拟南芥AAP基因家族的生物信息学分析

氨基酸通透酶(amino acid permease,AAP)是一种跨膜转运蛋白,广泛参与氨基酸的吸收、转运。本研究采用生物信息学方法对拟南芥AAP基因家族8个成员(编号AtAAP1~At AAP8)进行染色体定位、蛋白质理化性质、亚细胞定位、三级结构、跨膜区、基因结构及调控元件等进行分析,为探索AAP基因在拟南芥氨基酸吸收转运中的作用及进一步研究AtAAP基因家族的功能提供理论依据。     

高粱高亲和硝酸盐转运蛋白NRT2/3基因家族鉴定、表达与DNA变异分析

硝态氮是作物吸收无机氮素的主要形态,硝酸盐转运蛋白2(nitrate transporter 2,NRT2)作为高亲和性的转运蛋白,以硝酸盐作为特异性底物,在可利用的硝酸盐受限时,高亲和性转运系统被激活,在硝酸盐吸收、转运过程中发挥着重要作用。大多数NRT2不能单独转运硝酸盐,需在硝酸盐同化相关蛋白2(nitrate assimilation related protein 2,NAR2)的协助下才能完成硝酸盐的吸收或转运。作物氮利用效率受环境条件影响,品种间存在差异,因此培育高氮素利用效率品种有重大意义。高粱(Sorghum bicolor)具有耐贫瘠特性,对土壤中的氮素吸收和利用效率较高。本研究结合高粱基因组数据库对NRT2/3基因家族成员基因结构、染色体定位、理化性质、二级结构与跨膜结构域、信号肽与亚细胞定位、启动子区顺式作用元件、系统进化、单核苷酸多态性(single nucleotide polymorphism,SNP)的识别与注释及选择压力进行了全面分析。通过生物信息学分析,筛选出5个NRT2s(命名为SbNRT2-1a、2-1b、SbNRT2-2–4)基因和2个NAR2s(SbNRT3-1–2)基因,较谷子略少。分布在3条染色体上,分为4个亚家族,同一亚族中基因结构高度相似;高粱NRT2/3亲水性平均值均为正值,表明均为疏水性蛋白;α-螺旋和无规则卷曲占二级结构总量的比例大于70%;亚细胞定位均在质膜上,其中NRT2s蛋白不含信号肽,NRT3s蛋白含信号肽;进一步对其跨膜结构域进行分析,发现NRT2s家族成员跨膜结构域个数均大于10个,而NRT3s家族成员跨膜结构域个数为2个;高粱与玉米(Zea mays)NRT2/3s的共线性较好;蛋白结构域显示存在MFS_1和NAR2蛋白结构域,可执行高亲和力硝酸盐转运;系统进化树分析可知,高粱与玉米和谷子的NRT2/3基因亲缘关系更近;基因启动子顺式作用元件分析发现,SbNRT2/3基因的启动子区均具有数个植物激素和逆境应答元件,可以响应高粱生长和环境变化;基因表达热图显示低氮条件下在根诱导表达的是SbNRT2-1a、SbNRT2-1b和SbNRT3-1,推测可在高粱根部表达并调控对硝酸盐的吸收或转运过程。在SbNRT2-4和SbNRT2-1a等发现多个非同义SNP变异;选择压力分析表明,高粱NRT2/3基因家族在进化过程中受纯化选择作用。SbNRT2/3基因表达及蚜虫侵染影响与基因在不同组织中的表达分析结果一致,SbNRT2-1b和SbNRT3-1在感染蚜虫品系5-27sug根部表达显著,高粱蚜虫侵染叶片显著降低了SbNRT2-3、SbNRT2-4和SbNRT3-2的表达水平。本研究初步对高粱全基因组NRT2/3基因家族进行鉴定、表达与DNA变异分析,为高粱氮高效研究提供了基础。     

植物铜转运蛋白的结构和功能

铜(Cu)是植物必需的微量营养元素, 参与植物生长发育过程中的许多生理生化反应。Cu缺乏或过量都会影响植物的正常新陈代谢过程。因此, 植物需要一系列Cu转运蛋白协同作用以保持体内Cu离子的稳态平衡。通常, Cu转运蛋白可分为两类, 即吸收型Cu转运蛋白(如COPT、ZIP和YSL蛋白家族)和排出型Cu转运蛋白(如HMA蛋白家族), 主要负责Cu离子的跨膜转运及调节Cu离子的吸收和排出。然而, 最近有研究表明, 有些Cu伴侣蛋白家族可能是从Cu转运蛋白家族进化而来, 且它们在维持植物细胞Cu离子稳态平衡中也具重要功能。该文对Cu转运蛋白和Cu伴侣蛋白的表达、结构、定位及功能等研究进展进行综述。     

葡萄中蔗糖转运蛋白家族的序列分析及功能推测

以葡萄中的蔗糖转运蛋白为主要研究对象,结合了功能研究较为深入的拟南芥和水稻蔗糖转运蛋白家族序列,分析讨论了这些基因在启动子区域顺式作用元件的异同,以及这些差异可能对mRNA的转录带来的影响;同时,根据蔗糖转运蛋白氨基酸序列对其家族进行了分类,分析了不同亚类蔗糖转运蛋白基因结构的特点;最后还对蔗糖转运蛋白家族中氨基酸的保守性进行了分析。这些分析将为后续蔗糖转运蛋白功能基因组学的研究以及通过基因工程技术精确调节植物代谢提供一定的依据。     

水稻蔗糖转运载体蛋白(OsSUT2)非跨膜区域特征性序列的融合表达及其抗体制备

植物光合作用产生的蔗糖是植物生长发育的主要碳源物质,还是诱导植物生长发育过程中诸多相关基因表达的特异信号分子[1].蔗糖分子在植物器官及组织间的生理分配维持着整个植物体的正常生长发育[2].植物蔗糖转运载体(sucrosetransporter,SUT)是一类担负着蔗糖分子在细胞间的转运及信号转导的功能性蛋白家族,它在蔗糖的韧皮部装载、沿韧皮部的再吸收、韧皮部卸载和向库器官的转运等跨膜运输以及蔗糖特异信号感应过程中发挥着重要的生理功能[3～5].植物蔗糖转运载体蛋白分布于植物细胞质膜上,该转运载体蛋白含有12个疏水性跨膜结构域,在其氨…     

植物ABCB亚家族生物学功能研究进展

ABC转运蛋白超家族结构和功能复杂多样, 包含ABCA-ABCH八个亚家族。ABCB是ABC转运蛋白的一个亚家族, 多数定位于质膜, 少数定位于线粒体膜或叶绿体膜。ABCB与其它生长素转运蛋白(AUX1/LAX、PIN)共同参与调控植物生长素的极性运输, 在植物生长发育的各个阶段发挥作用。此外, ABCB转运蛋白还调控植物的向性运动和重金属抗性等过程。近年来, 随着越来越多植物全基因组测序的完成, ABCB亚家族在禾谷类单子叶植物水稻(Oryza sativa)、玉米(Zea mays)和高粱(Sorghum bicolor)中的生物学功能开始有少量报道, 然而多数ABCB转运蛋白的功能尚未得到阐释。该文对拟南芥(Arabidopsis thaliana)和禾谷类作物ABCB转运蛋白的研究进展进行综述, 以期为全面揭示ABCB亚家族生物学功能提供线索。     

植物ABCB亚家族生物学功能研究进展

ABC转运蛋白超家族结构和功能复杂多样, 包含ABCA-ABCH八个亚家族。ABCB是ABC转运蛋白的一个亚家族, 多数定位于质膜, 少数定位于线粒体膜或叶绿体膜。ABCB与其它生长素转运蛋白(AUX1/LAX、PIN)共同参与调控植物生长素的极性运输, 在植物生长发育的各个阶段发挥作用。此外, ABCB转运蛋白还调控植物的向性运动和重金属抗性等过程。近年来, 随着越来越多植物全基因组测序的完成, ABCB亚家族在禾谷类单子叶植物水稻(Oryza sativa)、玉米(Zea mays)和高粱(Sorghum bicolor)中的生物学功能开始有少量报道, 然而多数ABCB转运蛋白的功能尚未得到阐释。该文对拟南芥(Arabidopsis thaliana)和禾谷类作物ABCB转运蛋白的研究进展进行综述, 以期为全面揭示ABCB亚家族生物学功能提供线索。     

植物蔗糖转运蛋白及其生理功能的研究进展

高等植物通过光合作用产生蔗糖,并以蔗糖形式将同化碳源分配到植物各组织器官中,蔗糖的跨膜转运需要蔗糖转运蛋白的参与。目前,已有研究表明蔗糖转运蛋白广泛存在于高等植物体内,主要在种子、花器、叶肉细胞、韧皮部和根等器官中发挥作用,而通过亚细胞定位可以发现,蔗糖转运蛋白主要定位在细胞的液泡膜及细胞质膜上。综述了国内外对蔗糖转运蛋白的发现、结构、分类、生理功能、功能验证及定位等方面的研究内容,分析了研究蔗糖转运蛋白的意义及目前存在的一些问题,旨为更好地理解蔗糖转运蛋白在植物体内的作用机制做铺垫。     

SMR蛋白:一种跨膜蛋白演化研究的模式蛋白

多药抗性小蛋白(small multidrug resistance protein,SMR)是一类由4个跨膜螺旋束组成的跨膜质子梯度驱动的内膜转运蛋白,一般由100~140个氨基酸残基组成,结构相对简单,在特定去垢剂中相对稳定并具有功能活性,是研究细菌的耐药机制、跨膜转运蛋白的结构功能以及膜蛋白演化的一类很好的模式蛋白家族。同时,由于其灵活多变的拓扑学分布以及在膜转运蛋白演化中节点的位置,成为研究和追寻膜蛋白演化路径的理想的模式蛋白。现分别从SMR蛋白家族的系统发生分布与进化、SMR蛋白的耐药性与转运机制、SMR蛋白的结构等3个方面对本领域的研究近况进行分析和总结。     

溶质转运蛋白超家族的功能及结构研究进展

溶质转运蛋白(solute carriers,SLC)超家族是人类细胞膜(含胞内膜)上最重要的膜转运蛋白家族之一,它参与了细胞间的物质运输、能量传递、营养代谢、信号传导等重要生理活动。SLC转运蛋白超家族包含52个亚家族,共有400多名成员。研究表明,人类基因突变所致SLC蛋白表达异常或功能缺陷与糖尿病、高血压、抑郁症等多种重大疾病密切相关,使得该家族蛋白的功能研究近年来备受关注。SLC转运家族蛋白三维结构的解析有助于阐述其底物选择性结合与转运的精确分子机制,为研究该家族功能相关疾病的分子机理以及针对理性药物研发奠定了精细的三维结构基础。本文对近年来溶质转运蛋白超家族的结构及功能研究进展进行了总结,试图对该家族的共性规律进行阐述。     

Domain structure and pore loops in the 2-hydroxycarboxylate transporter family

The 2-hydroxycarboxylate transporter (2HCT) family is a family of bacterial secondary transporters for substrates like citrate, malate and lactate. The family is in class ST[3] of the MemGen classification system that groups membrane proteins in structural classes based on hydropathy profile analysis. The combination of computational analysis of the proteins in class ST[3] and available experimental data on members of the 2HCT family has yielded a detailed structural model of the transporters. The core of the model is formed by two homologous domains with opposite orientation in the membrane. Each domain consists of 5 trans membrane segments and contains a pore loop between the 4th and 5th segment. The two pore loops enter the membrane-embedded part from opposite sides of the membrane (trans pore loops) and are believed to form the translocation pathway in the 3D structure. A genome wide study of the cellular location of the C-terminus of all Escherichia coli membrane proteins [Daley et al., 2005. Science 308:1321-1323] showed that the C-termini of the 19 E. coli proteins in class ST[3] were correctly predicted by the structural model.     

Structural elucidation of transmembrane domain zero (TMD0) of EcdL: A multidrug resistance-associated protein (MRP) family of ATP-binding cassette transporter protein revealed by atomistic simulation

ATP-Binding cassette (ABC) transporters play an extensive role in the translocation of diverse sets of biologically important molecules across membrane. EchnocandinB (antifungal) and EcdL protein of Aspergillus rugulosus are encoded by the same cluster of genes. Co-expression of EcdL and echinocandinB reflects tightly linked biological functions. EcdL belongs to Multidrug Resistance associated Protein (MRP) subfamily of ABC transporters with an extra transmembrane domain zero (TMD0). Complete structure of MRP subfamily comprising of TMD0 domain, at atomic resolution is not known. We hypothesized that the transportation of echonocandinB is mediated via EcdL protein. Henceforth, it is pertinent to know the topological arrangement of TMD0, with other domains of protein and its possible role in transportation of echinocandinB. Absence of effective template for TMD0 domain lead us to model by I-TASSER, further structure has been refined by multiple template modelling using homologous templates of remaining domains (TMD1, NBD1, TMD2, NBD2). The modelled structure has been validated for packing, folding and stereochemical properties. MD simulation for 0.1 μs has been carried out in the biphasic environment for refinement of modelled protein. Non-redundant structures have been excavated by clustering of MD trajectory. The structural alignment of modelled structure has shown Z-score -37.9; 31.6, 31.5 with RMSD; 2.4, 4.2, 4.8 with ABC transporters; PDB ID 4F4C, 4M1 M, 4M2T, respectively, reflecting the correctness of structure. EchinocandinB has been docked to the modelled as well as to the clustered structures, which reveals interaction of echinocandinB with TMD0 and other TM helices in the translocation path build of TMDs.     

An orphaned Mce‐associated membrane protein of Mycobacterium tuberculosis is a virulence factor that stabilizes Mce transporters

Mycobacterium tuberculosis proteins that are exported out of the bacterial cytoplasm are ideally positioned to be virulence factors; however, the functions of individual exported proteins remain largely unknown. Previous studies identified Rv0199 as an exported membrane protein of unknown function. Here, we characterized the role of Rv0199 in M. tuberculosis virulence using an aerosol model of murine infection. Rv0199 appears to be a member of a Mce‐associated membrane (Mam) protein family leading us to rename it OmamA, for orphaned Mam protein A. Consistent with a role in Mce transport, we showed OmamA is required for cholesterol import, which is a Mce4‐dependent process. We further demonstrated a function for OmamA in stabilizing protein components of the Mce1 transporter complex. These results indicate a function of OmamA in multiple Mce transporters and one that may be analogous to the role of VirB8 in stabilizing Type IV secretion systems, as structural similarities between Mam proteins and VirB8 proteins are predicted by the Phyre 2 program. In this study, we provide functional information about OmamA and shed light on the function of Mam family proteins in Mce transporters.     

The 2-hydroxycarboxylate transporter family: physiology, structure, and mechanism.

The 2-hydroxycarboxylate transporter family is a family of secondary transporters found exclusively in the bacterial kingdom. They function in the metabolism of the di- and tricarboxylates malate and citrate, mostly in fermentative pathways involving decarboxylation of malate or oxaloacetate. These pathways are found in the class Bacillales of the low-CG gram-positive bacteria and in the gamma subdivision of the Proteobacteria. The pathways have evolved into a remarkable diversity in terms of the combinations of enzymes and transporters that built the pathways and of energy conservation mechanisms. The transporter family includes H+ and Na+ symporters and precursor/product exchangers. The proteins consist of a bundle of 11 transmembrane helices formed from two homologous domains containing five transmembrane segments each, plus one additional segment at the N terminus. The two domains have opposite orientations in the membrane and contain a pore-loop or reentrant loop structure between the fourth and fifth transmembrane segments. The two pore-loops enter the membrane from opposite sides and are believed to be part of the translocation site. The binding site is located asymmetrically in the membrane, close to the interface of membrane and cytoplasm. The binding site in the translocation pore is believed to be alternatively exposed to the internal and external media. The proposed structure of the 2HCT transporters is different from any known structure of a membrane protein and represents a new structural class of secondary transporters.     

Molecular characterization and expression patterns of sucrose transport-related genes in sweet sorghum under defoliation

Sucrose is the principal form of photosynthesis products, and long-distance transport of sucrose requires sucrose transporters (SUTs) to perform loading and unloading functions. SUTs play an important role in plant growth, development and reproduction. In this study, five unique sucrose transporter (SbSUT) genes that contain full-length cDNA sequences were cloned from sweet sorghum, and these SbSUT genes were clustered into four different clades: SUT1, SUT3, SUT4 and SUT5. Heterologous expression of SbSUTs in yeast demonstrated that they were functional sucrose transporters. Tissue-specific expression profiles showed that sorghum SUT genes had different tissue-specific expression patterns, suggesting that sorghum SUT genes may play an important role in plant growth and developmental processes. After defoliation, expression patterns of SbSUT1, SbSUT2 and SbSUT4 were different in leaf sheaths, leaves and roots. Taken together, the results indicate that the above mentioned five unique sucrose transporter genes may play important roles in performing sucrose loading and unloading functions and that they exhibit different expression in response to leaf blade removal.     

水稻与拟南芥跨膜蛋白14家族的分子进化特征与功能分析

脂类既是植物生命活动重要的能量来源,也是细胞膜系统不可或缺的结构成分,在植物生长发育和逆境反应等生命活动过程中都起到至关重要的作用。随着脂类代谢研究的不断深入,植物脂类合成通路已渐渐明晰,其中连通不同细胞器间脂类合成中间物质运送的膜蛋白也正被不断发现,但对质体脂类转运蛋白还鲜有报道。跨膜蛋白14家族(Transmembrane 14 family, Tmemb14 family)是一个新发现的跨膜蛋白家族,目前只有拟南芥FAX1 (Fatty Acid Export 1)和斑马鱼TMEM14已被克隆鉴定,该家族其他成员的生物学功能还未见报道。AtFAX1参与植物质体长链脂肪酸的跨膜外运,其功能丧失显著降低植物生物量并影响花粉发育和育性。本研究通过生物信息手段对拟南芥和水稻中的跨膜蛋白14家族成员的进化关系、蛋白理化性质、结构域功能和编码基因的表达模式进行了分析,揭示了Tmemb14家族成员在单、双子叶植物进化中的功能分化,为进一步研究跨膜蛋白14家族成员的生理功能提供了理论依据。     

Structural Features of the Glutamate Transporter Family

Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft and thus prevent neurotoxicity. The proteins belong to a large and widespread family of secondary transporters, including bacterial glutamate, serine, and C₄-dicarboxylate transporters; mammalian neutral-amino-acid transporters; and an increasing number of bacterial, archaeal, and eukaryotic proteins that have not yet been functionally characterized. Sixty members of the glutamate transporter family were found in the databases on the basis of sequence homology. The amino acid sequences of the carriers have diverged enormously. Homology between the members of the family is most apparent in a stretch of approximately 150 residues in the C-terminal part of the proteins. This region contains four reasonably well-conserved sequence motifs, all of which have been suggested to be part of the translocation pore or substrate binding site. Phylogenetic analysis of the C-terminal stretch revealed the presence of five subfamilies with characterized members: (i) the eukaryotic glutamate transporters, (ii) the bacterial glutamate transporters, (iii) the eukaryotic neutral-amino-acid transporters, (iv) the bacterial C₄-dicarboxylate transporters, and (v) the bacterial serine transporters. A number of other subfamilies that do not contain characterized members have been defined. In contrast to their amino acid sequences, the hydropathy profiles of the members of the family are extremely well conserved. Analysis of the hydropathy profiles has suggested that the glutamate transporters have a global structure that is unique among secondary transporters. Experimentally, the unique structure of the transporters was recently confirmed by membrane topology studies. Although there is still controversy about part of the topology, the most likely model predicts the presence of eight membrane-spanning α-helices and a loop-pore structure which is unique among secondary transporters but may resemble loop-pores found in ion channels. A second distinctive structural feature is the presence of a highly amphipathic membrane-spanning helix that provides a hydrophilic path through the membrane. Recent data from analysis of site-directed mutants and studies on the mechanism and pharmacology of the transporters are discussed in relation to the structural model.     

Integration of Evolutionary Features for the Identification of Functionally Important Residues in Major Facilitator Superfamily Transporters

The identification of functionally important residues is an important challenge for understanding the molecular mechanisms of proteins. Membrane protein transporters operate two-state allosteric conformational changes using functionally important cooperative residues that mediate long-range communication from the substrate binding site to the translocation pathway. In this study, we identified functionally important cooperative residues of membrane protein transporters by integrating sequence conservation and co-evolutionary information. A newly derived evolutionary feature, the co-evolutionary coupling number, was introduced to measure the connectivity of co-evolving residue pairs and was integrated with the sequence conservation score. We tested this method on three Major Facilitator Superfamily (MFS) transporters, LacY, GlpT, and EmrD. MFS transporters are an important family of membrane protein transporters, which utilize diverse substrates, catalyze different modes of transport using unique combinations of functional residues, and have enough characterized functional residues to validate the performance of our method. We found that the conserved cores of evolutionarily coupled residues are involved in specific substrate recognition and translocation of MFS transporters. Furthermore, a subset of the residues forms an interaction network connecting functional sites in the protein structure. We also confirmed that our method is effective on other membrane protein transporters. Our results provide insight into the location of functional residues important for the molecular mechanisms of membrane protein transporters.     

Structural features of the glutamate transporter family.

Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft and thus prevent neurotoxicity. The proteins belong to a large and widespread family of secondary transporters, including bacterial glutamate, serine, and C4-dicarboxylate transporters; mammalian neutral-amino-acid transporters; and an increasing number of bacterial, archaeal, and eukaryotic proteins that have not yet been functionally characterized. Sixty members of the glutamate transporter family were found in the databases on the basis of sequence homology. The amino acid sequences of the carriers have diverged enormously. Homology between the members of the family is most apparent in a stretch of approximately 150 residues in the C-terminal part of the proteins. This region contains four reasonably well-conserved sequence motifs, all of which have been suggested to be part of the translocation pore or substrate binding site. Phylogenetic analysis of the C-terminal stretch revealed the presence of five subfamilies with characterized members: (i) the eukaryotic glutamate transporters, (ii) the bacterial glutamate transporters, (iii) the eukaryotic neutral-amino-acid transporters, (iv) the bacterial C4-dicarboxylate transporters, and (v) the bacterial serine transporters. A number of other subfamilies that do not contain characterized members have been defined. In contrast to their amino acid sequences, the hydropathy profiles of the members of the family are extremely well conserved. Analysis of the hydropathy profiles has suggested that the glutamate transporters have a global structure that is unique among secondary transporters. Experimentally, the unique structure of the transporters was recently confirmed by membrane topology studies. Although there is still controversy about part of the topology, the most likely model predicts the presence of eight membrane-spanning alpha-helices and a loop-pore structure which is unique among secondary transporters but may resemble loop-pores found in ion channels. A second distinctive structural feature is the presence of a highly amphipathic membrane-spanning helix that provides a hydrophilic path through the membrane. Recent data from analysis of site-directed mutants and studies on the mechanism and pharmacology of the transporters are discussed in relation to the structural model.     

Eukaryotic major facilitator superfamily transporter modeling based on the prokaryotic GlpT crystal structure

The major facilitator superfamily (MFS) of transporters represents the largest family of secondary active transporters and has a diverse range of substrates. With structural information for four MFS transporters, we can see a strong structural commonality suggesting, as predicted, a common architecture for MFS transporters. The rate for crystal structure determination of MFS transporters is slow, making modeling of both prokaryotic and eukaryotic transporters more enticing. In this review, models of eukaryotic transporters Glut1, G6PT, OCT1, OCT2 and Pho84, based on the crystal structures of the prokaryotic GlpT, based on the crystal structure of LacY are discussed. The techniques used to generate the different models are compared. In addition, the validity of these models and the strategy of using prokaryotic crystal structures to model eukaryotic proteins are discussed. For comparison, E. coli GlpT was modeled based on the E. coli LacY structure and compared to the crystal structure of GlpT demonstrating that experimental evidence is essential for accurate modeling of membrane proteins.