A distinct potassium channel polypeptide encoded by the Drosophila eag locus.

Many of the signaling properties of neurons and other electrically excitable cells are determined by a diverse family of potassium channels. A number of genes that encode potassium channel polypeptides have been cloned from various organisms on the basis of their sequence similarity to the Drosophila Shaker (Sh) locus. As an alternative strategy, a molecular analysis of other Drosophila genes that were defined by mutations that perturb potassium channel function was undertaken. Sequence analysis of complementary DNA from the ether à go-go (eag) locus revealed that it encodes a structural component of potassium channels that is related to but is distinct from all identified potassium channel polypeptides.     

Intracellular functions of N-linked glycans

N-linked oligosaccharides arise when blocks of 14 sugars are added cotranslationally to newly synthesized polypeptides in the endoplasmic reticulum (ER). These glycans are then subjected to extensive modification as the glycoproteins mature and move through the ER via the Golgi complex to their final destinations inside and outside the cell. In the ER and in the early secretory pathway, where the repertoire of oligosaccharide structures is still rather small, the glycans play a pivotal role in protein folding, oligomerization, quality control, sorting, and transport. They are used as universal "tags" that allow specific lectins and modifying enzymes to establish order among the diversity of maturing glycoproteins. In the Golgi complex, the glycans acquire more complex structures and a new set of functions. The division of synthesis and processing between the ER and the Golgi complex represents an evolutionary adaptation that allows efficient exploitation of the potential of oligosaccharides.     

X-ray structure of the EmrE multidrug transporter in complex with a substrate

EmrE is a prototype of the Small Multidrug Resistance family of efflux transporters and actively expels positively charged hydrophobic drugs across the inner membrane of Escherichia coli. Here, we report the x-ray crystal structure, at 3.7 angstrom resolution, of one conformational state of the EmrE transporter in complex with a translocation substrate, tetraphenylphosphonium. Two EmrE polypeptides form a homodimeric transporter that binds substrate at the dimerization interface. The two subunits have opposite orientations in the membrane and adopt slightly different folds, forming an asymmetric antiparallel dimer. This unusual architecture likely confers unidirectionality to transport by creating an asymmetric substrate translocation pathway. On the basis of available structural data, we propose a model for the proton-dependent drug efflux mechanism of EmrE.     

Golgin tethers define subpopulations of COPI vesicles

Coiled-coil proteins of the golgin family have been implicated in intra-Golgi transport through tethering coat protein complex I (COPI) vesicles. The p115-golgin tether is the best studied, and here we characterize the golgin-84-CASP tether. The vesicles bound by this tether were strikingly different from those bound by the p115-golgin tether in that they lacked members of the p24 family of putative cargo receptors and contained enzymes instead of anterograde cargo. Microinjected golgin-84 or CASP also inhibited Golgi-enzyme transport to the endoplasmic reticulum, further implicating this tether in retrograde transport. These and other golgins may modulate the flow patterns within the Golgi stack.     

Signal-mediated dynamic retention of glycosyltransferases in the Golgi

Golgi-resident glycosyltransferases are a family of enzymes that sequentially modify glycoproteins in a subcompartment-specific manner. These type II integral membrane proteins are characterized by a short cytoplasmically exposed amino-terminal tail and a luminal enzymatic domain. The cytoplasmic tails play a role in the localization of glycosyltransferases, and coat protein complex I (COPI) vesicle-mediated retrograde transport is also involved in their Golgi localization. However, the tails of these enzymes lack known COPI-binding motifs. Here, we found that Vps74p bound to a pentameric motif present in the cytoplasmic tails of the majority of yeast Golgi-localized glycosyltransferases, as well as to COPI. We propose that Vps74p maintains the steady-state localization of Golgi glycosyltransferases dynamically, by promoting their incorporation into COPI-coated vesicles.     

家蚕核酸糖转运蛋白BmSlc35b3基因的克隆与序列分析

在电子克隆的基础上,利用RT-PCR方法克隆了家蚕的同源基因BmSlc35b3(GenBank登录号:GU244352)。生物信息学分析结果显示,该基因编码区长1128bp,包含5个外显子,由基因推定的蛋白质由375个氨基酸组成,内有9个跨膜结构域,8个N-糖基化位点,与家蚕溶质运载蛋白家族B3(ABD36159)有4个氨基酸的差异,与赤拟谷道(EFA00972)、金小蜂(XP_001601459)、致倦库蚊(EDS30138)、疟蚊(EAA11308)等的同源性均在70%以上。RT-PCR对BmSlc35b3基因在五龄3d家蚕幼虫9种组织中的表达进行检测,结果表明,其在中肠和精巢中表达,在其他组织中不表达。     

Mitotic Golgi partitioning is driven by the membrane-fissioning protein CtBP3/BARS

Organelle inheritance is an essential feature of all eukaryotic cells. As with other organelles, the Golgi complex partitions between daughter cells through the fission of its membranes into numerous tubulovesicular fragments. We found that the protein CtBP3/BARS (BARS) was responsible for driving the fission of Golgi membranes during mitosis in vivo. Moreover, by in vitro analysis, we identified two stages of this Golgi fragmentation process: disassembly of the Golgi stacks into a tubular network, and BARS-dependent fission of these tubules. Finally, this BARS-induced fission of Golgi membranes controlled the G2-to-prophase transition of the cell cycle, and hence cell division.     

胆固醇平衡调控中的SREBP途径

胆固醇在生物体内发挥着极其重要的作用。细胞主要通过调节胆固醇的合成、吸收、酯化及外流等途径之间的平衡以维持正常的胆固醇浓度。本文介绍甾醇调控元件结合蛋白(SREBP,Sterolregulatoryelement-bindingproteins)转录因子对胆固醇平衡的调控。SREBP主要调节胆固醇的合成、LDL受体介导的胆固醇的吸收和脂肪酸的合成。SREBP前体是一内质网膜蛋白,SREBP经两次蛋白酶水解后释放出其N端结构域,进入细胞核,激活靶基因的转录。随着对SREBP领域研究的深入,人们对胆固醇平衡调节将认识更多,并有可能开发出更为有效的控制胆固醇的药物。     

Leucine-tRNA initiates at CUG start codons for protein synthesis and presentation by MHC class I

Effective immune surveillance by cytotoxic T cells requires newly synthesized polypeptides for presentation by major histocompatibility complex (MHC) class I molecules. These polypeptides are produced not only from conventional AUG-initiated, but also from cryptic non-AUG-initiated, reading frames by distinct translational mechanisms. Biochemical analysis of ribosomal initiation complexes at CUG versus AUG initiation codons revealed that cells use an elongator leucine-bound transfer RNA (Leu-tRNA) to initiate translation at cryptic CUG start codons. CUG/Leu-tRNA initiation was independent of the canonical initiator tRNA (AUG/Met-tRNA(i)(Met)) pathway but required expression of eukaryotic initiation factor 2A. Thus, a tRNA-based translation initiation mechanism allows non-AUG-initiated protein synthesis and supplies peptides for presentation by MHC class I molecules.     

Receptor-mediated transport of insulin across endothelial cells

Hormones such as insulin are transported from the interior to the exterior of blood vessels. Whether endothelial cells, which line the inner walls of blood vessels have a role in this transport of hormones is not clear, but it is known that endothelial cells can internalize and release insulin rapidly with little degradation. The transport of iodine-125-labeled insulin was measured directly through the use of dual chambers separated by a horizontal monolayer of cultured bovine aortic endothelial cells. In this setting, endothelial cells took up and released the labeled insulin, thereby transporting it across the cells. The transport of insulin across the endothelial cells was temperature sensitive and was inhibited by unlabeled insulin and by antibody to insulin receptor in proportion to the ability of these substances to inhibit insulin binding to its receptor. More than 80 percent of the transported insulin was intact. These data suggest that insulin is rapidly transported across endothelial cells by a receptor-mediated process.     

Interaction of Hsp 70 with newly synthesized proteins: implications for protein folding and assembly

The 70-kilodalton family of heat shock proteins (Hsp 70) has been implicated in posttranslational protein assembly and translocation. Binding of cytosolic forms of Hsp 70 (Hsp 72,73) with nascent proteins in the normal cell was investigated and found to be transient and adenosine triphosphate (ATP)-dependent. Interaction of Hsp 72,73 with newly synthesized proteins appeared to occur cotranslationally, because nascent polypeptides released prematurely from polysomes in vivo can be isolated in a complex with Hsp 72,73. Moreover, isolation of polysomes from short-term [35S]Met-labeled cells (pulsed) revealed that Hsp 72,73 associated with nascent polypeptide chains. In cells experiencing stress, newly synthesized proteins coimmunoprecipitated with Hsp 72,73; however, in contrast to normal cells, interaction with Hsp 72,73 was not transient. A model consistent with these data suggests that under normal growth conditions, cytosolic Hsp 72,73 interact transiently with nascent polypeptides to facilitate proper folding, and that metabolic stress interferes with these events.     

Recombinant antibodies to the small GTPase Rab6 as conformation sensors

Here we report an approach, based on antibody phage display, to generate molecular conformation sensors. Recombinant antibodies specific to the guanosine triphosphate (GTP)-bound conformation of the small guanosine triphosphatase (GTPase) Rab6, a regulator of membrane traffic, were generated and used to locate Rab6.GTP in fixed cells, and, after green fluorescent protein (GFP) tagging and intracellular expression, to follow Rab6.GTP in vivo. Rab6 was in its GTP-bound conformation on the Golgi apparatus and transport intermediates, and the geometry of transport intermediates was modulated by Rab6 activity. More generally, the same approach could be applied to other molecules that can be locked in a particular conformation in vitro.     

Transmembrane molecular pump activity of Niemann-Pick C1 protein

Niemann-Pick C1 (NPC1) disease is characterized by cholesterol accumulation in lysosomes and aberrant feedback regulation of cellular cholesterol homeostasis. We provide evidence that the NPC1 protein has homology with the resistance-nodulation-division (RND) family of prokaryotic permeases and may normally function as a transmembrane efflux pump. Studies of acriflavine loading in normal and NPC1 fibroblasts indicated that NPC1 uses a proton motive force to remove accumulated acriflavine from the endosomal/lysosomal system. Expression of NPC1 in Escherichia coli (i) facilitated the transport of acriflavine across the plasma membrane, causing cytosolic accumulation, and (ii) resulted in transport of oleic acid but not cholesterol or cholesterol-oleate across the plasma membrane. These studies establish NPC1 as a eukaryotic member of the RND permease family.     

Golgi duplication in Trypanosoma brucei requires Centrin2

Centrins are highly conserved components of the centrosome, which in the parasitic protozoan T. brucei comprises the basal body and nucleates the flagellum used for locomotion. Here, we found TbCentrin2 in an additional bi-lobed structure near to the Golgi apparatus. One lobe was associated with the old Golgi, and the other became associated with the newly forming Golgi as the cell grew. Depletion of TbCentrin1 inhibited duplication of the basal body, whereas depletion of TbCentrin2 also inhibited duplication of the Golgi. Thus, a Centrin2-containing structure distinct from the basal body appears to mark the site for new Golgi assembly.     

Synapses onto different morphological types of retinal ganglion cells

The percentage of bipolar and amacrine synapses onto ganglion cell dendrites of the ground squirrel has been determined by electron microscopy of cells impregnated by the Golgi method. One group of ganglion cells has mainly amacrine input (approximately 97 percent); the other group has an approximately equal bipolar and amacrine input. Morphologically distinct types of ganglion cells usually have a consistent synaptic input, but exceptions may exist.     

Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmunity

Apoptosis can be triggered by members of the Bcl-2 protein family, such as Bim, that share only the BH3 domain with this family. Gene targeting in mice revealed important physiological roles for Bim. Lymphoid and myeloid cells accumulated, T cell development was perturbed, and most older mice accumulated plasma cells and succumbed to autoimmune kidney disease. Lymphocytes were refractory to apoptotic stimuli such as cytokine deprivation, calcium ion flux, and microtubule perturbation but not to others. Thus, Bim is required for hematopoietic homeostasis and as a barrier to autoimmunity. Moreover, particular death stimuli appear to activate apoptosis through distinct BH3-only proteins.     

Role of Rab9 GTPase in facilitating receptor recruitment by TIP47

Mannose 6-phosphate receptors (MPRs) deliver lysosomal hydrolases from the Golgi to endosomes and then return to the Golgi complex. TIP47 recognizes the cytoplasmic domains of MPRs and is required for endosome-to-Golgi transport. Here we show that TIP47 also bound directly to the Rab9 guanosine triphosphatase (GTPase) in its active, GTP-bound conformation. Moreover, Rab9 increased the affinity of TIP47 for its cargo. A functional Rab9 binding site was required for TIP47 stimulation of MPR transport in vivo. Thus, a cytosolic cargo selection device may be selectively recruited onto a specific organelle, and vesicle budding might be coupled to the presence of an active Rab GTPase.     

A vital stain for the Golgi apparatus

The Golgi complex, a membranous organelle with important functions in membrane traffic and macromolecular synthesis, has been stained in living cells with a fluorescent sphingolipid. Cells were first incubated with liposomes containing N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproyl sphingosine (C6-NBD-ceramide), or with a bovine serum albumin complex of the fluorescent lipid, and then examined by fluorescence microscopy. An intensely fluorescent perinuclear structure was identified as the Golgi apparatus by its colocalization with known Golgi markers in fixed cells. C6-NBD-ceramide was used to observe the morphology of the Golgi apparatus in living cells in the presence or absence of monensin or Colcemid, and during mitosis. In all cases, C6-NBD-ceramide revealed a Golgi apparatus in the living cell that was identical to that obtained with conventional procedures that require fixation.     

Partitioning of the matrix fraction of the Golgi apparatus during mitosis in animal cells

The Golgi apparatus is partitioned during mitosis in animal cells by a process of fragmentation, dispersal, and reassembly in each daughter cell. We fractionated the Golgi apparatus in vivo using the drug brefeldin A or a dominant-negative mutant of the Sar1p protein. After these treatments, Golgi enzymes moved back to the endoplasmic reticulum, leaving behind a matrix of Golgi structural proteins. Under these conditions, cells still entered and exited mitosis normally, and their Golgi matrix partitioned in a manner very similar to that of the complete organelle. Thus, the matrix may be the partitioning unit of the Golgi apparatus and may carry the Golgi enzyme-containing membranes into the daughter cells.