Signal for attachment of a phospholipid membrane anchor in decay accelerating factor

Decay accelerating factor (DAF) belongs to a novel group of membrane proteins anchored to the cell surface by a glycophospholipid membrane anchor that is covalently attached to the carboxyl terminus of the protein. The last 37 amino acids of membrane DAF, when fused to the carboxyl terminus of a secreted protein, are sufficient to target the fusion protein to the plasma membrane by means of a glycophospholipid anchor. This approach provides a novel means of targeting proteins to the cell-surface membrane.     

A microdomain for protein secretion in Gram-positive bacteria

Gram-positive bacteria face unique challenges in generating biologically active conformations for their exported proteins because they lack a dedicated compartment for folding secreted polypeptides. We have discovered that protein secretion by way of the general secretory (Sec) pathway in the important human pathogen Streptococcus pyogenes proceeds through a single microdomain. Unlike other mechanisms for asymmetry involving the Sec pathway, proteins destined for secretion are targeted to a single locus distal to either cell pole that has specialized to contain the Sec translocons. This subcellular organization may represent a paradigm for secretion common to other Gram-positive pathogens with profound implications for pathogenesis.     

Synthetic amphiphilic peptide models for protein ion channels

Ion channel proteins are important for the conduction of ions across biological membranes. Recent analyses of their sequences have suggested that they are composed of bundles of alpha-helices that associate to form ion-conducting channels. To gain insight into the mechanisms by which alpha-helices can aggregate and conduct ions, three model peptides containing only leucine and serine residues were synthesized and characterized. A 21-residue peptide, H2N-(Leu-Ser-Ser-Leu-Leu-Ser-Leu)3-CONH2, which was designed to be a membrane-spanning amphiphilic alpha-helix, formed well-defined ion channels with ion permeability and lifetime characteristics resembling the acetylcholine receptor. In contrast, a 14-residue version of this peptide, which was too short to span the phospolipid bilayer as an alpha-helix, failed to form discrete, stable channels. A third peptide, H2N-(Leu-Ser-Leu-Leu-Leu-Ser-Leu)3-CONH2, in which one serine per heptad repeat was replaced by leucine, produced proton-selective channels. Computer graphics and energy minimization were used to create molecular models that were consistent with the observed properties of the channels.     

NMR structure of Mistic, a membrane-integrating protein for membrane protein expression

Although structure determination of soluble proteins has become routine, our understanding of membrane proteins has been limited by experimental bottlenecks in obtaining both sufficient yields of protein and ordered crystals. Mistic is an unusual Bacillus subtilis integral membrane protein that folds autonomously into the membrane, bypassing the cellular translocon machinery. Using paramagnetic probes, we determined by nuclear magnetic resonance (NMR) spectroscopy that the protein forms a helical bundle with a surprisingly polar lipid-facing surface. Additional experiments suggest that Mistic can be used for high-level production of other membrane proteins in their native conformations, including many eukaryotic proteins that have previously been intractable to bacterial expression.     

Structural basis for tail-anchored membrane protein biogenesis by the Get3-receptor complex

Tail-anchored (TA) proteins are involved in cellular processes including trafficking, degradation, and apoptosis. They contain a C-terminal membrane anchor and are posttranslationally delivered to the endoplasmic reticulum (ER) membrane by the Get3 adenosine triphosphatase interacting with the hetero-oligomeric Get1/2 receptor. We have determined crystal structures of Get3 in complex with the cytosolic domains of Get1 and Get2 in different functional states at 3.0, 3.2, and 4.6 angstrom resolution. The structural data, together with biochemical experiments, show that Get1 and Get2 use adjacent, partially overlapping binding sites and that both can bind simultaneously to Get3. Docking to the Get1/2 complex allows for conformational changes in Get3 that are required for TA protein insertion. These data suggest a molecular mechanism for nucleotide-regulated delivery of TA proteins.     

A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A

Vitamin A has diverse biological functions. It is transported in the blood as a complex with retinol binding protein (RBP), but the molecular mechanism by which vitamin A is absorbed by cells from the vitamin A-RBP complex is not clearly understood. We identified in bovine retinal pigment epithelium cells STRA6, a multitransmembrane domain protein, as a specific membrane receptor for RBP. STRA6 binds to RBP with high affinity and has robust vitamin A uptake activity from the vitamin A-RBP complex. It is widely expressed in embryonic development and in adult organ systems. The RBP receptor represents a major physiological mediator of cellular vitamin A uptake.     

CRACM1 is a plasma membrane protein essential for store-operated Ca2+ entry

Store-operated Ca2+ entry is mediated by Ca2+ release-activated Ca2+ (CRAC) channels following Ca2+ release from intracellular stores. We performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that inhibit store-operated Ca2+ influx. A secondary patch-clamp screen identified CRACM1 and CRACM2 (CRAC modulators 1 and 2) as modulators of Drosophila CRAC currents. We characterized the human ortholog of CRACM1, a plasma membrane-resident protein encoded by gene FLJ14466. Although overexpression of CRACM1 did not affect CRAC currents, RNAi-mediated knockdown disrupted its activation. CRACM1 could be the CRAC channel itself, a subunit of it, or a component of the CRAC signaling machinery.     

Comment on "A G protein coupled receptor is a plasma membrane receptor for the plant hormone abscisic acid"

Liu et al. (Reports, 23 March 2007, p. 1712) reported that the Arabidopsis thaliana gene GCR2 encodes a seven-transmembrane, G protein-coupled receptor for abscisic acid. We argue that GCR2 is not likely to be a transmembrane protein nor a G protein-coupled receptor. Instead, GCR2 is most likely a plant homolog of bacterial lanthionine synthetases.     

甘蔗宿根矮化病病原菌一推测膜蛋白基因(Lxx18460)单克隆抗体的制备和检测

[目的]对甘蔗宿根矮化病病原菌(Leifsonia xyli subsp.xyli,Lxx)一推测为抗o'k因子的膜蛋白基因(Lxx18460)进行研究,为今后研究该基因在感病甘蔗体内表达打下基础.[方法]诱导表达并纯化含有推测为抗o'k因子的膜蛋白Lxx1.8460基因的pET-30a质粒菌液,委托南京金斯瑞生物科技有限公司制备单克隆抗体;用Western blotting对细菌及感病和健康甘蔗样品总蛋白进行检测.[结果]Lxx18460基因具有特异性,只存在于甘蔗Lxx中.ELISA单克隆抗体效价大于1∶512000,能够灵敏地检测细菌总蛋白和甘蔗样品蛋白.Western blotting检测结果表明,Lxx18460基因在细菌中和感病甘蔗样品中表达,在健康甘蔗样品中几乎不表达.[结论]Lxx18460基因具有特异性,是Lxx中的抗o'k因子的膜蛋白基因,能够在细菌和感病甘蔗样品体外进行表达.     

胎膜早破孕妇白细胞介素8、C反应蛋白及白细胞计数的检测对绒毛膜羊膜炎的诊断意义

目的了解胎膜早破孕妇母血中白细胞介素8(IL-8)、C反应蛋白(CRP)及白细胞计数对绒毛膜羊膜炎的诊断意义.方法采用放射免疫法测定54例胎膜早破(早破组)和40例正常足月孕妇(对照组)血中的IL-8水平,并同步检测CRP水平及白细胞计数;每例组织绒毛膜羊膜炎通过产后胎盘病理证实,按病理结果将早破组分为绒毛膜羊膜炎组(n＝36)及非绒毛膜羊膜炎组(n＝18).结果早破组和对照组的IL-8、CRP及白细胞计数的差异均有非常显著性(P＜0.01)[(130.4±25.6)ng·L-1vs(75.2±15.8)ng·L-1、(125.4±10.4) mg·L-1vs(1.70±1.1) mg·L-1 (11.5±3.6)×109/Lvs(8.9±1.9)×109/L],而绒毛膜羊膜炎组和非绒毛膜羊膜炎组比较,IL-8、CRP及白细胞计数的差异也有显著性和非常显著性(P＜0.05～0.01)[(138.1±27.2)ng·L-1vs(100.7±20.6)ng·L-1、(127.6±3.2) mg·L-1vs(1.9±1.4)mg·L-1、(12.8±4.0)×109/Lvs(8.9±2.7)×109/L].结论检测胎膜早破孕妇母血中的IL-8、CRP及白细胞计数可早期发现组织绒毛膜羊膜炎,这对及早处理、改善妊娠预后有重要临床意义.