Molecular convergence of bacterial and eukaryotic surface order

The conservation of fluidity is a theme common to all cell membranes. In this study, an analysis of lipid packing was conducted via C-laurdan spectroscopy of cell surface membranes prepared from representative species of Bacteria and Eukarya. We found that despite their radical differences in composition (namely the presence and absence of membrane-rigidifying sterol) the membrane order of all taxa converges on a remarkably similar level. To understand how this similarity is constructed, we reconstituted membranes with either bacterial or eukaryotic components. We found that transmembrane segments of proteins have an important role in buffering lipid-mediated packing. This buffering ensures that sterol-free and sterol-containing membranes exhibit similar barrier properties.     

Cooperative Recruitment of Dynamin and BIN/Amphiphysin/Rvs (BAR) Domain-containing Proteins Leads to GTP-dependent Membrane Scission

Dynamin mediates various membrane fission events, including the scission of clathrin-coated vesicles. Here, we provide direct evidence for cooperative membrane recruitment of dynamin with the BIN/amphiphysin/Rvs (BAR) proteins, endophilin and amphiphysin. Surprisingly, endophilin and amphiphysin recruitment to membranes was also dependent on binding to dynamin due to auto-inhibition of BAR-membrane interactions. Consistent with reciprocal recruitment in vitro, dynamin recruitment to the plasma membrane in cells was strongly reduced by concomitant depletion of endophilin and amphiphysin, and conversely, depletion of dynamin dramatically reduced the recruitment of endophilin. In addition, amphiphysin depletion was observed to severely inhibit clathrin-mediated endocytosis. Furthermore, GTP-dependent membrane scission by dynamin was dramatically elevated by BAR domain proteins. Thus, BAR domain proteins and dynamin act in synergy in membrane recruitment and GTP-dependent vesicle scission.     

The Golgi protein p115 associates with gamma-tubulin and plays a role in Golgi structure and mitosis progression

The Golgi apparatus is a network of polarized cisternae localized to the perinuclear region in mammalian cells. It undergoes extensive vesiculation at the onset of mitosis and its reassembly requires factors that are in part segregated via the mitotic spindle. Here we show that unlike typical Golgi markers, the Golgi-protein p115 partitioned with the spindle poles throughout mitosis. An armadillo-fold in its N terminus mediated a novel interaction between p115 and γ-tubulin and functioned in its centrosomal targeting. Both the N- and C-terminal regions of p115 were required to maintain Golgi structure. Strikingly, p115 was essential for mitotic spindle function and the resolution of the cytokinetic bridge because its depletion resulted in spindle collapse, chromosome missegregation, and failed cytokinesis. We demonstrate that p115 plays a critical role in mitosis progression, implicating it as the only known golgin to regulate both mitosis and apoptosis.     

膜生物反应器的研究进展

膜生物反应器是近年来发展的废水处理新技术,具有活性污泥浓度高、污泥龄长、占地面积小、投资省的特点。利用膜生物反应器进行污水处理不仅可以大大节约水资源,还可以大大节约能源,节省设备和运行费用,已成为二十一世纪研究热点。膜生物反应器是通过高效膜分离技术与活性污泥相结合,增大污泥中的特效菌来加快生化反应速率,提高废水处理效果。目前处理对象已从生活污水扩展到高浓度的有机废水和难降解的工业废水。本文综述了膜生物反应器在废水中的应用研究情况,并分析比较了各种膜材质的特点、适用范围以及膜的污染因素和清洗方法,展望了膜生物反应器的应用前景及进一步研究方向。     

The Viroporin Activity of the Minor Structural Proteins VP2 and VP3 Is Required for SV40 Propagation

For nonenveloped viruses such as Simian Virus 40, the mechanism used to translocate viral components across membranes is poorly understood. Previous results indicated that the minor structural proteins, VP2 and VP3, might act as membrane proteins during infection. Here, purified VP2 and VP3 were found to form pores in host cell membranes. To identify possible membrane domains, individual hydrophobic domains from VP2 and VP3 were expressed in a model protein and tested for their ability to integrate into membranes. Several domains from the late proteins supported endoplasmic reticulum membrane insertion as transmembrane domains. Mutations in VP2 and VP3 were engineered that inhibited membrane insertion and pore formation. When these mutations were introduced into the viral genome, viral propagation was inhibited. This comprehensive approach revealed that the viroporin activity of VP2 and VP3 was inhibited by targeted disruptions of individual hydrophobic domains and the loss of membrane disruption activity impaired viral infection.     

The N- and C-terminal Domains of Tomosyn Play Distinct Roles in Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Binding and Fusion Regulation

Tomosyn negatively regulates SNARE-dependent exocytic pathways including insulin secretion, GLUT4 exocytosis, and neurotransmitter release. The molecular mechanism of tomosyn, however, has not been fully elucidated. Here, we reconstituted SNARE-dependent fusion reactions in vitro to recapitulate the tomosyn-regulated exocytic pathways. We then expressed and purified active full-length tomosyn and examined how it regulates the reconstituted SNARE-dependent fusion reactions. Using these defined fusion assays, we demonstrated that tomosyn negatively regulates SNARE-mediated membrane fusion by inhibiting the assembly of the ternary SNARE complex. Tomosyn recognizes the t-SNARE complex and prevents its pairing with the v-SNARE, therefore arresting the fusion reaction at a pre-docking stage. The inhibitory function of tomosyn is mediated by its C-terminal domain (CTD) that contains an R-SNARE-like motif, confirming previous studies carried out using truncated tomosyn fragments. Interestingly, the N-terminal domain (NTD) of tomosyn is critical (but not sufficient) to the binding of tomosyn to the syntaxin monomer, indicating that full-length tomosyn possesses unique features not found in the widely studied CTD fragment. Finally, we showed that the inhibitory function of tomosyn is dominant over the stimulatory activity of the Sec1/Munc18 protein in fusion. We suggest that tomosyn uses its CTD to arrest SNARE-dependent fusion reactions, whereas its NTD is required for the recruitment of tomosyn to vesicle fusion sites through syntaxin interaction.     

Synip Arrests Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE)-dependent Membrane Fusion as a Selective Target Membrane SNARE-binding Inhibitor

The vesicle fusion reaction in regulated exocytosis requires the concerted action of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) core fusion engine and a group of SNARE-binding regulatory factors. The regulatory mechanisms of vesicle fusion remain poorly understood in most exocytic pathways. Here, we reconstituted the SNARE-dependent vesicle fusion reaction of GLUT4 exocytosis in vitro using purified components. Using this defined fusion system, we discovered that the regulatory factor synip binds to GLUT4 exocytic SNAREs and inhibits the docking, lipid mixing, and content mixing of the fusion reaction. Synip arrests fusion by binding the target membrane SNARE (t-SNARE) complex and preventing the initiation of ternary SNARE complex assembly. Although synip also interacts with the syntaxin-4 monomer, it does not inhibit the pairing of syntaxin-4 with SNAP-23. Interestingly, synip selectively arrests the fusion reactions reconstituted with its cognate SNAREs, suggesting that the defined system recapitulates the biological functions of the vesicle fusion proteins. We further showed that the inhibitory function of synip is dominant over the stimulatory activity of Sec1/Munc18 proteins. Importantly, the inhibitory function of synip is distinct from how other fusion inhibitors arrest SNARE-dependent membrane fusion and therefore likely represents a novel regulatory mechanism of vesicle fusion.     

Munc18-1 Protein Molecules Move between Membrane Molecular Depots Distinct from Vesicle Docking Sites

Four evolutionarily conserved proteins are required for mammalian regulated exocytosis: three SNARE proteins, syntaxin, SNAP-25, and synaptobrevin, and the SM protein, Munc18-1. Here, using single-molecule imaging, we measured the spatial distribution of large cohorts of single Munc18-1 molecules correlated with the positions of single secretory vesicles in a functionally rescued Munc18-1-null cellular model. Munc18-1 molecules were nonrandomly distributed across the plasma membrane in a manner not directed by mode of interaction with syntaxin1, with a small mean number of molecules observed to reside under membrane resident vesicles. Surprisingly, we found that the majority of vesicles in fully secretion-competent cells had no Munc18-1 associated within distances relevant to plasma membrane-vesicle SNARE interactions. Live cell imaging of Munc18-1 molecule dynamics revealed that the density of Munc18-1 molecules at the plasma membrane anticorrelated with molecular speed, with single Munc18-1 molecules displaying directed motion between membrane hotspots enriched in syntaxin1a. Our findings demonstrate that Munc18-1 molecules move between membrane depots distinct from vesicle morphological docking sites.     

Recent advances in membrane technologies for biorefining and bioenergy production

The bioeconomy, and in particular, biorefining and bioenergy production, have received considerable attention in recent years as a shift to renewable bioresources to produce similar energy and chemicals derived from fossil energy sources, represents a more sustainable path. Membrane technologies have been shown to play a key role in process intensification and products recovery and purification in biorefining and bioenergy production processes. Among the various separation technologies used, membrane technologies provide excellent fractionation and separation capabilities, low chemical consumption, and reduced energy requirements. This article presents a state-of-the-art review on membrane technologies related to various processes of biorefining and bioenergy production, including: (i) separation and purification of individual molecules from biomass, (ii) removal of fermentation inhibitors, (iii) enzyme recovery from hydrolysis processes, (iv) membrane bioreactors for bioenergy and chemical production, such as bioethanol, biogas and acetic acid, (v) bioethanol dehydration, (vi) bio-oil and biodiesel production, and (vii) algae harvesting. The advantages and limitations of membrane technologies for these applications are discussed and new membrane-based integrated processes are proposed. Finally, challenges and opportunities of membrane technologies for biorefining and bioenergy production in the coming years are addressed.     

Modeling membrane shaping by proteins: Focus on EHD2 and N-BAR domains

Cellular membranes are highly dynamic, undergoing both persistent and dynamic shape changes driven by specialized proteins. The observed membrane shaping can be simple deformations of existing shapes or membrane remodeling involving fission or fusion. Here we describe several mechanistic principles by which membrane shaping proteins act. We especially consider models for membrane bending and fission by EHD2 proteins and membrane bending by N-BAR domains. There are major challenges ahead to understand the general principles by which diverse membrane bending proteins act and to understand how some proteins appear to span multiple modes of action from driving curvature to inducing membrane remodeling.     

Structure of the membrane-tethering GRASP domain reveals a unique PDZ ligand interaction that mediates Golgi biogenesis

Biogenesis of the ribbon-like membrane network of the mammalian Golgi requires membrane tethering by the conserved GRASP domain in GRASP65 and GRASP55, yet the tethering mechanism is not fully understood. Here, we report the crystal structure of the GRASP55 GRASP domain, which revealed an unusual arrangement of two tandem PDZ folds that more closely resemble prokaryotic PDZ domains. Biochemical and functional data indicated that the interaction between the ligand-binding pocket of PDZ1 and an internal ligand on PDZ2 mediates the GRASP self-interaction, and structural analyses suggest that this occurs via a unique mode of internal PDZ ligand recognition. Our data uncover the structural basis for ligand specificity and provide insight into the mechanism of GRASP-dependent membrane tethering of analogous Golgi cisternae.     

膜融合诱导因子能使细胞质膜表面水化程度降低

研究了几种膜融合诱导因子,包括介质ｐＨ,温度以及钙离子等对小鼠艾氏腹水癌细胞质膜表面介电常数的影响．结果表明,酸性介质ｐＨ、温度降低或钙离子浓度增加,均可使其膜表面介电常数下降。从而提示,膜表面水化力下降和局部脱水可能是酸诱导膜融合,钙离子诱导膜融合及冰冻融化引起的膜融合等均需经过的关键步骤。     

The Role of the Strictly Conserved Positively Charged Residue Differs among the Gram-positive,Gram-negative,and Chloroplast YidC Homologs

Recently, the structure of YidC2 from Bacillus halodurans revealed that the conserved positively charged residue within transmembrane segment one (at position 72) is located in a hydrophilic groove that is embedded in the inner leaflet of the lipid bilayer. The arginine residue was essential for the Bacillus subtilis SpoIIIJ (YidC1) to insert MifM and to complement a SpoIIIJ mutant strain. Here, we investigated the importance of the conserved positively charged residue for the function of the Escherichia coli YidC, Streptococcus mutans YidC2, and the chloroplast Arabidopsis thaliana Alb3. Like the Gram-positive B. subtilis SpoIIIJ, the conserved arginine was required for functioning of the Gram-positive S. mutans YidC2 and was necessary to complement the E. coli YidC depletion strain and to promote insertion of a YidC-dependent membrane protein synthesized with one but not two hydrophobic segments. In contrast, the conserved positively charged residue was not required for the E. coli YidC or the A. thaliana Alb3 to functionally complement the E. coli YidC depletion strain or to promote insertion of YidC-dependent membrane proteins. Our results also show that the C-terminal half of the helical hairpin structure in cytoplasmic loop C1 is important for the activity of YidC because various deletions in the region either eliminate or impair YidC function. The results here underscore the importance of the cytoplasmic hairpin region for YidC and show that the arginine is critical for the tested Gram-positive YidC homolog but is not essential for the tested Gram-negative and chloroplast YidC homologs.     

A Toxin-based Probe Reveals Cytoplasmic Exposure of Golgi Sphingomyelin

Although sphingomyelin is an important cellular lipid, its subcellular distribution is not precisely known. Here we use a sea anemone cytolysin, equinatoxin II (EqtII), which specifically binds sphingomyelin, as a new marker to detect cellular sphingomyelin. A purified fusion protein composed of EqtII and green fluorescent protein (EqtII-GFP) binds to the SM rich apical membrane of Madin-Darby canine kidney (MDCK) II cells when added exogenously, but not to the SM-free basolateral membrane. When expressed intracellularly within MDCK II cells, EqtII-GFP colocalizes with markers for Golgi apparatus and not with those for nucleus, mitochondria, endoplasmic reticulum or plasma membrane. Colocalization with the Golgi apparatus was confirmed by also using NIH 3T3 fibroblasts. Moreover, EqtII-GFP was enriched in cis-Golgi compartments isolated by gradient ultracentrifugation. The data reveal that EqtII-GFP is a sensitive probe for membrane sphingomyelin, which provides new information on cytosolic exposure, essential to understand its diverse physiological roles.     

Membrane protein expression and production: effects of polyhistidine tag length and position

Polyhistidine tags enable the facile purification of proteins by immobilized metal affinity chromatography (IMAC). Both the type and position of purification tags can affect significantly properties of a protein such as its expression level, behavior in solution, and its ability to form suitable samples (esp. suitable crystals for X-ray crystallography). We investigated systematically the effects of polyhistidine tag length and position on many properties related to expression and purification of recombinant integral membrane proteins. Specifically, modified Escherichia coli pET expression vectors were built that placed 6- or 10-histidine tags at the N- or C-termini of the subcloned gene. The E. coli water channel AqpZ was subcloned into this suite of vectors and its expression, purification, solution properties, and yield were characterized. These studies show that: (1) all vectors yield similar expression levels, (2) tag length has a greater effect than tag position upon yield, (3) neither tag length nor position affects significantly detergent solubilization of the protein, (4) the length of the tag affects the oligomerization state of the purified protein, and (5) the tag length and position change chromatographic behavior of the detergent-solubilized protein. In addition, substitution of the lysine codon AAA at the second position, previously shown to have some effect upon soluble protein expression levels, did not have a large effect on AqpZ production. We are currently producing approximately 12 mg of purified AqpZ per liter of shake-flask culture, and preliminary crystals that diffract to approximately 5A resolution have been obtained.     

A microbial rhodopsin with a unique retinal composition shows both sensory rhodopsin II and bacteriorhodopsin-like properties

Rhodopsins possess retinal chromophore surrounded by seven transmembrane α-helices, are widespread in prokaryotes and in eukaryotes, and can be utilized as optogenetic tools. Although rhodopsins work as distinctly different photoreceptors in various organisms, they can be roughly divided according to their two basic functions, light-energy conversion and light-signal transduction. In microbes, light-driven proton transporters functioning as light-energy converters have been modified by evolution to produce sensory receptors that relay signals to transducer proteins to control motility. In this study, we cloned and characterized two newly identified microbial rhodopsins from Haloquadratum walsbyi. One of them has photochemical properties and a proton pumping activity similar to the well known proton pump bacteriorhodopsin (BR). The other, named middle rhodopsin (MR), is evolutionarily transitional between BR and the phototactic sensory rhodopsin II (SRII), having an SRII-like absorption maximum, a BR-like photocycle, and a unique retinal composition. The wild-type MR does not have a light-induced proton pumping activity. On the other hand, a mutant MR with two key hydrogen-bonding residues located at the interaction surface with the transducer protein HtrII shows robust phototaxis responses similar to SRII, indicating that MR is potentially capable of the signaling. These results demonstrate that color tuning and insertion of the critical threonine residue occurred early in the evolution of sensory rhodopsins. MR may be a missing link in the evolution from type 1 rhodopsins (microorganisms) to type 2 rhodopsins (animals), because it is the first microbial rhodopsin known to have 11-cis-retinal similar to type 2 rhodopsins.     

Tailored protection against plasmalemmal injury by annexins with different Ca2+ sensitivities

The annexins, a family of Ca(2+)- and lipid-binding proteins, are involved in a range of intracellular processes. Recent findings have implicated annexin A1 in the resealing of plasmalemmal injuries. Here, we demonstrate that another member of the annexin protein family, annexin A6, is also involved in the repair of plasmalemmal lesions induced by a bacterial pore-forming toxin, streptolysin O. An injury-induced elevation in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) triggers plasmalemmal repair. The highly Ca(2+)-sensitive annexin A6 responds faster than annexin A1 to [Ca(2+)](i) elevation. Correspondingly, a limited plasmalemmal injury can be promptly countered by annexin A6 even without the participation of annexin A1. However, its high Ca(2+) sensitivity makes annexin A6 highly amenable to an unproductive binding to the uninjured plasmalemma; during an extensive injury accompanied by a massive elevation in [Ca(2+)](i), its active pool is severely depleted. In contrast, annexin A1 with a much lower Ca(2+) sensitivity is ineffective at the early stages of injury; however, it remains available for the repair even at high [Ca(2+)](i). Our findings highlight the role of the annexins in the process of plasmalemmal repair; a number of annexins with different Ca(2+)-sensitivities provide a cell with the means to react promptly to a limited injury in its early stages and, at the same time, to withstand a sustained injury accompanied by the continuous formation of plasmalemmal lesions.     

Use of cyclodextrins to manipulate plasma membrane cholesterol content: Evidence, misconceptions and control strategies

The physiological importance of cholesterol in the cell plasma membrane has attracted increased attention in recent years. Consequently, the use of methods of controlled manipulation of membrane cholesterol content has also increased sharply, especially as a method of studying putative cholesterol-enriched cell membrane domains (rafts). The most common means of modifying the cholesterol content of cell membranes is the incubation of cells or model membranes with cyclodextrins, a family of compounds, which, due to the presence of relatively hydrophobic cavity, can be used to extract cholesterol from cell membranes. However, the mechanism of this activity of cyclodextrins is not completely established. Moreover, under conditions commonly used for cholesterol extraction, cyclodextrins may remove cholesterol from both raft and non-raft domains of the membrane as well as alter the distribution of cholesterol between plasma and intracellular membranes. In addition, other hydrophobic molecules such as phospholipids may also be extracted from the membranes by cyclodextrins. We review the evidence for the specific and non-specific effects of cyclodextrins and what is known about the mechanisms for cyclodextrin-induced cholesterol and phospholipid extraction. Finally, we discuss useful control strategies that may help to verify that the observed effects are due specifically to cyclodextrin-induced changes in cellular cholesterol.     

On vesicles and membrane compartments

Summary Two different mechanisms have been proposed to explain transport along the endocytic and biosynthetic transport routes in cells. The first involves stable compartments connected by vesicular traffic while the second argues that the key organelles (early endosomes or the cis Golgi) form de novo by fusion of vesicles and subsequently mature into later forms. In the first part of this article, I propose a classification that distinguishes between stable, preexisting membrane compartments and vesicles that are, by definition, transient organelles. In this scheme, compartments, but not vesicles, are capable of homotypic fusion while vesicles, but not compartments, are able to mature, a process defined as an irreversible set of biochemical events which lead to a physiologically distinct end-state of the vesicle prior to its vectorial fusion with a target compartment. In the second part, I summarize my current ideas about the ultrastructural organization of the ER-Golgi region. Finally, I review the cell biology of selected examples of different vesicle types in order to exemplify the fascinating diversity of functions that this class of membrane organelles has evolved.Abbreviations COP coatomer - ECV endosome carrier vesicle - ER endoplasmic reticulum - HRP horseradish peroxidase - IC intermediate compartment between ER and Golgi - MVB multivesicular body - NSF N-ethyl maleimide sensitive factor - SNAPS soluble NSF associated proteins - TGN trans Golgi network Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Influence of Membrane Lipid Composition on a Transmembrane Bacterial Chemoreceptor

Most bacterial chemoreceptors are transmembrane proteins. Although less than 10% of a transmembrane chemoreceptor is embedded in lipid, separation from the natural membrane environment by detergent solubilization eliminates most receptor activities, presumably because receptor structure is perturbed. Reincorporation into a lipid bilayer can restore these activities and thus functionally native structure. However, the extent to which specific lipid features are important for effective restoration is unknown. Thus we investigated effects of membrane lipid composition on chemoreceptor Tar from Escherichia coli using Nanodiscs, small (∼10-nm) plugs of lipid bilayer rendered water-soluble by an annulus of “membrane scaffold protein.” Disc-enclosed bilayers can be made with different lipids or lipid combinations. Nanodiscs carrying an inserted receptor dimer have high protein-to-lipid ratios approximating native membranes and in this way mimic the natural chemoreceptor environment. To identify features important for functionally native receptor structure, we made Nanodiscs using natural and synthetic lipids, assaying extents and rates of adaptational modification. The proportion of functionally native Tar was highest in bilayers closest in composition to E. coli cytoplasmic membrane. Some other lipid compositions resulted in a significant proportion of functionally native receptor, but simply surrounding the chemoreceptor transmembrane segment with a lipid bilayer was not sufficient. Membranes effective in supporting functionally native Tar contained as the majority lipid phosphatidylethanolamine or a related zwitterionic lipid plus a rather specific proportion of anionic lipids, as well as unsaturated fatty acids. Thus the chemoreceptor is strongly influenced by its lipid environment and is tuned to its natural one.