used in our simulation two complex model membranes, one resembling the plasma membrane (PM) and the other one the late endosome (LE) membrane. Our results support that EGCG tends to associate with the membrane and exists inside it in a relatively stable and steady location with a low propensity to be associated with other EGCG molecules. Interestingly, EGCG forms hydrogen bonds with POPC and POPE in the PM system but POPC and BMP and no POPE in the LE. These data suggest that the broad beneficial effects of EGCG could be mediated, at least in part, through its membranotropic effects and therefore membrane functioning.

Communicated by Ramaswamy H. Sarma     

Characterization of thermotropic structural transitions of the erythrocyte membrane: a biochemical and electron-paramagnetic resonance approach

The relationship between membrane structural properties and functions has been generally inferred from observed thermotropic phenomena. By the use of 16-dinyloxyl stearic acid spin probe we investigated the red blood cell membrane components involved in three characteristic thermotropic structural transitions occurring at 8, 20, and 40 degrees C. The transition at 8 degrees C is removed by chymotrypsin treatment at the cytoplasmic membrane layer. The 20 degrees C phase transition is unmodified after chymotrypsin treatment and occurs at 15 degrees C after complete proteolysis of intramembrane chymotrypsin-insensitive peptides. Liposomes from the total lipid extract of RBC show only one thermotropic transition at 15 degrees C. The 40 degrees C phase transition is absent in vesicles free of skeletal proteins, in vesicles obtained after RBC storage, and in low-ionic-strength resealed ghosts. Transitions at 8 degrees C and 40 degrees C appear to be due to the interactions of cytoplasmic exposed proteins with membrane, whereas the 20 degrees C transition is intrinsic to the lipid component.     

Role of curvature and phase transition in lipid sorting and fission of membrane tubules

We have recently developed a minimal system for generating long tubular nanostructures that resemble tubes observed in vivo with biological membranes. Here, we studied membrane tube pulling in ternary mixtures of sphingomyelin, phosphatidylcholine and cholesterol. Two salient results emerged: the lipid composition is significantly different in the tubes and in the vesicles; tube fission is observed when phase separation is generated in the tubes. This shows that lipid sorting may depend critically on both membrane curvature and phase separation. Phase separation also appears to be important for membrane fission in tubes pulled out of giant liposomes or purified Golgi membranes.