Disulphide-isomerase-enabled shedding of tumour-associated NKG2D ligands

Tumour-associated ligands of the activating NKG2D (natural killer group 2, member D; also called KLRK1) receptor-which are induced by genotoxic or cellular stress-trigger activation of natural killer cells and co-stimulation of effector T cells, and may thus promote resistance to cancer. However, many progressing tumours in humans counter this anti-tumour activity by shedding the soluble major histocompatibility complex class-I-related ligand MICA, which induces internalization and degradation of NKG2D and stimulates population expansions of normally rare NKG2D+CD4+ T cells with negative regulatory functions. Here we show that on the surface of tumour cells, MICA associates with endoplasmic reticulum protein 5 (ERp5; also called PDIA6 or P5), which, similar to protein disulphide isomerase, usually assists in the folding of nascent proteins inside cells. Pharmacological inhibition of thioreductase activity and ERp5 gene silencing revealed that cell-surface ERp5 function is required for MICA shedding. ERp5 and membrane-anchored MICA form transitory mixed disulphide complexes from which soluble MICA is released after proteolytic cleavage near the cell membrane. Reduction of the seemingly inaccessible disulphide bond in the membrane-proximal alpha3 domain of MICA must involve a large conformational change that enables proteolytic cleavage. These results uncover a molecular mechanism whereby domain-specific deconstruction regulates MICA protein shedding, thereby promoting tumour immune evasion, and identify surface ERp5 as a strategic target for therapeutic intervention.     

Structure of the Sec23/24-Sar1 pre-budding complex of the COPII vesicle coat

COPII-coated vesicles form on the endoplasmic reticulum by the stepwise recruitment of three cytosolic components: Sar1-GTP to initiate coat formation, Sec23/24 heterodimer to select SNARE and cargo molecules, and Sec13/31 to induce coat polymerization and membrane deformation. Crystallographic analysis of the Saccharomyces cerevisiae Sec23/24-Sar1 complex reveals a bow-tie-shaped structure, 15 nm long, with a membrane-proximal surface that is concave and positively charged to conform to the size and acidic-phospholipid composition of the COPII vesicle. Sec23 and Sar1 form a continuous surface stabilized by a non-hydrolysable GTP analogue, and Sar1 has rearranged from the GDP conformation to expose amino-terminal residues that will probably embed in the bilayer. The GTPase-activating protein (GAP) activity of Sec23 involves an arginine side chain inserted into the Sar1 active site. These observations establish the structural basis for GTP-dependent recruitment of a vesicular coat complex, and for uncoating through coat-controlled GTP hydrolysis.     

Glycoproteins form mixed disulphides with oxidoreductases during folding in living cells

The formation of intra- and interchain disulphide bonds constitutes an integral part of the maturation of most secretory and membrane-bound proteins in the endoplasmic reticulum. Evidence indicates that members of the protein disulphide isomerase (PDI) superfamily are part of the machinery needed for proper oxidation and isomerization of disulphide bonds. Models based on in vitro studies predict that the formation of mixed disulphide bonds between oxidoreductase and substrate is intermediate in the generation of the native intrachain disulphide bond in the substrate polypeptide. Whether this is how thiol oxidoreductases work inside the endoplasmic reticulum is not clear. Nor has it been established which of the many members of the PDI superfamily interacts directly with newly synthesized substrate proteins, because transient mixed disulphides have never been observed in the mammalian endoplasmic reticulum during oxidative protein folding. Here we describe the mechanisms involved in co- and post-translational protein oxidation in vivo. We show that the endoplasmic-reticulum-resident oxidoreductases PDI and ERp57 are directly involved in disulphide oxidation and isomerization, and, together with the lectins calnexin and calreticulin, are central in glycoprotein folding in the endoplasmic reticulum of mammalian cells.     

Identification of a ribosome receptor in the rough endoplasmic reticulum

Attachment of ribosomes to the membrane of the endoplasmic reticulum is one of the crucial first steps in the transport and secretion of intracellular proteins in mammalian cells. The process is mediated by an integral membrane protein of relative molecular mass 180,000 (Mr 180K), having a large (at least 160K) cytosolic domain that, when proteolytically detached from the membrane, can competitively inhibit the binding of ribosomes to intact membranes. Isolation of this domain has led to the identification, purification and characterization of the intact ribosome receptor, as well as its functional reconstitution into lipid vesicles.     

Reconstitution of a phospholipid flippase from rat liver microsomes

The endoplasmic reticulum is the principal site of synthesis and initial incorporation of membrane lipids in eukaryotic cells; the enzymes of glycerolipid biosynthesis are exclusively located on its cytoplasmic surface. To maintain a phospholipid bilayer in this organelle, newly synthesized phospholipids must be translocated to the lumenal surface. Consistent with this are measurements indicating that movement of phospholipids across microsomal membranes is rapid, with a half-time less than 5 min (refs 3 and 4). Rapid movement of phospholipids has also been detected across the plasma membrane of Bacillus megaterium, another site of de novo lipid biosynthesis. The rapid transmembrane movement of phosphatidylcholine has not been detected, however, in vesicles prepared from microsomal lipids. These latter data suggest involvement in the endoplasmic reticulum of a phospholipid-translocating protein, as was first proposed by Bretscher who called it 'flippase'. Here we report reconstitution of a phospholipid flippase from rat liver microsomes into lipid vesicles.     

家蚕后部丝腺细胞粗面内质网膜系统的多态性研究

应用电镜技术,对家蚕后部丝腺细胞粗面内质网膜系统在5龄生长期的超微形态变化,进行了连续观察与研究.结果发现,粗面内质网膜系统的超微形态呈现出扁平囊状、管泡状和同心圆状等规律性变化,这种多态性现象与丝腺细胞的生长发育状态及生理机能的变化密切相关,并受蚕体内分泌环境的调节.     

甘薯珠心细胞衰退过程的超微结构研究

阐述了甘薯珠心细胞衰退过程中的超微结构变化。发育成熟的珠心细胞，随着胚囊的发育和扩展以不同的方式衰退。在细胞质原位自溶方式，细胞质中的核糖核蛋白体密度迅速下降；细胞中可见许多被溶解的细胞组分的碎片；细胞核的染色质团块逐渐减少，核膜消失。最后在细胞中仅剩下细胞核和淀粉粒残体。在内质网对细胞质的吞噬和分隔方式中，同心环状和平行叠置的粗糙内质网大量增生，同时槽库膨大，并对细胞质组分进行分隔和吞噬；内质网膜膨大、断裂、分解，核糖体仍然存在，原生质体对电子染料的亲和性增强；接着质膜、液泡膜消失，原生质变为电子致密的物质。     

Action of brefeldin A blocked by activation of a pertussis-toxin-sensitive G protein.

In many mammalian cells brefeldin A interferes with mechanisms that keep the Golgi appartus separate from the endoplasmic reticulum. The earliest effect of brefeldin A is release of the coat protein beta-COP from the Golgi. This release is blocked by pretreatment with GTP-gamma S or AlF4- (ref. 12). The AlF4- ion activates heterotrimeric G proteins but not proteins of the ras superfamily, suggesting that a heterotrimeric G protein might control membrane transfer from the endoplasmic reticulum to the Golgi. We report here that mastoparan, a peptide that activates heterotrimeric G proteins, promotes binding of beta-COP to Golgi membranes in vitro and antagonizes the effect of brefeldin A on beta-COP in perforated cells and on isolated Golgi membranes. This inhibition is greatly diminished if cells are pretreated with pertussis toxin before perforation. Thus, a heterotrimeric G protein of the Gi/Go subfamily regulates association of coat components with Golgi membranes.     

Inositol 1,4,5-trisphosphate receptor localized to endoplasmic reticulum in cerebellar Purkinje neurons

Inositol 1,4,5-trisphosphate (InsP3) mediates the effects of several neurotransmitters, hormones and growth factors by mobilizing Ca2+ from a vesicular, non-mitochondrial intracellular store. Many studies have indirectly suggested the endoplasmic reticulum (ER) to be the site of InsP3 action, though some have implicated the plasma membrane or a newly described smooth surfaced structure, termed the calciosome. Using antibodies directed against a purified InsP3-receptor glycoprotein, of relative molecular mass 260,000, in electron microscope immunocytochemical studies of rat cerebellar Purkinje cells, we have now localized the InsP3 receptor to ER, including portions of the rough endoplasmic reticulum, a population of smooth-membrane-bound organelles (smooth ER), a portion of subplasmalemmal cisternae and the nuclear membrane, but not to mitochondria or the cell membrane. These results suggest that in cerebellar Purkinje cells, InsP3-induced intracellular calcium release is not the property of a single organelle, but is effected by specialized portions of both rough and smooth ER, and possibly by other smooth surfaced structures. The present findings are the first immunocytochemical demonstration of an InsP3 receptor within a cell.     

滞育和非滞育棉铃虫脑的组织解剖学研究

滞育和非滞育棉铃虫在不同的发育阶段，脑的形态组织学结构存在着一定的差异，主要表现在：非滞育脑的神经纤维体发达，神经分泌细胞中的核较大，且细胞质中除了有线粒体外，还有大量的粗面内质网及游离的核糖体；而滞育脑的神经纤维体相对固缩、不发达，神经分泌细胞中核较小，细胞质中含有线粒体、光滑内质网和特有的脂滴，不具粗面内质网．这些证据表明滞育棉铃虫的脑活性相对较低．     

Functional genomics reveals genes involved in protein secretion and Golgi organization

Yeast genetics and in vitro biochemical analysis have identified numerous genes involved in protein secretion. As compared with yeast, however, the metazoan secretory pathway is more complex and many mechanisms that regulate organization of the Golgi apparatus remain poorly characterized. We performed a genome-wide RNA-mediated interference screen in a Drosophila cell line to identify genes required for constitutive protein secretion. We then classified the genes on the basis of the effect of their depletion on organization of the Golgi membranes. Here we show that depletion of class A genes redistributes Golgi membranes into the endoplasmic reticulum, depletion of class B genes leads to Golgi fragmentation, depletion of class C genes leads to aggregation of Golgi membranes, and depletion of class D genes causes no obvious change. Of the 20 new gene products characterized so far, several localize to the Golgi membranes and the endoplasmic reticulum.     

Model for signal sequence recognition from amino-acid sequence of 54K subunit of signal recognition particle

Protein targeting to the endoplasmic reticulum in mammalian cells is catalysed by signal recognition particle (SRP). Cross-linking experiments have shown that the subunit of relative molecular mass 54,000 (Mr 54K; SRP54) interacts directly with signal sequences as they emerge from the ribosome. Here we present the sequence of a complementary DNA clone of SRP54 which predicts a protein that contains a putative GTP-binding domain and an unusually methionine-rich domain. The properties of this latter domain suggest that it contains the signal sequence binding site. A previously uncharacterized Escherichia coli protein has strong homology to both domains. Closely homologous GTP-binding domains are also found in the alpha-subunit of the SRP receptor (SR alpha, docking protein) in the endoplasmic reticulum membrane and in a second E. coli protein, ftsY, which resembles SR alpha. Recent work has shown that SR alpha is a GTP-binding protein and that GTP is required for the release of SRP from the signal sequence and the ribosome on targeting to the endoplasmic reticulum membrane. We propose that SRP54 and SR alpha use GTP in sequential steps of the targeting reaction and that essential features of such a pathway are conserved from bacteria to mammals.     

Dissection of COPI and Arf1 dynamics in vivo and role in Golgi membrane transport

Cytosolic coat proteins that bind reversibly to membranes have a central function in membrane transport within the secretory pathway. One well-studied example is COPI or coatomer, a heptameric protein complex that is recruited to membranes by the GTP-binding protein Arf1. Assembly into an electron-dense coat then helps in budding off membrane to be transported between the endoplasmic reticulum (ER) and Golgi apparatus. Here we propose and corroborate a simple model for coatomer and Arf1 activity based on results analysing the distribution and lifetime of fluorescently labelled coatomer and Arf1 on Golgi membranes of living cells. We find that activated Arf1 brings coatomer to membranes. However, once associated with membranes, Arf1 and coatomer have different residence times: coatomer remains on membranes after Arf1-GTP has been hydrolysed and dissociated. Rapid membrane binding and dissociation of coatomer and Arf1 occur stochastically, even without vesicle budding. We propose that this continuous activity of coatomer and Arf1 generates kinetically stable membrane domains that are connected to the formation of COPI-containing transport intermediates. This role for Arf1/coatomer might provide a model for investigating the behaviour of other coat protein systems within cells.     

Xenopus oocytes can secrete bacterial beta-lactamase

Most secretory proteins are synthesized as precursor polypeptides carrying N-terminal, hydrophobic sequences which, by means of a signal recognition particle (SRP), trigger the membrane transfer of the polypeptide and are subsequently cleaved off. The signal sequences appear to be interchangeable between prokaryotes and eukaryotes. In bacteria, secretion only involves the crossing of a membrane, whereas in eukaryotes the secretory process can be separated into two distinct phases: translocation across the membrane of the rough endoplasmic reticulum and subsequent intraluminal transport by processes involving vesicle budding and fusion. Since secretory proteins must be distinguished from other soluble proteins destined for various sites in the reticular system, it is conceivable that eukaryotic secretory proteins possess additional markers distinct from the signal peptide to guide the polypeptide after its transfer through the membrane. Proteins are secreted at different rates from a eukaryotic cell, suggesting a role in intracellular transport for receptors with differing affinities for some topogenic features in secretory proteins. We have tested this possibility by introducing into the lumen of eukaryotic rough endoplasmic reticulum a prokaryotic protein which, by virtue of its origin, had not been adapted to the eukaryotic secretory pathway. We reasoned that secretion of the bacterial protein would indicate that after membrane transfer no topogenic signal(s) and corresponding recognition system(s) are required. We report here that this is indeed the case.     

Putative receptor for inositol 1,4,5-trisphosphate similar to ryanodine receptor

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) serves as an intracellular second messenger for several neurotransmitters, hormones and growth factors by initiating calcium release from intracellular stores. A cerebellar Ins(1,4,5)P3 receptor has been characterized biochemically and shown by immunocytochemistry to be present in intracellular membranes in Purkinje cells. We show that a previously described Purkinje-cell messenger RNA encodes a protein of relative molecular mass 260,000 (260 K) with the same properties as the cerebellar Ins(1,4,5)P3 receptor. Its sequence is partially homologous to the skeletal muscle ryanodine receptor. By immunocytochemistry and electron microscopy the protein is shown to be present in all parts of the endoplasmic reticulum, including those that extend into axon terminals and dendritic spines. Our results indicate that gated calcium release from intracellular stores in muscle and Purkinje cells uses similar calcium-channel proteins localized in analogous intracellular compartments. This implies that the intracellular calcium stores in the endoplasmic reticulum of neurons extend into presynaptic terminals and dendritic spines where they may play a direct role in regulating the efficacy of neurotransmission.     

Signal-sequence recognition by an Escherichia coli ribonucleoprotein complex.

Hydrophobic signal-sequences direct the transfer of secretory proteins across the inner membrane of prokaryotes and the endoplasmic reticulum membranes of eukaryotes. In mammalian cells, signal-sequences are recognized by the 54K protein (M(r) 54,000) of the signal recognition particle (SRP) which is believed to hold the nascent chain in a translocation-competent conformation until it contacts the endoplasmic reticulum membrane. The SRP consists of a 7S RNA and six different polypeptides. The 7S RNA and the 54K signal-sequence-binding protein (SRP54) of mammalian SRP exhibit strong sequence similarity to the 4.5S RNA and P48 protein (Ffh) of Escherichia coli which form a ribonucleoprotein particle. Depletion of 4.5S RNA or overproduction of P48 causes the accumulation of the beta-lactamase precursor, although not of other secretory proteins. Whether 4.5S RNA and P48 are part of an SRP-like complex with a role in protein export is controversial. Here we show that the P48/4.5S RNA ribonucleoprotein complex interacts specifically with the signal sequence of a nascent secretory protein and therefore is a signal recognition particle.     

Evidence for the involvement of ATP in co-translational protein translocation

Identification of the source of energy for protein translocation across biological membranes is important in understanding the mechanism of this process. In eukaryotic cells, the tight coupling between translation and translocation and firm attachment of the secreting ribosomes to membranes, as well as theoretical calculations, have led to the suggestion that energy derived from protein synthesis is sufficient for protein translocation. On the other hand, in bacterial systems neither the attachment of ribosomes to membrane (other than nascent chains) nor tight coupling of translocation to translocation has been observed. Moreover, certain proteins can be translocated across membranes either at the time of, or after, translation. The separation of protein translocation from translation has made possible the demonstration that ATP hydrolysis is essential for post-translational protein translocation across bacterial membranes and, more recently, also across canine and yeast endoplasmic reticulum membranes. Here we report that certain ATP analogues inhibit co-translational protein translocation at concentrations that do not interfere with protein synthesis, suggesting that ATP is also required for co-translational protein translocation.     

Topology of signal recognition particle receptor in endoplasmic reticulum membrane

The signal recognition particle (SRP) receptor is an integral membrane protein of the endoplasmic reticulum which, in conjunction with SRP, ensures the correct targeting of nascent secretory proteins to this membrane system. From the complementary DNA sequence we have deduced the complete primary structure of the SRP receptor and established that its amino-terminal region is anchored in the membrane. The anchor fragment and the cytoplasmic fragment contribute jointly to a functionally important region which is highly charged and may function in nucleic acid binding.     

团头鲂脑垂体GTH细胞钙调素免疫电镜定位

本文运用免疫胶体金电镜定位技术对团头鲂(Megalobrama Ambly cephala)(?)垂体(?)性腺激素细胞进行了钙素(CaM)的超微结构定位。钙调素分别标记在与激素蛋白合成及分泌有关的细胞核、粗面内质网和分泌颗粒中,表明 CaM 参与激素的合成和转运作用。此外,促性腺激素分泌细胞中的大颗粒和小颗粒上均有 CaM,说明这两种颗粒具有共性,均参与激素的分泌活动,这一结果进一步弄清了大、小颗粒的性质,为鱼类的生殖生理及人工催产机理的研究提供了有价值的资料。