Phase transitions and heterogeneity in lipid bilayers

The optical reflectivity of several well-characterized lipid bilayer systems has been correlated with calorimetric studies of the membrane components. There is a large increase in mean membrane thickness when a bilayer is cooled below the transition temperature of the membrane lipid. Similar studies on membranes generated from a mixture of two lipids possessing different degrees of unsaturation suggest that between the characteristic transition temperatures of the two lipids, the bilayer contains clusters of gel and liquid crystalline lipid which coexist within the plane of the membrane.     

Protein translocation across biological membranes

Subcellular compartments have unique protein compositions, yet protein synthesis only occurs in the cytosol and in mitochondria and chloroplasts. How do proteins get where they need to go? The first steps are targeting to an organelle and efficient translocation across its limiting membrane. Given that most transport systems are exquisitely substrate specific, how are diverse protein sequences recognized for translocation? Are they translocated as linear polypeptide chains or after folding? During translocation, how are diverse amino acyl side chains accommodated? What are the proteins and the lipid environment that catalyze transport and couple it to energy? How is translocation coordinated with protein synthesis and folding, and how are partially translocated transmembrane proteins released into the lipid bilayer? We review here the marked progress of the past 35 years and salient questions for future work. Subcellular compartments have unique protein compositions, yet protein synthesis only occurs in the cytosol and in mitochondria and chloroplasts. How do proteins get where they need to go? The first steps are targeting to an organelle and efficient translocation across its limiting membrane. Given that most transport systems are exquisitely substrate specific, how are diverse protein sequences recognized for translocation? Are they translocated as linear polypeptide chains or after folding? During translocation, how are diverse amino acyl side chains accommodated? What are the proteins and the lipid environment that catalyze transport and couple it to energy? How is translocation coordinated with protein synthesis and folding, and how are partially translocated transmembrane proteins released into the lipid bilayer? We review here the marked progress of the past 35 years and salient questions for future work.     

Red cell membrane glycophorin labeling from within the lipid bilayer

Human red blood cell membranes were labeled from within the lipid bilayer by the apolar photosensitive reagent, 5-[125I]iodonaphthyl-1-azide. Glycophorin, the major sialoglycoprotein of the red cell membrane, was purified by two different methods; it contained approximately half of the total label incorporated into membrane proteins. The label was confined to the trypsin-insoluble peptide of glycophorin that includes a sequence of 20, mainly apolar, amino acids. These findings provide direct evidence that the labeled segment resides within the membrane in direct contact with the lipid bilayer, and support the suggestion that glycophorin spans the bilayer through its hydrophobic domain.     

植物油脂体及其结构蛋白研究

高等植物在其种子中储藏大量的脂类,为种子萌发及后续的籽苗生长提供碳源和能源。储藏脂类均贮存于油脂体（oil body,OB）中。油脂体的结构简单,核心部分是TAG组成的不透明结构,外层为磷脂单分子层,表面有多种膜蛋白,其中含量最丰富的是Oleosin。Oleosin和Caleosin作为2种结构蛋白在维持油脂体稳定性以及控制油脂体大小方面发挥重要的作用,对于种子质量的提高即种子油类产量和优化储存脂类及蛋白的提取过程具有重要的研究价值。油脂体表面还存在一些酶类如Steroleosin,可能与甾类化合物的合成相关。对油脂体形成的机制及相关蛋白结构与功能的研究进展进行了概述。     

The E. coli BtuCD structure: a framework for ABC transporter architecture and mechanism

The ABC transporters are ubiquitous membrane proteins that couple adenosine triphosphate (ATP) hydrolysis to the translocation of diverse substrates across cell membranes. Clinically relevant examples are associated with cystic fibrosis and with multidrug resistance of pathogenic bacteria and cancer cells. Here, we report the crystal structure at 3.2 angstrom resolution of the Escherichia coli BtuCD protein, an ABC transporter mediating vitamin B12 uptake. The two ATP-binding cassettes (BtuD) are in close contact with each other, as are the two membrane-spanning subunits (BtuC); this arrangement is distinct from that observed for the E. coli lipid flippase MsbA. The BtuC subunits provide 20 transmembrane helices grouped around a translocation pathway that is closed to the cytoplasm by a gate region whereas the dimer arrangement of the BtuD subunits resembles the ATP-bound form of the Rad50 DNA repair enzyme. A prominent cytoplasmic loop of BtuC forms the contact region with the ATP-binding cassette and appears to represent a conserved motif among the ABC transporters.     

Membrane insertion of a potassium-channel voltage sensor

The mechanism of voltage gating in K+ channels is controversial. The paddle model posits that highly charged voltage-sensor domains move relatively freely across the lipid bilayer in response to membrane depolarization; competing models picture the charged S4 voltage-sensor helix as being shielded from lipid contact by other parts of the protein. We measured the apparent free energy of membrane insertion of a K+-channel S4 helix into the endoplasmic reticulum membrane and conclude that S4 is poised very near the threshold of efficient bilayer insertion. Our results suggest that the paddle model is not inconsistent with the high charge content of S4.     

Calorimetric detection of a membrane-lipid phase transition in living cells

The membrane lipids in living Mycoplasma laidlawii exhibit a phase transition characteristic of that from crystal to liquid crystal within the bilayer conformation. The transition occurs at the same temperature in viable organisms, membranes isolated from the organisms, and isolated membrane lipids. The enthalpy of the transition in the membrane is compared with that of an aqueous suspension of isolated membrane lipids. The result is consistent with presence of an extended lipid bilayer in the native membrane.     

Leader peptidase of Escherichia coli: critical role of a small domain in membrane assembly

Leader peptidase spans the Escherichia coli plasma membrane with its amino-terminal domain facing the cytoplasm and its carboxyl terminus facing the periplasm. It is made without a cleavable leader sequence. The three apolar domains near the amino terminus of the peptidase are candidates for internal "signal sequences" and they anchor the protein to the lipid bilayer. Oligonucleotide-directed deletion was used to show that only the second domain has an essential function in membrane assembly. While this second apolar domain is crucial for membrane assembly, its continued function when disrupted by arginine suggests that its apolar character per se is not its only important feature.     

Lateral movements of membrane glycoproteins restricted by dynamic cytoplasmic barriers.

Cell membranes often are patchy, composed of lateral domains. These domains may be formed by barriers within or on either side of the membrane bilayer. Major histocompatibility complex (MHC) class 1 molecules that were either transmembrane- (H-2Db) or glycosylphosphatidylinositol (GPI)-anchored (Qa2) were labeled with antibody-coated gold particles and moved across the cell surface with a laser optical tweezers until they encountered a barrier, the barrier-free path length (BFP). At room temperature, the BFPs of Qa2 and H-2Db were 1.7 +/- 0.2 and 0.6 +/- 0.1 (micrometers +/- SEM), respectively. Barriers persisted at 34 degrees C, although the BFP for both MHC molecules was fivefold greater at 34 degrees C than at 23 degrees C. This indicates that barriers to lateral movement are primarily on the cytoplasmic half of the membrane and are dynamic.     

玉米S型细胞质雄性不育与恢复花粉中基因表达差异分析

以近等基因系S-Mo17Rf3Rf3和S-Mo17rf3rf3为材料,通过抑制消减杂交对S(Rf3)和S(rf3)花粉中差异表达的基因进行了研究.结果表明,玉米(Zea mays L.)S(Rf3)和S(rf3)花粉中存在与信号传导、膜系统形成、花粉的成熟以及抗细胞衰老等细胞和生理活动相关的基因表达的差异,但没有发现参与糖代谢、淀粉合成及其转运相关基因的表达差异.O-乙酰半乳糖胺转移酶(O-linked GlcNAc transferase,OGT)基因在Rf3花粉中表达累积.依据OGT基因的功能及其在玉米不同育性花粉中的表达模式,推测OGT受糖类物质诱导表达并与S(Rf3)配子的育性恢复有关.这一假说还有待进一步实验证实.     

How electric fields modify alkane solubility in lipid bilayers

The planar lipid bilayer membrane is assumed to be in osmotic equilibrium with the surrounding Plateau-Gibbs border (annulus) and entrapped microlenses. An electric field applied across the membrane raises the chemical potential of the alkane in the bilayer, causing it to shift from the bilayer to the annulus and microlenses. This shift results in a decrease in thickness.     

The fluid mosaic model of the structure of cell membranes

A fluid mosaic model is presented for the gross organization and structure of the proteins and lipids of biological membranes. The model is consistent with the restrictions imposed by thermodynamics. In this model, the proteins that are integral to the membrane are a heterogeneous set of globular molecules, each arranged in an amphipathic structure, that is, with the ionic and highly polar groups protruding from the membrane into the aqueous phase, and the nonpolar groups largely buried in the hydrophobic interior of the membrane. These globular molecules are partially embedded in a matrix of phospholipid. The bulk of the phospholipid is organized as a discontinuous, fluid bilayer, although a small fraction of the lipid may interact specifically with the membrane proteins. The fluid mosaic structure is therefore formally analogous to a two-dimensional oriented solution of integral proteins (or lipoproteins) in the viscous phospholipid bilayer solvent. Recent experiments with a wide variety of techniqes and several different membrane systems are described, all of which abet consistent with, and add much detail to, the fluid mosaic model. It therefore seems appropriate to suggest possible mechanisms for various membrane functions and membrane-mediated phenomena in the light of the model. As examples, experimentally testable mechanisms are suggested for cell surface changes in malignant transformation, and for cooperative effects exhibited in the interactions of membranes with some specific ligands. Note added in proof: Since this article was written, we have obtained electron microscopic evidence (69) that the concanavalin A binding sites on the membranes of SV40 virus-transformed mouse fibroblasts (3T3 cells) are more clustered than the sites on the membranes of normal cells, as predicted by the hypothesis represented in Fig. 7B. T-here has also appeared a study by Taylor et al. (70) showing the remarkable effects produced on lymphocytes by the addition of antibodies directed to their surface immunoglobulin molecules. The antibodies induce a redistribution and pinocytosis of these surface immunoglobulins, so that within about 30 minutes at 37 degrees C the surface immunoglobulins are completely swept out of the membrane. These effects do not occur, however, if the bivalent antibodies are replaced by their univalent Fab fragments or if the antibody experiments are carried out at 0 degrees C instead of 37 degrees C. These and related results strongly indicate that the bivalent antibodies produce an aggregation of the surface immunoglobulin molecules in the plane of the membrane, which can occur only if the immunoglobulin molecules are free to diffuse in the membrane. This aggregation then appears to trigger off the pinocytosis of the membrane components by some unknown mechanism. Such membrane transformations may be of crucial importance in the induction of an antibody response to an antigen, as well as iv other processes of cell differentiation.     

PI4P and PI(4,5)P2 are essential but independent lipid determinants of membrane identity

The quantitatively minor phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P(2)] fulfills many cellular functions in the plasma membrane (PM), whereas its synthetic precursor, phosphatidylinositol 4-phosphate (PI4P), has no assigned PM roles apart from PI(4,5)P(2) synthesis. We used a combination of pharmacological and chemical genetic approaches to probe the function of PM PI4P, most of which was not required for the synthesis or functions of PI(4,5)P(2). However, depletion of both lipids was required to prevent PM targeting of proteins that interact with acidic lipids or activation of the transient receptor potential vanilloid 1 cation channel. Therefore, PI4P contributes to the pool of polyanionic lipids that define plasma membrane identity and to some functions previously attributed specifically to PI(4,5)P(2), which may be fulfilled by a more general polyanionic lipid requirement.     

Regulation of SREBP processing and membrane lipid production by phospholipids in Drosophila

Animal cells exert exquisite control over the physical and chemical properties of their membranes, but the mechanisms are obscure. We show that phosphatidylethanolamine, the major phospholipid in Drosophila, controls the release of sterol regulatory element-binding protein (SREBP) from Drosophila cell membranes, exerting feedback control on the synthesis of fatty acids and phospholipids. The finding that SREBP processing is controlled by different lipids in mammals and flies (sterols and phosphatidylethanolamine, respectively) suggests that an essential function of SREBP is to monitor cell membrane composition and to adjust lipid synthesis accordingly.     

The effect of GTPase activating protein upon ras is inhibited by mitogenically responsive lipids

Bacterially synthesized c-Ha-ras protein (Ras) was incubated with guanosine triphosphatase (GTPase) activating (GA) protein in the presence of various phospholipids. The stimulation of Ras GTPase activity by GA protein was inhibited in some cases. Among the lipids most active in blocking GA protein activity were lipids that show altered metabolism during mitogenic stimulation. These included phosphatidic acid (containing arachidonic acid), phosphatidylinositol phosphates, and arachidonic acid. Other lipids, including phosphatidic acid with long, saturated side chains, diacylglycerols, and many other common phospholipids, were unable to alter GA protein activity. The interaction of lipids with GA protein might be important in the regulation of Ras activity during mitogenic stimulation.     

Role of a highly conserved bacterial protein in outer membrane protein assembly

After transport across the cytoplasmic membrane, bacterial outer membrane proteins are assembled into the outer membrane. Meningococcal Omp85 is a highly conserved protein in Gram-negative bacteria, and its homolog Toc75 is a component of the chloroplast protein-import machinery. Omp85 appeared to be essential for viability, and unassembled forms of various outer membrane proteins accumulated upon Omp85 depletion. Immunofluorescence microscopy revealed decreased surface exposure of outer membrane proteins, which was particularly apparent at the cell-division planes. Thus, Omp85 is likely to play a role in outer membrane protein assembly.     

Structure of MsbA from E. coli: a homolog of the multidrug resistance ATP binding cassette (ABC) transporters

Multidrug resistance (MDR) is a serious medical problem and presents a major challenge to the treatment of disease and the development of novel therapeutics. ABC transporters that are associated with multidrug resistance (MDR-ABC transporters) translocate hydrophobic drugs and lipids from the inner to the outer leaflet of the cell membrane. To better elucidate the structural basis for the "flip-flop" mechanism of substrate movement across the lipid bilayer, we have determined the structure of the lipid flippase MsbA from Escherichia coli by x-ray crystallography to a resolution of 4.5 angstroms. MsbA is organized as a homodimer with each subunit containing six transmembrane alpha-helices and a nucleotide-binding domain. The asymmetric distribution of charged residues lining a central chamber suggests a general mechanism for the translocation of substrate by MsbA and other MDR-ABC transporters. The structure of MsbA can serve as a model for the MDR-ABC transporters that confer multidrug resistance to cancer cells and infectious microorganisms.     

血液和组织标本中脂肪酸组成的毛细管柱气相色谱分析

用氯仿甲醇抽提、三氟化硼－乙醚甲醇酯化，然后利用毛细管柱和程序升温的方法进行气相色谱分析，建立了血浆和红细胞膜总脂以及组织磷脂和中性脂脂肪酸组成的毛细管柱气相色谱分析法。图谱表明各峰间分离效果良好、峰形尖锐、干扰少。各种脂肪酸的最小检测限均可达１０－（－１１）ｇ／ｓ，ＦＩＤ对各脂肪酸的响应均呈线性。实验重复性好，回收率达定量分析要求．     

Advances on Current Techniques and Methodologies in Milk Lipidomics

Milk is a complex biological fluid containing lipids, proteins, carbohydrates and minerals, which are essential for infant growth. While the lipid portion constitutes only 3％-5％ of the total milk composition, it accounts for over 50％ of the infant's daily energy intake. The dominant portion (approximately 98％) is in the form of triacylglycerols and polar lipids, such as glycerophospholipids and sphingolipids, forming minor components. Recently, with the development of lipidomics, important progresses have been made in milk lipidomics, and the identification and quantification of several milk lipids at the group and molecular species level has become a reality, thereby providing useful information for the infant formula industry. In this review, an overview of the separation of the main components of milk lipids was presented, including glycerolipids, phospholipids and sphingolipids. The analytical methods and strategies for milk lipidomics, including gas chromatography-mass spectrometry (MS), capillary electrophoresis MS, nuclear magnetic resonance, matrix-assisted laser desorption ionization-MS, electrospray ionization-MS, shotgun lipidomics and liquid chromatography-MS, were reviewed. Additionally, the bioinformatics of lipidomics for milk lipid determination, including lipid classification, lipid databases and lipid analysis software, were investigated. This review would aid future investigations of the nutrition of milk lipids and refined researches on formula milk powder.     

细胞脂滴的物理结构及影响其稳定的因素

脂滴是储存中性脂质的细胞器,对能量代谢至关重要,它们广泛存在于存在于动物、植 物、真菌,甚至细菌中。脂滴是油包水乳液在细胞水溶液中的分散相,乳液的基本生物物理原理 对于脂滴生物学的重要性正在被人们所重视。由于其存在于分散油相和水胞质之间的独特结 构,其形成,生长和收缩的具体机制尤为复杂,这种机制使细胞能够在代谢能量或膜合成需求发 生变化时使用乳化油,有利于为细胞新陈代谢提供更有利的途径。此外,脂滴表面的磷脂作为 表面活性剂组成的调控对脂滴的稳态和表面蛋白靶向至关重要。在这里,我们回顾脂滴的乳液 结构及其在