共查询到20条相似文献，搜索用时 578 毫秒
1.
Copic A Latham CF Horlbeck MA D'Arcangelo JG Miller EA 《Science (New York, N.Y.)》2012,335(6074):1359-1362
Eukaryotic secretory proteins exit the endoplasmic reticulum (ER) via transport vesicles generated by the essential coat protein complex II (COPII) proteins. The outer coat complex, Sec13-Sec31, forms a scaffold that is thought to enforce curvature. By exploiting yeast bypass-of-sec-thirteen (bst) mutants, where Sec13p is dispensable, we probed the relationship between a compromised COPII coat and the cellular context in which it could still function. Genetic and biochemical analyses suggested that Sec13p was required to generate vesicles from membranes that contained asymmetrically distributed cargoes that were likely to confer opposing curvature. Thus, Sec13p may rigidify the COPII cage and increase its membrane-bending capacity; this function could be bypassed when a bst mutation renders the membrane more deformable. 相似文献
2.
【目的】对小反刍兽疫病毒（Peste des petits ruminants virus,PPRV）H蛋白胞外区（tH）细胞膜受体进行鉴定,为PPRV致病机制的研究奠定基础。【方法】克隆PPRV H基因胞外区（tH）,在毕赤酵母(Pichia pastoris)中进行真核表达,纯化后免疫家兔,获得兔抗PPRVtH蛋白特异性抗体;提取山羊外周血淋巴细胞膜蛋白,经SDS-PAGE检测后,湿转印法转印至NC膜,分别利用纯化的重组tH蛋白和PPRV进行病毒铺覆蛋白结合试验（VOPBA）,对受体进行鉴定。【结果】成功克隆了1 653 bp的PPRV tH基因,构建其重组酵母表达质粒pPIC9K-tH,诱导表达后获得了60 ku目的蛋白。重组tH蛋白免疫家兔后获得了效价为1∶200的抗血清。Westernblotting分析发现,该重组蛋白可与PPRV多克隆抗体发生特异性反应,山羊外周血淋巴细胞膜蛋白上有2个与PPRV和重组tH蛋白结合的蛋白带,分子质量约为38和100 ku。【结论】从山羊外周血淋巴细胞膜蛋白上鉴定到了2种与PPRV结合的蛋白组分,其特性有待于进一步研究。 相似文献
3.
The endoplasmic reticulum (ER) supports disulfide bond formation by a poorly understood mechanism requiring protein disulfide isomerase (PDI) and ERO1. In yeast, Ero1p-mediated oxidative folding was shown to depend on cellular flavin adenine dinucleotide (FAD) levels but not on ubiquinone or heme, and Ero1p was shown to be a FAD-binding protein. We reconstituted efficient oxidative folding in vitro using FAD, PDI, and Ero1p. Disulfide formation proceeded by direct delivery of oxidizing equivalents from Ero1p to folding substrates via PDI. This kinetic shuttling of oxidizing equivalents could allow the ER to support rapid disulfide formation while maintaining the ability to reduce and rearrange incorrect disulfide bonds. 相似文献
4.
Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1 总被引：2，自引：0，他引：2
Urano F Wang X Bertolotti A Zhang Y Chung P Harding HP Ron D 《Science (New York, N.Y.)》2000,287(5453):664-666
Malfolded proteins in the endoplasmic reticulum (ER) induce cellular stress and activate c-Jun amino-terminal kinases (JNKs or SAPKs). Mammalian homologs of yeast IRE1, which activate chaperone genes in response to ER stress, also activated JNK, and IRE1alpha-/- fibroblasts were impaired in JNK activation by ER stress. The cytoplasmic part of IRE1 bound TRAF2, an adaptor protein that couples plasma membrane receptors to JNK activation. Dominant-negative TRAF2 inhibited activation of JNK by IRE1. Activation of JNK by endogenous signals initiated in the ER proceeds by a pathway similar to that initiated by cell surface receptors in response to extracellular signals. 相似文献
5.
Biosynthesis of the Torpedo californica acetylcholine receptor alpha subunit in yeast 总被引：4，自引：0，他引：4
N Fujita N Nelson T D Fox T Claudio J Lindstrom H Riezman G P Hess 《Science (New York, N.Y.)》1986,231(4743):1284-1287
Yeast cells were transformed with a plasmid containing complementary DNA encoding the alpha subunit of the Torpedo californica acetylcholine receptor. These cells synthesized a protein that had the expected molecular weight, antigenic specificity, and ligand-binding properties of the alpha subunit. The subunit was inserted into the yeast plasma membrane, demonstrating that yeast has the apparatus to express a membrane-bound receptor protein and to insert such a foreign protein into its plasma membrane. The alpha subunit constituted approximately 1 percent of the total yeast membrane. The alpha subunit constituted approximately 1 percent of the total yeast membrane proteins, and its density was about the same in the plasma membrane of yeast and in the receptor-rich electric organ of Electrophorus electricus. In view of the available technology for obtaining large quantities of yeast proteins, it may now be possible to obtain amplified amounts of interesting membrane-bound proteins for physical and biochemical studies. 相似文献
6.
The unfolded protein response (UPR) detects the accumulation of unfolded proteins in the endoplasmic reticulum (ER) and adjusts the protein-folding capacity to the needs of the cell. Under conditions of ER stress, the transmembrane protein Ire1 oligomerizes to activate its cytoplasmic kinase and ribonuclease domains. It is unclear what feature of ER stress Ire1 detects. We found that the core ER-lumenal domain (cLD) of yeast Ire1 binds to unfolded proteins in yeast cells and to peptides primarily composed of basic and hydrophobic residues in vitro. Mutation of amino acid side chains exposed in a putative peptide-binding groove of Ire1 cLD impaired peptide binding. Peptide binding caused Ire1 cLD oligomerization in vitro, suggesting that direct binding to unfolded proteins activates the UPR. 相似文献
7.
目的构建喉癌(Hep-2)细胞的酵母双杂交cDNA文库,为筛选包涵体膜蛋白分子的作用配体提供前期工作基础.方法从Hep-2细胞中提取总RNA,应用SMARTTM技术,构建以pGADT7-Rec为载体的Hep-2细胞酵母GAL4AD融合cDNA文库.结果所构建的文库展现出良好的多态性.结论可用于酵母双杂交系统的筛选. 相似文献
8.
Starting with purified, bacterially produced protein, we have created a [PSI(+)]-inducing agent based on an altered (prion) conformation of the yeast Sup35 protein. After converting Sup35p to its prion conformation in vitro, we introduced it into the cytoplasm of living yeast using a liposome transformation protocol. Introduction of substoichiometric quantities of converted Sup35p greatly increased the rate of appearance of the well-characterized epigenetic factor [PSI+], which results from self-propagating aggregates of cellular Sup35p. Thus, as predicted by the prion hypothesis, proteins can act as infectious agents by causing self-propagating conformational changes. 相似文献
9.
10.
ER tubules mark sites of mitochondrial division 总被引：1，自引：0，他引：1
Friedman JR Lackner LL West M DiBenedetto JR Nunnari J Voeltz GK 《Science (New York, N.Y.)》2011,334(6054):358-362
Mitochondrial structure and distribution are regulated by division and fusion events. Mitochondrial division is regulated by Dnm1/Drp1, a dynamin-related protein that forms helices around mitochondria to mediate fission. Little is known about what determines sites of mitochondrial fission within the mitochondrial network. The endoplasmic reticulum (ER) and mitochondria exhibit tightly coupled dynamics and have extensive contacts. We tested whether ER plays a role in mitochondrial division. We found that mitochondrial division occurred at positions where ER tubules contacted mitochondria and mediated constriction before Drp1 recruitment. Thus, ER tubules may play an active role in defining the position of mitochondrial division sites. 相似文献
11.
The Caenorhabditis elegans excretory canal is composed of a single elongated and branched cell that is tunneled by an inner lumen of apical character. Loss of the exc-4 gene causes a cystic enlargement of this intracellular tube. exc-4 encodes a member of the chloride intracellular channel (CLIC) family of proteins. EXC-4 protein localizes to various tubular membranes in distinct cell types, including the lumenal membrane of the excretory tubes. A conserved 55-amino acid domain enables EXC-4 translocation from the cytosol to the lumenal membrane. The tubular architecture of this membrane requires EXC-4 for both its formation and maintenance. 相似文献
12.
【目的】研究MbNramp1基因的功能。【方法】通过异源互补试验鉴定该基因转运铁的功能,并对MbNRAMP1蛋白进行亚细胞定位研究。【结果】MbNramp1基因转化酵母突变株DDY4在缺铁的培养基上恢复生长。在供试BPDS浓度的培养基上,MbNramp1基因转化酵母突变株DDY4生长状况均明显好于空载体转化后的突变株。培养基中BPDS增加到15μmol·L-1时,转化了MbNramp1的酵母细胞生长状况与野生型相差无几。在30μmol·L-1BPDS时,MbNramp1转化的细胞与低浓度BPDS相比生长延缓,但是明显优于空载体转化的DDY4突变株。与较低浓度BPDS相比,野生型酵母(DY1457)的生长量也减少了。亚细胞定位表明,MbNRAMP1主要位于部分质膜上而非整个膜上。【结论】初步证明MbNramp1编码具有功能的铁转运蛋白,能够使酵母吸收铁的突变株恢复突变,MbNRAMP1主要位于部分细胞膜上行使铁营养转运功能。 相似文献
13.
【目的】筛选与FWL1互作的蛋白,为研究FWL1基因调节果实大小机理提供依据。【方法】以杜梨、库尔勒香梨、鸭梨、早美香为材料,提取4个梨品种果实细胞分裂关键时期的果肉组织混样RNA。构建梨幼果FWL1膜系统酵母双杂交三框cDNA文库,并鉴定文库质量。构建诱饵载体并转化酵母菌,筛选与FWL1互作的蛋白,对初步筛选出的阳性酵母克隆进行测序并在NCBI中进行Blast比对,确定候选互作蛋白。【结果】文库库容约为3×107 CFU,大于1×107 CFU,平均插入片段大于1 000 bp,阳性率为≥98%。诱饵载体无自激活功能,利用共转化方法,筛选出272个与FWL1互作的蛋白质。【结论】梨幼果膜系统酵母双杂交三框cDNA文库符合酵母双杂文库的基本条件,适用于后期筛选相互作用的蛋白。筛选出的272个互作蛋白在果实发育过程中表达不同,其中collagen and calcium-binding EGF domain-containing protein 1和metallothionein-like protein可能与梨果实大小发育有关。 相似文献
14.
Isolation and characterization of a novel protein (X-ORF product) from SIV and HIV-2 总被引：27，自引：0，他引：27
L E Henderson R C Sowder T D Copeland R E Benveniste S Oroszlan 《Science (New York, N.Y.)》1988,241(4862):199-201
A protein designated p14 was purified from a simian immunodeficiency virus (SIVMne) and was shown by amino acid sequence analysis to be nearly identical to the predicted translational product of a unique open reading frame (X-ORF) in the nucleotide sequences of SIVmac and human immunodeficiency virus type 2 (HIV-2). Thus the X-ORF is proven to be a new retroviral gene. The p14 is present in SIVMne in molar amounts equivalent to those of the gag proteins. This is the first example of a retrovirus that contains a substantial quantity of a viral protein that is not a product of the gag, pro, pol, or env genes. SIV p14 and its homolog in HIV-2 may function as nucleic acid binding proteins since purified p14 binds to single-stranded nucleic acids in vitro. Antisera to the purified protein detected p14 in SIVMne, SIVmac, and a homologous protein (16 kilodaltons) in HIV-2 but did not react with HIV-1. Diagnostic procedures based on this novel protein will distinguish between HIV-1 and HIV-2. 相似文献
15.
Young L Leonhard K Tatsuta T Trowsdale J Langer T 《Science (New York, N.Y.)》2001,291(5511):2135-2138
ATP-binding cassette (ABC) adenosine triphosphatases actively transport a wide variety of compounds across biological membranes. Here, the ABC protein Mdl1 was identified as an intracellular peptide transporter localized in the inner membrane of yeast mitochondria. Mdl1 was required for mitochondrial export of peptides with molecular masses of approximately 2100 to 600 daltons generated by proteolysis of inner-membrane proteins by the m-AAA protease in the mitochondrial matrix. Proteolysis by the i-AAA protease in the intermembrane space led to the release of similar-sized peptides independent of Mdl1. Thus, two pathways of peptide efflux from mitochondria exist that may allow communication between mitochondria and their cellular environment. 相似文献
16.
目前在针对鱼类神经坏死病毒的疫苗研究中,主要是将神经坏死病毒某些蛋白作为抗原进行注射免疫,但是传统的注射免疫并不能有效地激发黏膜免疫。笔者将鱼类神经坏死病毒的衣壳蛋白（MCP）与鮰爱德华氏菌的跨粘膜蛋白ompN1融合表达,拟制备能够抵抗神经坏死病毒的粘膜疫苗;利用从NCBI GenBank库里获得的鱼类神经坏死病毒的外壳蛋白MCP和鮰爱德华氏菌的外膜蛋白ompN1的基因序列,将两者进行序列优化与全基因合成,分别构建原核表达载体:MCP-ompN1 pET28a和MCP pET28a和ompN1 pET28a,并在大肠杆菌内分别诱导表达融合蛋白MCP-ompN1,MCP,ompN1后,再利用包涵体纯化及透析复性获得MCP-ompN1,MCP,ompN1蛋白。SDS-PAGE结果显示,原核表达纯化得到了较纯的MCP-ompN1 融合蛋白,Western Blotting结果表明,纯化得到的MCP-ompN1 融合蛋白不仅具有MCP抗原性,还具有ompN1抗原性。本实验通过原核表达纯化得到了鱼类神经坏死病毒衣壳蛋白MCP和鮰爱德华氏菌外膜蛋白ompN1的融合蛋白MCP-ompN1,为进一步验证融合蛋白MCP-ompN1能否作为抵抗神经坏死病毒的粘膜疫苗奠定了基础。 相似文献
17.
拟南芥Ran2基因的原核表达及产物的纯化 总被引：4，自引：0，他引：4
采用RT-PCR方法扩增Ran2cDNA完整的编码区序列,构建pTYB2-Ran2重组质粒,转化大肠杆菌ER2566,经IPTG诱导蛋白表达后SDS-PAGE分析表达产物及存在形式。结果表明,表达的蛋白26ku与预期的理论值一致,并以包涵体形式存在;包涵体经纯化、破碎变性、复性及PBS透析,得到纯度较高的表达蛋白(Ran2)。 相似文献
18.
Two cytosolic neutrophil oxidase components absent in autosomal chronic granulomatous disease 总被引：31，自引：0，他引：31
Neutrophils kill microorganisms with oxygen radicals generated by an oxidase that uses the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) as substrate. This system requires both membrane and cytosolic components and is defective in patients with chronic granulomatous disease. A cytosolic complex capable of activating latent membrane oxidase was eluted from guanosine triphosphate-agarose and was used to raise polyclonal antiserum that recognized 47- and 67-kilodalton proteins. These proteins were restricted to the cytosol of myeloid cells. Both proteins were associated with NADPH oxidase-activating capacity when neutrophil cytosol was purified on nucleotide affinity matrices or molecular sizing columns. Neutrophils from patients with two different forms of autosomal chronic granulomatous disease lacked either the 47- or 67-kilodalton protein. 相似文献
19.
将获得的拟南芥黑芥子酶结合蛋白PBP1基因表达载体pTYB2-PBP1导入大肠杆菌ER2566细胞,经IPTG诱导,SDS-PAGE电泳显示,PBP1-CBD融合蛋白获得了较好的表达。进一步对PBP1蛋白进行分离纯化,SDS-PAGE电泳结果显示分离纯化效果较好,所获得的纯化的PBP1蛋白可进一步用于研究PBP1与MBP6的酵母双杂交技术。 相似文献
20.
[目的]对橡胶树不同品系橡胶粒子蛋白进行比较研究。[方法]以11个橡胶树品系为试材,采用不同离心速度分级分离方法,获得不同直径大小的纯化橡胶粒子,分别洗脱其膜蛋白并作电泳SDS-PAGE分析,并比较了高产品系和低产品系胶乳中的大橡胶粒子膜蛋白质谱。[结果]14.5、24.02、7.0、32.0、34.0和49.0 kD等蛋白条带的含量在不同直径大小的橡胶粒子上有明显变化,其中14.5 kD蛋白是橡胶延长因子,24.0 kD蛋白是小橡胶粒子蛋白。橡胶树各个品系具有各自独立的特征蛋白谱带,主要表现为在品系之间存在某些谱带表达量上的差异,其中27.0 kD橡胶粒子膜蛋白在耐乙稀利刺激的PR107中的丰度要显著高于不耐乙稀利刺激的海垦1号。[结论]橡胶粒子膜表面的一些蛋白条带可能跟橡胶粒子直径大小或发育相关。 相似文献