高尔基体堆叠蛋白GRASP55结构折叠变化研究

哺乳动物细胞中,高尔基体是由数个扁平囊泡堆叠形成的高度有极性的细胞器,其堆叠主要通过GRASP55和GRASP65蛋白的寡聚化来介导.GRASP55由N端两个串联的PDZ结构域和C端脯氨酸丰富结构域组成.前期工作中发现PDZ结构域末尾存在一段内部配体序列在GRASP55寡聚过程中有重要作用.为进一步验证该内部配体的作用,实验中以不含内部配体的截短型GRASP55为研究对象进行结构学研究,发现GRASP55中存在包含此内部配体的3处在构象上易变的区域,通过这些区域在折叠上的变化而影响GRASP55的寡聚化,进而影响高尔基体的堆叠.     

烟草叶肉原生质体再生壁形成的电镜研究

电镜观察表明,新分离的烟草叶肉原生质体表面光滑,在质膜外没有纤维状物质,内质网和高尔基体几乎没有发育:培养1天后,线粒体明显增加,粗面内质网和高尔基体沿质膜发育,质膜变得粗糙;2天后,质膜发生折皱,高尔基囊泡向质膜外分泌它的内含物;6天后,质膜表面出现明显松散分布的再生壁。在培养基中附加200 ppm 香豆素处理,在培养1天后,质膜仍然光滑,未出现内质网和高尔基体;2天后,质膜开始变粗糙,内质网开始发育。试验表明,质膜、内质网、高尔基体在原生质体再生壁形成中起重要作用,并讨论了它们的可能作用。香豆索可能是在一定时同内,通过抑制质膜的活动和内质网、高尔基体的发育而抑制壁的再生。     

真菌Rab蛋白功能的研究进展

Rab家族蛋白是小G蛋白Ras超家族最大的亚家族,不同Rab家族蛋白定位在特定的细胞内膜上.研究结果表明,作为真核生物细胞中各细胞器之间囊泡运输的分子开关,Rab蛋白通过其上游的调节子蛋白和下游的效应子蛋白调控着细胞内囊泡的形成和运输,调节生物体内各种蛋白在细胞内外的运输和分配,在真菌的生长发育和致病过程中起到关键作用.     

植物细胞高尔基体的研究进展

随着研究的深入,对于高尔基体在植物细胞中的作用,已有了一定的认识。普遍认为植物细胞高尔基体的功能主要有：１）高尔基体与植物细胞多糖类分泌物形成有关;２）植物细胞中高尔基体不参与糖蛋白的合成与运输;３）植物细胞中,高尔基体与胞内运输有一定联系;４）在植物细胞壁的形成过程中,高尔基体发挥重要作用。     

果蝇锌离子转运蛋白Catsup亚细胞定位分析

Catsup是果蝇体内一个功能重要的基因,其突变会导致体内多巴胺含量过高,引起果蝇致死及生殖障碍.绿色荧光蛋白(green fluorescent protein,GFP)在确定蛋白的亚细胞定位进而在研究蛋白功能方面起着重要的作用.为了探索Catsup的亚细胞定位进而能够更好地研究Catsup的功能,文章利用基因工程技术和生物学方法,聚合酶链式反应(polymerase chain reaction,PCR)扩增Catsup基因全长,与GFP一起连接至pUAST质粒,构建载体pUAST-Catsup-GFP,通过显微注射技术导入果蝇,获得UAS-Catsup-GFP转基因果蝇.通过克隆验证了转基因果蝇构建成功.利用该果蝇进行Catsup的亚细胞定位,确定其定位在高尔基体上,为进一步研究该基因的功能奠定基础.     

酵母Mn-transporter基因研究进展

在酵母中有两个锰离子的转运系统 :即高亲和力系统和低亲和力系统 .本文简要介绍了酵母中与锰离子转运有关的基因 :SMF1、BSD2、ATX2、CCC1、PMR1和MNR1及它们所编码蛋白的大小、细胞定位、跨膜域及他们在锰转运中的功能 .文中还比较了几种蛋白之间的关系 .     

拟南芥RabA2b互作蛋白的筛选与鉴定

Rab蛋白家族在调控细胞囊泡的运输过程中发挥着重要作用.拟南芥中存在数量庞大的RabA亚族成员,它们参与调控植物不同阶段的生长发育过程.为了寻找拟南芥RabA亚族下游的调节蛋白或效应蛋白,进而阐明RabA在细胞活动中的角色,本研究选取RabA2b蛋白进行了深入研究.本研究首先构建过表达RabA2b的拟南芥植株,同时结合免疫共沉淀方法和液相色谱-串联质谱法(LC-MS/MS)检测到多个Hsp70蛋白家族成员与RabA2b蛋白共沉淀.为了进一步研究Hsp70成员与RabA2b的相互关系,将5个细胞质和细胞核定位的Hsp70s蛋白分别构建于植物表达载体,通过在烟草叶表皮细胞中瞬时表达Hsp70s和RabA2b发现它们共定位于细胞质膜、细胞质以及细胞核周围的丝状结构.同时,体外pull-down实验证明了5个Hsp70s蛋白都能与RabA2b蛋白直接相互作用.此外,双分子荧光互补实验(BiFC)进一步证实了Hsp70s与RabA2b在烟草叶表皮细胞中的相互作用,说明拟南芥中细胞质和细胞核定位的Hsp70s可以作为RabA2b的调节蛋白或效应蛋白一同调控植物的生长发育过程.     

胜利二元复合驱体系界面扩张流变研究

利用Langmuir槽法,采用正弦周期振荡和界面张力弛豫两种方式,研究了胜利稀释原油的界面扩张流变性质,考察了现场表面活性剂及聚合物对其界面吸附膜特性的影响.研究结果表明,胜利石油磺酸盐在较低浓度时可增加原油界面膜强度,非离子表面活性剂由于空间效应无法增大膜强度;而聚合物分子链段插入界面上的活性组分分子间,对界面膜强度有较明显的削弱作用.     

SNX12调控BACE1的细胞内定位和Aβ的产生

不溶性β淀粉样蛋白(β-amyloid,Aβ)的沉积是阿尔茨海默症(Alzheimer′s disease,AD)的中心环节,Aβ是由β-分泌酶(BACE1)和γ-分泌酶顺序切割β淀粉样蛋白前体蛋白(β-amyloid precursor protein,APP)产生的,BACE1与APP在质膜、内含体/溶酶体、高尔基体反面膜囊(TGN)以及早期分泌途径之间的转运很大程度上影响了Aβ的产生.通过Western blot和免疫荧光的方法探讨了Sorting nexin12(SNX12)对Aβ产生的影响及其作用机制.Western blot结果显示过表达SNX12后,分泌到细胞外的Aβ减少,γ-分泌酶的活性及PS1、NCT、pen-2、APP和BACE1的蛋白水平没有明显变化,APP-βCTF水平降低,而下调SNX12时Aβ水平增加,与对照组相比,差异均具有统计学意义(p<0.05);免疫荧光结果表明,SNX12与BACE1在细胞内存在共定位,并且SNX12表达增加可以使BACE1和APP在细胞内处于同一区域的数量减少.这些结果表明,过表达SNX12通过调节BACE1在细胞内的定位,使细胞内处于同一区域的BACE1与APP的数量减少,导致经BACE1切割的APP减少,从而降低Aβ的产生.因此SNX12可能在AD的发病过程中起着一定的调节作用.     

大鼠中性氨基酸转运蛋白SNAT1-HA融合蛋白的构建和表达(英文)

Na+偶联的中性氨基酸转运蛋白SNAT1是一种膜蛋白,属于转运蛋白第38家族(SLC38),在中枢神经系统(CNS)中起到重要的生理学作用.为了更容易地检测SNAT1在细胞膜上的表达,以pBK-CMVΔ-SNAT1为模板,用PCR,双酶切和T4连接酶连接的方法在SNAT1的C端加上了一个HA标签蛋白,构建了一个新质粒pBK-CMVΔ-SNAT1-HA.用该质粒瞬时转染人胚胎肾细胞(HEK293T),转染36 h以后,融合蛋白SNAT1-HA可以用HA抗体检测.Western blot结果显示在约54 kDa处有一条特异性条带,与预期分子量大小一致.利用重组质粒pBK-CMVΔ-SNAT1-HA可以更方便地检测到SNAT1在细胞膜上的表达与定位,为进一步研究SNAT1结构与功能奠定了基础.     

CtBP/BARS induces fission of Golgi membranes by acylating lysophosphatidic acid

Membrane fission is essential in intracellular transport. Acyl-coenzyme As (acyl-CoAs) are important in lipid remodelling and are required for fission of COPI-coated vesicles. Here we show that CtBP/BARS, a protein that functions in the dynamics of Golgi tubules, is an essential component of the fission machinery operating at Golgi tubular networks, including Golgi compartments involved in protein transport and sorting. CtBP/BARS-induced fission was preceded by the formation of constricted sites in Golgi tubules, whose extreme curvature is likely to involve local changes in the membrane lipid composition. We find that CtBP/BARS uses acyl-CoA to selectively catalyse the acylation of lysophosphatidic acid to phosphatidic acid both in pure lipidic systems and in Golgi membranes, and that this reaction is essential for fission. Our results indicate a key role for lipid metabolic pathways in membrane fission.     

Receptor-rich intracellular membrane vesicles transporting asialotransferrin and insulin in liver

A wide range of receptors are located at the blood sinusoidal aspect of the hepatocyte plasma membrane. Many circulating ligands that bind to receptors on the cell surfaces are interiorized along two pathways. Asialoglycoproteins are transferred from the plasma membrane to lysosomes and degraded, whereas immunoglobulin A and bile acids are transported across the hepatocyte interior and released into bile. Asialotransferrin type 3 (ref. 6) follows a further pathway termed diacytosis. After binding to the asialoglycoprotein receptor, asialotransferrin is endocytosed and then returned to blood with a proportion of its carbohydrate side chains resialylated. We now describe in liver the properties of intracellular asialotransferrin-enclosing vesicles (diacytosomes) and show that they differ from Golgi, lysosome and plasma membrane fractions. Furthermore, we show that the asialoglycoprotein binding sites are located on the cytoplasmic (outer) surface of diacytosomes.     

Sequence and topology of a model intracellular membrane protein, E1 glycoprotein, from a coronavirus

In the eukaryotic cell, both secreted and plasma membrane proteins are synthesized at the endoplasmic reticulum, then transported, via the Golgi complex, to the cell surface. Each of the compartments of this transport pathway carries out particular metabolic functions, and therefore presumably contains a distinct complement of membrane proteins. Thus, mechanisms must exist for localizing such proteins to their respective destinations. However, a major obstacle to the study of such mechanisms is that the isolation and detailed analysis of such internal membrane proteins pose formidable technical problems. We have therefore used the E1 glycoprotein from coronavirus MHV-A59 as a viral model for this class of protein. Here we present the primary structure of the protein, determined by analysis of cDNA clones prepared from viral mRNA. In combination with a previous study of its assembly into the endoplasmic reticulum membrane, the sequence reveals several unusual features of the protein which may be related to its intracellular localization.     

Live imaging of yeast Golgi cisternal maturation

There is a debate over how protein trafficking is performed through the Golgi apparatus. In the secretory pathway, secretory proteins that are synthesized in the endoplasmic reticulum enter the early compartment of the Golgi apparatus called cis cisternae, undergo various modifications and processing, and then leave for the plasma membrane from the late (trans) cisternae. The cargo proteins must traverse the Golgi apparatus in the cis-to-trans direction. Two typical models propose either vesicular transport or cisternal progression and maturation for this process. The vesicular transport model predicts that Golgi cisternae are distinct stable compartments connected by vesicular traffic, whereas the cisternal maturation model predicts that cisternae are transient structures that form de novo, mature from cis to trans, and then dissipate. Technical progress in live-cell imaging has long been awaited to address this problem. Here we show, by the use of high-speed three-dimensional confocal microscopy, that yeast Golgi cisternae do change the distribution of resident membrane proteins from the cis nature to the trans over time, as proposed by the maturation model, in a very dynamic way.     

Chloroquine and ammonium chloride prevent terminal glycosylation of immunoglobulins in plasma cells without affecting secretion

The generation of an acidic pH in intracellular organelles is required for several membrane and protein recycling processes. For instance, the internalization of ligands by receptor-mediated endocytosis is followed by the development of an acidic pH inside endosomes; this allows dissociation of the ligand, which is then transported to the lysosomes, from the receptor, which is recycled to the cell surface. There is evidence that part of this recycling process involves the distal region of the Golgi complex, where terminal glycosylation occurs: when the plasma membrane transferrin receptor is desialylated by neuraminidase treatment, it acquires new sialic acid molecules after endocytosis and before cell-surface re-expression. Golgi membranes have been shown to contain a proton pump and the distal Golgi cisternae appear to have an acidic content. Here, we have studied the effects of chloroquine and ammonium chloride, which raise the pH of acidic intracellular compartments, on the processing and secretion of immunoglobulins by plasma cells. Sialic acid transfer to terminal galactose residues, a reaction known to occur in the distal Golgi shortly before secretion, is completely and rapidly inhibited in the presence of these drugs, without significant modification of the secretion rate. This effect is accompanied by a dilatation of the Golgi cisternae and is not rapidly reversible.     

Brefeldin A inhibits Golgi membrane-catalysed exchange of guanine nucleotide onto ARF protein.

The fungal metabolite brefeldin A is a powerful tool for investigating membrane traffic in eukaryotic cells. The effects of brefeldin A on traffic are partly explained by its ability to prevent binding of cytosolic coat proteins onto membranes. The non-clathrin coatomer complex binds reversibly to Golgi membranes in a GTP-controlled cycle. The low-molecular-mass GTP-binding protein ADP-ribosylation factor (ARF), which also associates reversibly with Golgi membranes, is required for coatomer binding and probably accounts for the control by guanine nucleotide of the coatomer-membrane interaction. Brefeldin A prevents the assembly of coatomer onto the membrane by inhibiting the GTP-dependent interaction of ARF with the Golgi membrane, but the nature of this interaction has not been established. Here we demonstrate that Golgi membranes can specifically catalyse the exchange of GTP onto ARF and that brefeldin A prevents this function.     

Topological restriction of SNARE-dependent membrane fusion

To fuse transport vesicles with target membranes, proteins of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) complex must be located on both the vesicle (v-SNARE) and the target membrane (t-SNARE). In yeast, four integral membrane proteins, Sed5, Bos1, Sec22 and Bet1 (refs 2-6), each probably contribute a single helix to form the SNARE complex that is needed for transport from endoplasmic reticulum to Golgi. This generates a four-helix bundle, which ultimately mediates the actual fusion event. Here we explore how the anchoring arrangement of the four helices affects their ability to mediate fusion. We reconstituted two populations of phospholipid bilayer vesicles, with the individual SNARE proteins distributed in all possible combinations between them. Of the eight non-redundant permutations of four subunits distributed over two vesicle populations, only one results in membrane fusion. Fusion only occurs when the v-SNARE Bet1 is on one membrane and the syntaxin heavy chain Sed5 and its two light chains, Bos1 and Sec22, are on the other membrane where they form a functional t-SNARE. Thus, each SNARE protein is topologically restricted by design to function either as a v-SNARE or as part of a t-SNARE complex.     

An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells

Circumstantial evidence suggests that intracellular membrane trafficking pathways diversified independently in the plant kingdom, but documented examples are rare. ARF-GEFs (guanine-nucleotide exchange factors for ADP-ribosylation factor GTPases) are essential for vesicular trafficking in all eukaryotic kingdoms, but of the eight ARF-GEF families, only the ancestral BIG and GBF types are found in plants. Whereas fungal and animal GBF proteins perform conserved functions at the Golgi, the Arabidopsis thaliana GBF protein GNOM is thought to act in only the process of recycling from endosomes. We now show that the related Arabidopsis GBF protein GNOM-LIKE1 (GNL1) has an ancestral function at the Golgi but is also required for selective internalization from the plasma membrane in the presence of brefeldin A (BFA). We identified gnl1 mutants that accumulated biosynthetic and recycling endoplasmic reticulum markers in enlarged internal compartments. Notably, in the absence of functional GNL1, Golgi stacks were rendered sensitive to the selective ARF-GEF inhibitor BFA, which caused them to fuse with the endoplasmic reticulum. Furthermore, in BFA-treated gnl1 roots, the internalization of a polar plasma-membrane marker, the auxin efflux carrier PIN2, was selectively inhibited. Thus, GNL1 is a BFA-resistant GBF protein that functions with a BFA-sensitive ARF-GEF both at the Golgi and in selective endocytosis, but not in recycling from endosomes. We propose that the evolution of endocytic trafficking in plants was accompanied by neofunctionalization within the GBF family, whereas in other kingdoms it occurred independently by elaboration of additional ARF-GEF families.     

Carbon-free nanoporous gold based membrane electrocatalysts for fuel cells

Nanoporous gold (NPG) membranes made by dealloying consist of a bicontinuous network of Au ligaments and open pore channels, which have gained considerable attention as a platform for the design of carbon-free electrodes for proton exchange membrane fuel cells (PEMFCs). Benefiting from a unique combination of high electronic conductivity, high surface area, and modifiable surface chemistry, these self-supporting membrane type electrodes allow integration of various structural functions required for specific electrode reactions and simultaneously facilitate the entire fuel cell kinetic process. In this review, we summarize the major research progresses in this area, with an emphasis on how to customize the surface structures of these three-dimensional electrocatalysts for desired fuel molecule oxidation and oxidant reduction. We will also discuss these designed structural characteristics that can be readily accommodated in membrane electrode assemblies (MEA), thus effectively bridging the technological gap between electrocatalysts’ intrinsic activities and their actual performances in fuel cells.     

Morphology of the medullary visceral zone

Since 1990, a series of studies on rats, monkeys and human foetus showed that an arc-shaped zone is present in the middle-caudal segment of medulla oblongata, running from the dorsomedi-al part to the ventrolateral part and passing through the reticular formation. It was named the medullary visceral zone (MVZ). The MVZ has been investigated with various techniques (Golgi method, Nissl method, immunohistochemical method, in situ hybridization method, triple labelling method, neuro-physiological method, etc.), and the morphological features as well as the physiological functions of MVZ have been preliminarily understood. It is proved that the medullary life center is located in MVZ. An introduction and some comments are given on the location of MVZ, its shape and extent, cytoarchi-tecture and chemicoarchitecture, afferent and efferent fiber connections and their functions, and its important physiological functions.