Receptor activation alters inner surface potential during phagocytosis

The surface potential of biological membranes varies according to their lipid composition. We devised genetically encoded probes to assess surface potential in intact cells. These probes revealed marked, localized alterations in the charge of the inner surface of the plasma membrane of macrophages during the course of phagocytosis. Hydrolysis of phosphoinositides and displacement of phosphatidylserine accounted for the change in surface potential at the phagosomal cup. Signaling molecules such as K-Ras, Rac1, and c-Src that are targeted to the membrane by electrostatic interactions were rapidly released from membrane subdomains where the surface charge was altered by lipid remodeling during phagocytosis.     

An acylation cycle regulates localization and activity of palmitoylated Ras isoforms

We show that the specific subcellular distribution of H- and Nras guanosine triphosphate-binding proteins is generated by a constitutive de/reacylation cycle that operates on palmitoylated proteins, driving their rapid exchange between the plasma membrane (PM) and the Golgi apparatus. Depalmitoylation redistributes farnesylated Ras in all membranes, followed by repalmitoylation and trapping of Ras at the Golgi, from where it is redirected to the PM via the secretory pathway. This continuous cycle prevents Ras from nonspecific residence on endomembranes, thereby maintaining the specific intracellular compartmentalization. The de/reacylation cycle also initiates Ras activation at the Golgi by transport of PM-localized Ras guanosine triphosphate. Different de/repalmitoylation kinetics account for isoform-specific activation responses to growth factors.     

Cationic protein-bearing granules of polymorphonuclear leukocytes: separation from enzyme-rich granules

The cationic, antibacterial proteins of polymorphonuclear leukocytes are associated with a unique subcellular particle that is separable through zonal density gradient centrifugation from acid phosphatase-containing particles as well as from particles that contain alkaline phosphatase and lysozyme. Normal macrophages, macrophages stimulated by bacillus Calmette-Guérin, and liver cells lack this particle and the associated group of cationic proteins. Particles physically and biochemically similar to slower sedimenting enzyme-rich particles of polymorphonuclear leukocytes are shared by all the tlhree cell types.     

Cell corpse engulfment mediated by C. elegans phosphatidylserine receptor through CED-5 and CED-12

During apoptosis, phosphatidylserine, which is normally restricted to the inner leaflet of the plasma membrane, is exposed on the surface of apoptotic cells and has been suggested to act as an "eat-me" signal to trigger phagocytosis. It is unclear how phagocytes recognize phosphatidylserine. Recently, a putative phosphatidylserine receptor (PSR) was identified and proposed to mediate recognition of phosphatidylserine and phagocytosis. We report that psr-1, the Caenorhabditis elegans homolog of PSR, is important for cell corpse engulfment. In vitro PSR-1 binds preferentially phosphatidylserine or cells with exposed phosphatidylserine. In C. elegans, PSR-1 acts in the same cell corpse engulfment pathway mediated by intracellular signaling molecules CED-2 (homologous to the human CrkII protein), CED-5 (DOCK180), CED-10 (Rac GTPase), and CED-12 (ELMO), possibly through direct interaction with CED-5 and CED-12. Our findings suggest that PSR-1 is likely an upstream receptor for the signaling pathway containing CED-2, CED-5, CED-10, and CED-12 proteins and plays an important role in recognizing phosphatidylserine during phagocytosis.     

Vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells

Viruses employ many different strategies to enter host cells. Vaccinia virus, a prototype poxvirus, enters cells in a pH-dependent fashion. Live cell imaging showed that fluorescent virus particles associated with and moved along filopodia to the cell body, where they were internalized after inducing the extrusion of large transient membrane blebs. p21-activated kinase 1 (PAK1) was activated by the virus, and the endocytic process had the general characteristics of macropinocytosis. The induction of blebs, the endocytic event, and infection were all critically dependent on the presence of exposed phosphatidylserine in the viral membrane, which suggests that vaccinia virus uses apoptotic mimicry to enter cells.     

Assembly of clathrin-coated pits onto purified plasma membranes

During receptor-mediated endocytosis, coated pits invaginate to form coated vesicles, clathrin and associated proteins dissociate from the vesicle membrane, and these proteins form new coated pits at the cell surface. As a means of elucidating molecular mechanisms that govern the function of coated pits, the assembly phase of this cycle was reconstituted by incubating purified membranes that were treated to remove endogenous coated pits with cytoplasm extracted from cultured cells. The in vitro assembly of coated pits on these membranes satisfactorily mimics many features of coated pit formation in the intact cell. These studies indicate that: the membranes contain a limited number of coated pit assembly sites that bind clathrin with high affinity; the half-time for assembly is 5 minutes both at 4 degrees C and 37 degrees C; during assembly, proteins with molecular sizes of 180, 110, and 36 kilodaltons are recruited to the plasma membrane; and assembly is not dependent on adenosine triphosphate, but this nucleotide triggers a temperature-dependent loss of coated pits that are assembled in the absence of adenosine triphosphate.     

Role of C. elegans TAT-1 protein in maintaining plasma membrane phosphatidylserine asymmetry

The asymmetrical distribution of phospholipids on the plasma membrane is critical for maintaining cell integrity and physiology and for regulating intracellular signaling and important cellular events such as clearance of apoptotic cells. How phospholipid asymmetry is established and maintained is not fully understood. We report that the Caenorhabditis elegans P-type adenosine triphosphatase homolog, TAT-1, is critical for maintaining cell surface asymmetry of phosphatidylserine (PS). In animals deficient in tat-1, PS is abnormally exposed on the cell surface, and normally living cells are randomly lost through a mechanism dependent on PSR-1, a PS-recognizing phagocyte receptor, and CED-1, which contributes to recognition and engulfment of apoptotic cells. Thus, tat-1 appears to function in preventing appearance of PS in the outer leaflet of plasma membrane, and ectopic exposure of PS on the cell surface may result in removal of living cells by neighboring phagocytes.     

The insulin receptor contains a calmodulin-binding domain

Substantial evidence suggests that calcium has a pivotal role in regulating the initial events through which insulin alters plasma membrane metabolism. Because binding of insulin to its receptor represents the initial site of insulin action in the plasma membrane, studies were undertaken to determine whether the insulin receptor is a calmodulin-binding protein. Preparations enriched in the insulin receptor and calmodulin-binding proteins were isolated from detergent-solubilized rat adipocyte membranes by chromatography with wheat germ agglutinin agarose and calmodulin-conjugated Sepharose, respectively. Substantial purification of a manganese-dependent, insulin-sensitive phosphoprotein of 95K identified as the beta subunit of the insulin receptor was accomplished. Binding and photocovalent cross-linking of iodine-125-labeled calmodulin to these affinity-purified preparations and to isolated plasma membranes, followed by immunoadsorption with insulin receptor antibodies bound to protein A Sepharose, resulted in significant purification of a binding complex of 110K to 140K. These results indicate that the adipocyte insulin receptor or a polypeptide closely associated with the receptor is a calmodulin-binding protein.     

植物生长素结合蛋白ABP1的研究进展

ABP1(Auxin Binding Protein)是最早发现的生长素结合蛋白之一,作为一种生长素受体参与质膜上生长素的响应过程,在细胞周期、细胞扩增、质膜内吞作用和基于ROP的细胞骨架重建等快速反应中起着重要的作用。因此,对ABP1最新的研究进展进行了综述,以期为生长素受体作用机制的深度阐明提供参考。     

Influenza virus effects on cell membrane proteins

During infection by influenza virus, viral proteins become firmly attached to (or part of) host cell plasma membrane, nuclear membranes, mitochondrial, and microsomal membranes. Purified virus particles contain less than I percent host cell protein. Virus envelope proteins completely replace host membrane proteins in those discrete spots on the plasma membrane from which progeny virions bud out.     

Vesicle formation at the plasma membrane and trans-Golgi network: the same but different

An elaborate vesicle transport system supports the active exchange of membranes and protein cargo between the plasma membrane and the trans-Golgi network. Many observations suggest that highly conserved mechanisms are used in vesicle formation and scission. Such similarity is found both at the level of the receptor-ligand sequestration process that uses clathrin and associated polymeric and monomeric adaptor proteins, and in the machinery used to deform and vesiculate lipid membranes.     

A glycan-phosphatidylinositol-specific phospholipase D in human serum

A group of proteins anchored to the cell by phosphatidylinositol (PI) has recently been identified. The significance of this new class of membrane anchor is unknown; one possibility is that it facilitates release of the molecule by phospholipases. In fact, phospholipase C enzymes specific for the complex carboxyl-terminal glycolipids of these proteins have been isolated from African trypanosomes and from hepatocyte plasma membranes. This study reports the discovery of a glycan-PI-specific phospholipase D in human serum that cleaves both the membrane form of the variant surface glycoprotein of African trypanosomes and its glycolipid precursor, but not phosphatidylethanolamine, phosphatidylcholine, or phosphatidylinositol. Decay-accelerating factor, another PI-anchored molecule, is also cleaved by the enzyme and converted from a hydrophobic to a soluble protein. The enzyme is Ca2+-dependent, heat labile, and not affected by the inhibitor of serine proteases, phenylmethylsulfonylfluoride. Its function is not known, but the present findings indicate that it participates in the metabolism of glycolipid-anchored membrane proteins.     

Platelet membrane glycoprotein IIb/IIIa: member of a family of Arg-Gly-Asp--specific adhesion receptors

Adhesive interactions of the platelet surface with plasma proteins such as fibrinogen and fibronectin play an important role in thrombosis and hemostasis. The binding of both of these proteins to platelets is inhibited by synthetic peptides containing the sequence Arg-Gly-Asp, which corresponds to the cell adhesion site in fibronectin and is also present in the alpha chain of fibrinogen. An affinity matrix made of an insolubilized heptapeptide containing the Arg-Gly-Asp sequence selectively binds the platelet membrane glycoprotein IIb/IIIa from detergent extracts of platelets. When incorporated into liposome membranes, the isolated protein confers to the liposomes the ability to bind to surfaces coated with fibrinogen, fibronectin, and vitronectin but not to surfaces coated with thrombospondin or albumin. This platelet receptor is related to the previously identified fibronectin and vitronectin receptors in that it recognizes an Arg-Gly-Asp sequence but differs from the other receptors in its wider specificity toward various adhesive proteins. These results establish the existence of a family of adhesion receptors that recognize the sequence Arg-Gly-Asp.     

Chemoattractant-regulated mobilization of a novel intracellular compartment in human neutrophils

A novel mobilizable intracellular compartment was identified in human neutrophils by latent alkaline phosphatase activity. This compartment is mobilized to the plasma membrane much more readily than any identified granule subset and has kinetics of up-regulation in the membrane similar to those reported for a variety of receptor proteins. Triton X-100 permeabilization of both intact human neutrophils and subcellular fractions obtained by density-gradient centrifugation revealed that 70 percent of the alkaline phosphatase is located in an intracellular compartment distinct from primary, secondary, and gelatinase granules and from the plasma membrane. This compartment fully translocates to the plasma membrane after stimulation with nanomolar concentrations of the chemotactic peptide N-formylmethionylleucylphenylalanine.     

Membrane structure: some general principles

The arrangement of lipids and some proteins in the erythrocyte membrane has been discussed. The conclusions from this are listed here as a set of general guidelines for the structure of membranes of higher organisms: some of these rules may be wrong. But at this stage it seems useful to sharpen our thoughts in this way and thereby focus attention on various specific points. 1) The basis of a membrane is a lipid bilayer with (i) choline phospholipids and glycolipids in the external half and (ii) amino (and possibly some choline) phospholipids in the cytoplasmic half. There is effectively no lipid exchange across the bilayer (unless enzymatically catalyzed) (68). 2) Some proteins extend across the bilayer. Where this is so, they will in general have carbohydrate on their surface remote from the cytoplasm. This carbohydrate may prevent the protein diffusing out of the membrane into the cytoplasm; it acts as a lock on the protein. 3) Just as lipids do not flip-flop, proteins do not rotate across the membrane. Lateral motion or rotation of lipids and proteins in the plane of the bilayer may be expected. 4) Most membrane protein is associated with the inner, cytoplasmic, urface of the membrane. Proteins are not usually associated exclusively with the outer half of the lipid bilayer. 5) Membrane proteins are a special class of cytoplasmic proteins, not of secreted proteins.     

水稻抗白叶枯病相关基因的亚细胞定位

利用亚细胞定位技术对水稻抗白叶枯病相关基因的表达产物进行了分析,为基因功能分析提供了重要信息。通过前期基因芯片筛选、半定量/定量PCR的验证及基因功能注释,从高抗白叶枯病水稻品种Y73中筛选了18个潜在的与抗白叶枯病相关的基因,并在烟草叶片细胞中进行亚细胞定位分析。通过观察融合有绿色荧光标记(sGFP)的表达产物,初步了解这些基因在烟草细胞中的表达位置。经过激光共聚焦显微镜观察后发现: 其中4个基因的 sGFP 融合蛋白定位于细胞核上,可能发挥了转录因子的功能;4个基因的 sGFP 融合蛋白定位于细胞质膜上,推测可能与细胞质膜的稳定或控制蛋白转运相关;4个基因的 sGFP 融合蛋白同时定位于细胞核和细胞质膜上;另有6个基因的 sGFP 融合表达后没有观察到明显的定位信号。     

Mechanism of membrane anchoring affects polarized expression of two proteins in MDCK cells

The signals that direct membrane proteins to the apical or basolateral plasma membrane domains of polarized epithelial cells are not known. Several of the class of proteins anchored in the membrane by glycosyl-phosphatidylinositol (GPI) are expressed on the apical surface of such cells. However, it is not known whether the mechanism of membrane anchorage or the polypeptide sequence provides the sorting information. The conversion of the normally basolateral vesicular stomatitis virus glycoprotein (VSV G) to a GPI-anchored protein led to its apical expression. Conversely, replacement of the GPI anchor of placental alkaline phosphatase with the transmembrane and cytoplasmic domains of VSV G shifted its expression from the apical to the basolateral surface. Thus, the mechanism of membrane anchorage can determine the sorting of proteins to the apical or basolateral surface, and the GPI anchor itself may provide an apical transport signal.     

Role of ER export signals in controlling surface potassium channel numbers

Little is known about the identity of endoplasmic reticulum (ER) export signals and how they are used to regulate the number of proteins on the cell surface. Here, we describe two ER export signals that profoundly altered the steady-state distribution of potassium channels and were required for channel localization to the plasma membrane. When transferred to other potassium channels or a G protein-coupled receptor, these ER export signals increased the number of functional proteins on the cell surface. Thus, ER export of membrane proteins is not necessarily limited by folding or assembly, but may be under the control of specific export signals.     

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.     

The cytoskeletal protein vinculin contains transformation-sensitive, covalently bound lipid

Vinculin, which is associated with the cytoskeleton of many cells, has been suggested as a possible linker between microfilament bundles and the plasma membrane. Here it will be shown that fatty acid is covalently attached to vinculin in vivo. Furthermore, in chicken embryo fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus, tsNY68, the acylation of vinculin at the permissive temperature was less than one-third that at the nonpermissive temperature. Thus, the covalent binding of lipid to vinculin is a transformation-sensitive event. The covalent modification of vinculin by lipids could be directly or indirectly involved in its reversible association with membranes. This modification may also provide a mechanism to alter the organization of vinculin within cells and thereby play a regulatory role in anchoring or stabilizing microfilament bundles at plasma membranes.