EoRab43参与游仆虫细胞内大核周围的物质运输

Rab家族蛋白是真核细胞内膜泡运输途径中重要的调节因子。EoRab43是八肋游仆虫中一种编码非典型Rab蛋白的基因。本研究依据已获得的EoRab43基因序列设计引物．从八肋游仆虫大核DNA中扩增了EoRab43基因的3’端153bp片段,即EoRab43 153bp（对应于EoRab43蛋白的C末端50个氨基酸,EoRab43C）,构建重组表达质粒pGEX—EoRab43,53bp转化大肠杆菌BL21（DE3）进行表达．纯化后的融合蛋白GST—EoRab43C免疫BALB／c小鼠制备多克隆抗体。经检测,制备的抗体具有较高的效价及良好的特异性。利用制备的抗体对EoRab43在游仆虫细胞内进行免疫荧光定位．结果显示该蛋白主要定位于该生物细胞内大核染色体的周围。     

游仆虫EoRab2a蛋白的表达、纯化及定位分析

Rab GTPase家族蛋白是真核细胞内膜系统转运途径中重要的调控因子,不同的Rab家族成员在细胞具有功能多样性。为了解Rab2的功能,八肋游仆虫EoRab2a基因连接入原核表达质粒pGEX-6P-1中,获得重组表达质粒pGEX-6P-1-EoRab2a。质粒pGEX-6P-1-EoRab2a转化大肠杆菌BL21(DE3),经IPTG诱导,大肠杆菌BL21(DE3)/pGEX-6P-1-EoRab2a高效表达了可溶性GST-EoRab2a蛋白。融合蛋白GST-EoRab2a经亲和层析获得电泳纯蛋白。纯化后的GST-EoRab2a免疫BALB/c小鼠制备多克隆抗体。ELISA和Western blotting检测显示制备的抗体效价1∶25600,特异性良好。免疫荧光定位表明EoRab2a在游仆虫细胞质中点状分布,推测参与内质网与高尔基体间膜泡转运。       

EoRab43为八肋游仆虫中编码非典型Rab的基因

Rab蛋白是与真核细胞内的膜泡运输密切相关的调节分子。本研究运用简并引物PCR技术从原生动物八肋游仆虫大核基因组中克隆获得了一个全新的Rab基因,EoRab43 (GenBank登陆号为EU365391) ,该基因拟编码蛋白的氨基酸序列基本包括Rab蛋白保守的GTP结合区以及RabF模序。Blast结果显示,EoRab43序列与其它生物中Rab5A、Rab6和Rab13的一致性相对较高,但也仅为36·4 %-38·5 %,无法将其归类于任何现有的Rab蛋白亚家族。序列分析显示该基因拟编码的蛋白质属于非典型Rab,这是首次在游仆虫中发现的编码非典型Rab蛋白的基因,推测其在原生动物八肋游仆虫细胞内可能执行某些特殊的生理功能。     

EoRab11a在游仆虫及HEK293T细胞中的定位

Rab11是一种在真核生物细胞生命活动过程中发挥多种调控作用的小分子GTP酶.EoRab11a是八肋游仆虫中的Rab11蛋白同源物,为了解EoRab11a蛋白在细胞中的功能,本研究将EoRab11a基因克隆到哺乳动物表达载体pEGFP-C2中,构建重组表达质粒pEGFP-C2-EoRab11a,转染HEK293T细胞并观察其细胞定位.在间期HEK293T细胞中,EoRab11a定位于细胞核附近;在游仆虫细胞中,EoRab11a具有相似的分布模式.在HEK293T细胞的胞质分裂过程中,EoRab11a在分裂沟附近、分裂沟收缩区、以及最后形成的中间体处分布,提示EoRab11a可能参与了胞质分离过程中分裂沟及中间体处的膜泡运输事件.     

游仆虫Rab蛋白基因的克隆表达与纯化

为对单细胞原生动物纤毛虫中Rab蛋白的功能进行研究 ,进而探讨以胞吞和胞吐为主要物质交换途径的纤毛虫中囊泡定向运输的机理 .利用PCR技术从游仆虫大核DNA及cDNA中扩增出rab基因 ,并进行了序列分析 ,该基因全长为 783bp ,两端为端粒序列 ,编码框为 6 2 4bp ,编码 2 0 7个氨基酸 ,开放读框中有 3个TGA ,在此编码半胱氨酸 .利用定点突变将rab基因中 3个TGA突变为通用半胱氨酸密码子TGC .将游仆虫Rab蛋白基因构建于原核表达载体pGEX 4T 2中 ,得到的重组质粒pGEX Eorab1转化至大肠杆菌BL2 1(DE3)中 ,IPTG诱导表达 .表达产物与抗GST抗体在 4 9kD处有很强的交叉反应 .融合蛋白GST EoRab1通过亲和层析柱纯化和凝血酶的切割 ,再经两步纯化得到电泳纯的游仆虫Rab蛋白 .     

游仆虫Rab家族成员Eorab1f基因的克隆、表达及多克隆抗体制备

Rab家族蛋白在真核细胞囊泡转运过程中起关键作用。为进一步研究该家族成员的功能, 本研究从八肋游仆虫(Euplotes octocarinatus) 大核基因组中克隆得到Rab蛋白家族中一个新Rab蛋白基因(命名为Eorab1f)的编码区。该开放读框长624 bp, 含有两个通用终止密码子TGA。通过定点突变将TGA突变为TGC。突变后的Eorab1f克隆入原核表达载体pRSETc 中, 工程菌E coli BL21 (DE3) /pRSETc Eorab1f 经IPTG诱导表达,SDS- PAGE分析表明, 有一分子量约为26 kD的特异蛋白条带出现。表达产物经IMAC金属螯合亲和层析及Re source- Q阴离子交换层析纯化, 获得电泳纯的蛋白。Brandford法检测表明每升发酵液中可获得纯化的目的蛋白1 353 mg。Western blotting印迹分析表明该蛋白为融合有6个His的Eorab1f蛋白。纯化的Eorab1f融合蛋白免疫大鼠制备多克隆抗体, ELISA法测得抗体效价为1∶5 000。用制备的多克隆抗体检测八肋游仆虫的蛋白提取物,表明Eorab1f蛋白在游仆虫细胞内表达, 同时表明所得的多克隆抗体特异性良好。     

八肋游仆虫Rab家族新成员Eo-rab-1N基因的克隆与序列分析

Rab蛋白家族属于小分子GTP结合蛋白家族Ras超家族中最大的亚家族,主要在囊泡运输中起作用。本实验运用PCR、RT-PCR等技术,从八肋游仆虫中克隆到一种新的rab基因。序列分析结果表明：在大核中,该基因全长884bp,除去两端的端粒与非编码区,该基因在大核中由723bp组成。从小核中克隆相应的基因片段,此基因片段序列与大核中序列一致,表明该基因在小核中无内部删除序列的存在。通过RT-PCR,从mRNA获得的该基因的开放读框为663bp,表明该基因在转录过程中有内含子的删除。大核基因序列和cDNA序列比较,发现60bp的内含子序列位于大核基因的153~212bp之间,并符合一类内含子GU-AG剪切规则。在遗传密码使用上,该基因内部含有2个TGA,在游仆虫中编码半胱氨酸。同时首次发现,八肋游仆虫基因使用TAG作为终止密码子。NCBI上序列比对表明该基因翻译的蛋白与其它物种Rab1蛋白的同源性达49%~52%,因此我们将它命名为Eo-rab-1N,GenBank登录号为DQ105562。Eo-rab-1N与其他物种的Rab1蛋白构建进化树,发现该蛋白的进化与物种的进化保持一致,表明该基因在细胞中具有重要功能。     

八肋游仆虫微管蛋白基因家族的鉴定及进化分析

为了系统分析八肋游仆虫(Euplotes octocarinatus)微管蛋白基因家族, 从八肋游仆虫大核基因组中共鉴定得到20个微管蛋白基因, 基于同源比对及系统进化分析, 将其归入α、β、γ、δ、ε及η六个微管蛋白亚家族; 多序列比对及Western blot结果显示八肋游仆虫η微管蛋白基因在翻译过程中需发生一次+1位编程性核糖体移码, 其移码位点为AAA-TAA; 所有自由生纤毛虫都含有多个α和β微管蛋白基因亚型, 可能用于组成不同的微管结构。研究为后续深入探讨八肋游仆虫微管蛋白的生物学功能及微管多样性奠定了基础。     

Rab4：细胞囊泡循环运输过程的重要因子

囊泡运输是真核细胞中物质运输及信息交流的重要形式,Rab蛋白在这个过程中发挥着重要功能.Rab4是Rab蛋白家族的成员之一,参与调控早期内体的分选与内体循环途径.Rab4包括Rab4A、Rab4B和Rab4C 3个亚型.本文主要阐述了Rab4的结构特征、主要的效应蛋白和参与运输的货物蛋白以及影响细胞自噬、葡萄糖摄取、神经调节、心脏功能及肿瘤发生方面的功能.     

八肋游仆虫含绿色荧光蛋白基因的大核人工染色体的构建

为研究八肋游仆虫(Euplotes octocarinatus)相关基因的功能,构建了八肋游仆虫大核人工染色体(macronuclear artificial chromosome of E. octocarinatus,EoMAC-G),其两端为克隆自八肋游仆虫大核β2-微管蛋白基因的5′和3′非编码区和两侧的端粒序列,中间为多克隆位点和密码子优化后的增强型绿色荧光蛋白(enhanced green fluorescence protein, EGFP-Eo) 报道基因. 用脂质体转染方法将携带有EoMAC－G的pBTub-Tel载体转入八肋游仆虫大核,分析EGFP-Eo基因在八肋游仆虫细胞中的表达. 荧光显微镜观察发现,EGFP-Eo产生的荧光均匀分布于八肋游仆虫细胞质中. 在细胞进行有丝分裂的情况下,荧 光可持续20 d以上. 相比pEGFP-N1质粒转化的游仆虫,人工染色体中的EGFP-Eo基因表达的荧光亮度强、稳定且持续时间长. Western 杂交分析进一步证实,外源EGFP-Eo基因在细胞中过量表达. 通过细菌喂食法进行纤毛虫RNA干扰实验,抑制了外源EGFP-Eo基因在八肋游仆虫细胞中的表达. 利用构建的人工染色体不仅可以在八肋游仆虫细胞内表达外源基因,对目的蛋白质进行活细胞实时动态的定位分析,还可通过RNA干扰的方法调控外源基因在纤毛虫细胞中的表达,便于进一步分析目的蛋白质的功能.     

Characterization of a Rab11 homologue, EoRab11a, in Euplotes octocarinatus

Rab GTPases are crucial in the regulation of intracellular vesicular trafficking. A novel Rab GTPase gene, EoRab11a (GenBank accession no. EF061065 ), was isolated and identified from Euplotes octocarinatus cells in this study. It contains an ORF of 696-bp nucleotides, encoding 231 amino acids with a calculated molecular weight of 26.8 kDa. Alignment of EoRab11a with other Rab11 proteins from other eukaryotes demonstrated that these proteins shared 53–61% identity at the amino acid level. The recombinant EoRab11a was expressed in Escherichia coli and purified by immobilized metal chelate affinity chromatography and iron chromatography. The GTPase activity of EoRab11a was 0.0024 min⁻¹ detected by HPLC at 30 °C. Three mutations were generated at amino acids Ser21 and Gly22 positions in the G1 domain of EoRab11a. All three mutants, S21P, S21G and G22R, increased the GTPase activity in vitro . Immunofluorescence microscopy results indicated that EoRab11a was localized on the phagosomal membrane during phagocytosis of E. octocarinatus . These data show that EoRab11a possesses GTP hydrolysis activity and may participate in vesicle transport events during phagocytosis of E. octocarinatus .     

Versatile role of Rab27 in membrane trafficking: focus on the Rab27 effector families

Rab27A was the only Rab protein whose dysfunction was found to cause human immunodeficiency. Since Griscelli syndrome patients (i.e., Rab27A-deficient) exhibit silvery hair color (i.e., pigmentary dilution) in addition to loss of cytotoxic killing activity by cytotoxic T lymphocytes, and Rab27A protein is expressed in a wide variety of secretory cells, Rab27A (or its closely related isoform Rab27B) has been implicated in the regulation of different types of membrane trafficking, including melanosome transport and various regulated secretion events. How does Rab27 protein regulate these different types of membrane trafficking? Recent discoveries of three different families of Rab27-binding proteins (a total of eleven distinct proteins) have supplied an important clue to the answer of this question: different types of Rab27 effectors function in different cell types. In this review I describe the literature on the identification of Rab27-binding proteins (i.e., the synaptotagmin-like protein (Slp) family with tandem C2 Ca(2+)-binding motifs, the Slac2 family without any C2 motifs, and Munc13-4, a putative priming factor for exocytosis) and the current state of our understanding of the molecular mechanism of the Rab27-dependent membrane trafficking.     

Large scale screening for novel rab effectors reveals unexpected broad Rab binding specificity

Small GTPase Rab is generally thought to control intracellular membrane trafficking through interaction with specific effector molecules. Because of the large number of Rab isoforms in mammals, however, the effectors of most of the mammalian Rabs have never been identified, and the Rab binding specificity of the Rab effectors previously reported has never been thoroughly investigated. In this study we systematically screened for novel Rab effectors by a yeast two-hybrid assay with 28 different mouse or human Rabs (Rab1-30) as bait and identified 27 Rab-binding proteins, including 19 novel ones. We further investigated their Rab binding specificity by a yeast two-hybrid assay with a panel of 60 different GTP-locked mouse or human Rabs. Unexpectedly most (17 of 27) of the Rab-binding proteins we identified exhibited broad Rab binding specificity and bound multiple Rab isoforms. As an example, inositol-polyphosphate 5-phosphatase OCRL (oculocerebrorenal syndrome of Lowe) bound the greatest number of Rabs (i.e. 16 distinct Rabs). Others, however, specifically recognized only a single Rab isoform or only two closely related Rab isoforms. The interaction of eight of the novel Rab-binding proteins identified (e.g. INPP5E and Cog4) with a specific Rab isoform was confirmed by co-immunoprecipitation assay and/or colocalization analysis in mammalian cell cultures, and the novel Rab2B-binding domain of Golgi-associated Rab2B interactor (GARI) and GARI-like proteins was identified by deletion and homology search analyses. The findings suggest that most Rab effectors (or Rab-binding proteins) regulate intracellular membrane trafficking through interaction with several Rab isoforms rather than through a single Rab isoform.     

Ras-related proteins (Rab) are key proteins related to male fertility following a unique activation mechanism

Ras-related protein Rab (Rab) proteins, member of Ras superfamily of monomeric G proteins, are well known key regulators of intracellular vesicular transport. Recently, it has been reported that Rab 2A and 3A are related to acrosomal exocytosis in spermatozoa and Rab 2A can be used to fertility-related biomarker in male. However, the role and mechanism of Rab proteins in spermatozoa has not been fully understood yet. Therefore, the study to analyze the expression and location of various Rab proteins in spermatozoa is required to understand the role and mechanism of Rab proteins in spermatozoa. In present study, to analyze the expression level and location of various Rab proteins (Rab 2A, Rab3A, Rab4, Rab5, Rab8A, Rab9, Rab11, Rab14, Rab25, Rab27A, and Rab34) and Rab protein regulators (RabGAP, RabGDI, RabGEF) in spermatozoa following capacitation, immunofluorescence and western blot analysis were performed. All of 11 Rab proteins were expressed in acrosomal region and tail of spermatozoa. Furthermore, all Rab proteins and Rab protein regulators, except RabGAP, have decreased expression patterns after capacitation. Taken together, Rab proteins were located in sperm head and tail. In addition, expression patterns of Rab proteins in spermatozoa were altered following capacitation. Therefore, our results suggested that Rab proteins may be key proteins related with capacitation as well as playing important role with uniquely activation pathway for male fertility.     

Isoprenylation of Rab proteins possessing a C-terminal CaaX motif

Rab proteins are small GTPases highly related to the yeast Ypti and Sec4 proteins involved in secretion. The Rab proteins were found associated with membranes of different compartments along the secretory and endocytic pathways. They share distinct C-terminal cysteine motifs required for membrane association. Unlike the other Rab proteins, Rab8, Rab11 and Rab13 terminate with a C-terminal CaaX motif similar to those of Ras/Rho proteins. This report demonstrates that Rab8 and Rab13 proteins are isoprenylated in vivo and geranylgeranylated in vitro. Rab11 associates in vitro geranylgeranylpyrophosphate and farnesylpyrophosphate. Our study shows that the CaaX motif is required for isoprenylation.