酵母双杂交系统在膜蛋白相互作用研究中的应用

膜相关蛋白约占细胞总蛋白质中的1/3,它们大都参与了细胞的诸多生理、病理过程和药物反应机理。研究膜蛋白的相互作用对于揭示细胞的生命活动规律及寻找药物作用靶标都有重要的意义。由于膜蛋白本身的特性及其难以进入核内等原因,经典的酵母双杂交技术并不适用于检测膜蛋白间的相互作用。针对在活细胞中研究膜蛋白相互作用的需要,近年来国际上先后发展了一系列用于膜蛋白相互作用研究的酵母双杂交新系统,并取得了许多重要发现。     

酵母双杂交系统

就酵母双杂交系统的基本原理、主要进展和在蛋白质组研究中的应用作一综述。     

酵母双杂交系统研究进展

20世纪90年代新兴起的酵母双杂交系统,应用有效的酵母遗传学方法,快速、直接分析已知蛋白之间的相互作用,并能寻找、分离新的与已知蛋白相互作用的配体,发现新基因。在此基础上,相继出现了反向双杂交系统、三杂交系统等多种新技术,为探索蛋白质－蛋白质、蛋白质－核酸作用的奥秘开拓了更为广阔的天地。     

酵母双杂交衍生系统

酵母双杂交系统是在酵母体内分析蛋白质蛋白质相互作用的基因系统,由Fields等人于1989年首次建立并得到广泛地应用。十余年来,随着酵母双杂交迅速推广,不断涌现出一些衍生系统,其中包括酵母双杂交的二元诱饵系统,逆向双杂交系统,非转录读出特点的双杂交系统(如Sos蛋白招募系统、PI3K介导的靶蛋白识别系统和断裂泛素为基础的双杂交系统)以及转录激活因子与其相关蛋白之间的相互作用的双杂交系统(如以polⅢ为基础的杂交系统和RTA系统)等。它们的建立在很大程度上克服了传统酵母双杂交系统的局限性,扩大了被研究的蛋白质的范围,提高了系统的灵敏度。     

酵母双杂交及其衍生系统

酵母双杂交系统是一种研究蛋白质相互作用的分子生物学方法,过去20多年里,大量衍生系统的出现使得这套双杂交技术体系更加完善和高效,成为研究蛋白质-配体相互作用的重要技术手段,广泛应用于功能基因组学、蛋白质组学、病理学等研究领域。对酵母双杂交及其衍生系统的基本原理和应用进展进行综述。     

逆向酵母双杂交系统的应用

198 9年Ｆｉｅｌｄｓ等提出了酵母双杂交系统来研究真核细胞的蛋白质之间的相互作用。这一崭新手段迅速发展成为一种常规的分子生物学技术 ,并得到了广泛的应用。近来 ,国外学者为拓宽传统的双杂交系统的应用范围 ,在原有的酵母双杂交的基础上发展了大量衍生系统 ,包括用于研究蛋白质与ＤＮＡ之间相互作用的单杂交系统 (ｏｎｅ ｈｙｂｒｉｄｓｙｓ ｔｅｍ) [1] ;研究两个蛋白质与第三个成分间相互作用的三杂交系统 (ｔｈｒｅｅ ｈｙｂｒｉｄｓｙｓｔｅｍ) [2 ] ,第三个成分可以是蛋白质、ＲＮＡ或小分子药物等[3 ] 。另外 ,在通过双杂交体系…     

酵母双杂交系统研究进展

综述了酵母双杂交技术的基本原理和实际应用,以及该技术的发展,并展望了该系统的发展前景。     

酵母双杂交系统

酵母双杂交系统是研究细胞内蛋白质之间相互作用的一种分子遗传学技术,用已知的蛋白质作为诱饵来筛选其可以相互作用的伙伴蛋白。本文简要叙述了酵母双杂交系统的原理、基本方法,以及这个技术的发展和应用。     

大规模酵母双杂交技术研究蛋白质相互作用的应用

简介了酵母双杂交技术原理, 总结了酵母双杂交技术大规模筛选蛋白质相互作用的基础、应用及存在的问题。因为大规模酵母双杂交技术结果有大量假阳性及假阴性问题, 因此, 有条件情况下有必要同时开展其他方法的大规模蛋白相互作用研究, 以构建规模更大可信度更高的蛋白质相互作用网络图。     

酵母双杂交系统的发展及其衍生系统的比较

酵母双杂交及其衍生系统是鉴定及分析蛋白质-蛋白质、蛋白质-DNA、蛋白质-RNA相互作用的最常用、最有效的工具之一。本文系统介绍了该技术的背景、发展过程,以及由Fields和Song初次描述的由酵母双杂交系统衍生而来的几种主要的双杂交系统的特点,并简要比较了各系统的优缺点。     

类囊体膜蛋白质磷酸酶的研究进展

光合作用是地球上最重要的生命活动过程之一。近年来,围绕着光合机构运转的调节和控制问题,对叶绿体类囊体膜蛋白质的可逆磷酸化进行了广泛研究,发现类囊体膜蛋白质的磷酸化和去磷酸化是调控光合机构运转的步骤之一。现在已知,多种叶绿体类囊体收稿日期：１９９９－０６－２５作者简介：邹永龙（１９７４～）,男,博士生;王国强（１９３９～）,男,研究员。膜蛋白质都能进行可逆的磷酸化,这一反应参与了调节光合电子传递、光状态Ⅰ和状态Ⅱ的转换和编码光合机构的基因表达等。目前,研究人员将研究重点集中在捕光色素复合物Ⅱ（ＬＨ…     

Identification of Novel Protein-Protein Interactions of Yersinia pestis Type III Secretion System by Yeast Two Hybrid System

Type III secretion system (T3SS) of the plague bacterium Y. pestis encodes a syringe-like structure consisting of more than 20 proteins, which can inject virulence effectors into host cells to modulate the cellular functions. Here in this report, interactions among the possible components in T3SS of Yersinia pestis were identified using yeast mating technique. A total of 57 genes, including all the pCD1-encoded genes except those involved in plasmid replication and partition, pseudogenes, and the putative transposase genes, were subjected to yeast mating analysis. 21 pairs of interaction proteins were identified, among which 9 pairs had been previously reported and 12 novel pairs were identified in this study. Six of them were tested by GST pull down assay, and interaction pairs of YscG-SycD, YscG-TyeA, YscI-YscF, and YopN-YpCD1.09c were successfully validated, suggesting that these interactions might play potential roles in function of Yersinia T3SS. Several potential new interactions among T3SS components could help to understand the assembly and regulation of Yersinia T3SS.     

Chloroplast Chaperonin-Mediated Targeting of a Thylakoid Membrane Protein

Posttranslational protein targeting requires chaperone assistance to direct insertion-competent proteins to integration pathways. Chloroplasts integrate nearly all thylakoid transmembrane proteins posttranslationally, but mechanisms in the stroma that assist their insertion remain largely undefined. Here, we investigated how the chloroplast chaperonin (Cpn60) facilitated the thylakoid integration of Plastidic type I signal peptidase 1 (Plsp1) using in vitro targeting assays. Cpn60 bound Plsp1 in the stroma. In isolated chloroplasts, the membrane integration of imported Plsp1 correlated with its dissociation from Cpn60. When the Plsp1 residues that interacted with Cpn60 were removed, Plsp1 did not integrate into the membrane. These results suggested Cpn60 was an intermediate in thylakoid targeting of Plsp1. In isolated thylakoids, the integration of Plsp1 decreased when Cpn60 was present in excess of cpSecA1, the stromal motor of the cpSec1 translocon that inserts unfolded Plsp1 into the thylakoid. An excess of cpSecA1 favored integration. Introducing Cpn60’s obligate substrate RbcL displaced Cpn60-bound Plsp1; then, the released Plsp1 exhibited increased accessibility to cpSec1. These in vitro targeting experiments support a model in which Cpn60 captures and then releases insertion-competent Plsp1, whereas cpSecA1 recognizes free Plsp1 for integration. Thylakoid transmembrane proteins in the stroma can interact with Cpn60 to shield themselves from the aqueous environment.     

Lattice Models for Protein Organization throughout Thylakoid Membrane Stacks

Proteins in photosynthetic membranes can organize into patterned arrays that span the membrane’s lateral size. Attractions between proteins in different layers of a membrane stack can play a key role in this ordering, as was suggested by microscopy and fluorescence spectroscopy and demonstrated by computer simulations of a coarse-grained model. The architecture of thylakoid membranes, however, also provides opportunities for interlayer interactions that instead disfavor the high protein densities of ordered arrangements. Here, we explore the interplay between these opposing driving forces and, in particular, the phase transitions that emerge in the periodic geometry of stacked thylakoid membrane disks. We propose a lattice model that roughly accounts for proteins’ attraction within a layer and across the stromal gap, steric repulsion across the lumenal gap, and regulation of protein density by exchange with the stroma lamellae. Mean-field analysis and computer simulation reveal rich phase behavior for this simple model, featuring a broken-symmetry striped phase that is disrupted at both high and low extremes of chemical potential. The resulting sensitivity of microscopic protein arrangement to the thylakoid’s mesoscale vertical structure raises intriguing possibilities for regulation of photosynthetic function.     

应用酵母双杂交系统筛选AMPK相互作用蛋白

一磷酸腺苷激活蛋白激酶(AMPK)是调节体内代谢平衡的丝氨酸/苏氨酸蛋白激酶。应用酵母双杂交系统,以AMPKβ1亚基作为"诱饵"蛋白,筛选均一化的人源cDNA文库,寻找与AMPK相互作用的蛋白。通过对150个阳性克隆进行验证,最终得到了63个与AMPKβ1亚基相互作用的蛋白。其中,包括代谢酶、转录因子或转录相关蛋白、蛋白转运相关蛋白、GTP结合蛋白、支架蛋白、细胞周期调节蛋白、RNA结合蛋白等以及一些未知功能的蛋白。从酵母双杂交的结果来看,AMPK不仅在代谢领域,而且在许多非代谢领域,如核受体及其它转录因子的调节、信号转导、DNA修复及细胞周期调节等,可能都起到非常重要的作用。     

Protein Interactions and Membrane Geometry

The difficulty in growing crystals for x-ray diffraction analysis has hindered the determination of membrane protein structures. However, this is changing with the advent of a new method for growing high quality membrane protein crystals from the lipidic cubic phase. Although successful, the mechanism underlying this method has remained unclear. Here, we present a theoretical analysis of the process. We show that it is energetically favorable for proteins embedded in the highly curved cubic phase to cluster together in flattened regions of the membrane. This stabilizes the lamellar phase, permitting its outgrowth from the cubic phase. A kinetic barrier-crossing model is developed to determine the free energy barrier to crystallization from the time-dependent growth of protein clusters. Determining the values of key parameters provides both a rational basis for optimizing the experimental procedure for membrane proteins that have not yet been crystallized and insight into the analogous cubic to lamellar transitions in cells. We also discuss the implications of this mechanism for protein sorting at the exit sites of the Golgi and endoplasmic reticulum and the general stabilization of membrane structures.     

棉花锌指蛋白GhZFP1相互作用蛋白的酵母双杂交筛选

GhZFP1蛋白是从盐胁迫棉花幼苗cDNA文库中分离的一种CCCH型锌指蛋白.初步的生物学功能研究表明,过量表达该基因的转基因烟草耐盐性和抗病性显著提高.为深入研究GhZFP1蛋白的作用机制,构建pGBKT7-m1诱饵表达载体,利用酵母双杂交系统从盐胁迫诱导棉花cDNA文库中筛选与其相互作用的蛋白.通过阳性克隆的表型确定、PCR和限制性内切酶检测以及测序和生物信息学分析,获得9个与诱饵蛋白相互作用的靶蛋白.双分子荧光互补实验证明,GhZFP1与GZIRD19A确实存在互作关系.通过分析这些靶蛋白的已知功能,为研究GhZFP1锌指蛋白的未知生物学功能提供重要信息.     

利用酵母双杂交系统筛选与黄瓜花叶病毒外壳蛋白互作的寄主蛋白

利用酵母双杂交系统,以黄瓜花叶病毒(Cucumber mosaic virus,CMV)的外壳蛋白(coat protein,CP)为诱饵,从番茄叶片c DNA文库中筛选与其互作的蛋白。结果显示,诱饵载体pBT3-SUC-CMV-CP均能在酵母细胞中正确表达,无自激活活性而且对酵母无毒性;通过对酵母双杂交文库的筛选和回转验证,共获得了98个阳性克隆,分别编码67个可能与CMV-CP相互作用的蛋白,分别参与植物防御反应、光合作用、物质转运、信号转导、能量代谢、氨基酸代谢、细胞壁的形态建成、植物的激素代谢等。本研究结果表明,CMV CP可同时调控寄主的多个代谢过程,在CMV的致病过程中有多重功能。     

抗增殖蛋白在酵母双杂交系统中转录激活作用的检测

目的:检测抗增殖蛋白在酵母双杂交系统中是否具有转录自激活作用。方法:采用PCR方法扩增抗增殖蛋白基因全长序列,构建酵母双杂交系统的诱饵载体pGBKT7-PHB,制备酵母菌AH109感受态细胞,将pGBKT7-PHB转化感受态酵母菌AH109后培养于含缺失培养基的平板上,检测β-半乳糖苷酶活性,判定其是否具有转录自激活作用。结果:扩增出抗增殖蛋白基因全长序列,构建了诱饵载体pGBKT7-PHB,转化酵母菌AH109后在双缺和三缺培养基上未能使β-半乳糖苷酶活性滤纸片变蓝,说明报告基因未被激活。结论:抗增殖蛋白没有转录自激活作用,可用于酵母双杂交系统。