Subcellular Localization Directs Signaling Specificity of the Cryptococcus neoformans Ras1 Protein

In the human fungal pathogen Cryptococcus neoformans, Ras signaling mediates sexual differentiation, morphogenesis, and pathogenesis. By studying Ras prenylation and palmitoylation in this organism, we have found that the subcellular localization of this protein dictates its downstream signaling specificity. Inhibiting C. neoformans Ras1 prenylation results in the defective general membrane targeting of this protein and the loss of all Ras function. In contrast, palmitoylation mediates localization of Ras1 to the plasma membrane and is required for normal morphogenesis and survival at high temperatures. However, palmitoylation and plasma membrane localization are not required for Ras-dependent sexual differentiation. Likely as a result of its effect on thermotolerance, Ras1 palmitoylation is also required for the pathogenesis of C. neoformans. These data support an emerging paradigm of compartmentalized Ras signaling. However, our studies also demonstrate fundamental differences between the Ras pathways in different organisms that emphasize the functional flexibility of conserved signaling cascades.     

Perilipin5基因功能结构域载体的构建及亚细胞定位分析

目的:依据perilipin5的功能结构域,构建含perilipin5截断体的真核表达载体,并研究它们的亚细胞定位。方法:以小鼠肝脏cDNA文库为模板,PCR扩增出perilipin5的全长及功能结构域,将之分别装载入真核表达载体PCMV5中,并引入HA标签。酶切和测序鉴定,脂质体法将构建的质粒转染293T细胞,Western blot验证表达,免疫荧光检测标记HA,于荧光显微镜下观察perilipin5各结构域的亚细胞定位。结果:构建的质粒序列正确,转染细胞后可检测到HA-perilipin5融合蛋白的表达,免疫荧光显示含有1-188aa结构域的perilipin5截断体可定位于脂滴表面,1-188aa一旦缺失perilipin5的截断体则弥散于胞内。结论:包含perilipin5功能结构域的真核表达载体构建成功,perilipin5的1-188aa与其脂滴定位密切相关。     

拟南芥血红蛋白1的亚细胞定位

为研究拟南芥的血红蛋白1(AtGLB1)基因的亚细胞定位,该实验构建了拟南芥血红蛋白1基因与绿色荧光蛋白基因融合的植物表达载体pUCGFP/ AtGLB1.利用基因枪转化法将重组载体转入洋葱表皮细胞瞬时表达,通过检测融合蛋白在洋葱表皮细胞中的分布来确定拟南芥血红蛋白1在细胞中的定位.荧光显微镜检测结果表明,AtGLB1基因表达产物主要定位在细胞核中,少量定位在细胞质中.     

Subcellular Localization and Functional Analysis of the Arabidopsis GTPase RabE

Membrane trafficking plays a fundamental role in eukaryotic cell biology. Of the numerous known or predicted protein components of the plant cell trafficking system, only a relatively small subset have been characterized with respect to their biological roles in plant growth, development, and response to stresses. In this study, we investigated the subcellular localization and function of an Arabidopsis (Arabidopsis thaliana) small GTPase belonging to the RabE family. RabE proteins are phylogenetically related to well-characterized regulators of polarized vesicle transport from the Golgi apparatus to the plasma membrane in animal and yeast cells. The RabE family of GTPases has also been proposed to be a putative host target of AvrPto, an effector protein produced by the plant pathogen Pseudomonas syringae, based on yeast two-hybrid analysis. We generated transgenic Arabidopsis plants that constitutively expressed one of the five RabE proteins (RabE1d) fused to green fluorescent protein (GFP). GFP-RabE1d and endogenous RabE proteins were found to be associated with the Golgi apparatus and the plasma membrane in Arabidopsis leaf cells. RabE down-regulation, due to cosuppression in transgenic plants, resulted in drastically altered leaf morphology and reduced plant size, providing experimental evidence for an important role of RabE GTPases in regulating plant growth. RabE down-regulation did not affect plant susceptibility to pathogenic P. syringae bacteria; conversely, expression of the constitutively active RabE1d-Q74L enhanced plant defenses, conferring resistance to P. syringae infection.     

KKXX-motif对VKORC1亚细胞定位的影响

目的研究细胞内质网蛋白贮留信号KKXX-motif,对维生素K环氧化物还原酶复合物亚基1（VKORC1）的亚细胞定位的作用。方法利用点突变试剂盒构建VKORC1的KKXX突变体——VKORC1-SSXX;同时应用pEGFP载体构建VKORC1-SSXX和VKORC1-KKXX的绿色荧光融合蛋白表达质粒。瞬时转染HEK293s细胞,观察野生融合蛋白和突变融合蛋白的表达情况。结果野生型融合蛋白荧光分布在胞浆内,而突变型融合蛋白聚集表达。结论KKXX-motif影响了VKORC1的亚细胞定位。     

Brassinosteroid Mutants Uncover Fine Tuning of Phytochrome Signaling

Phytochromes (phy) A and B provide higher plants the ability to perceive divergent light signals. phyB mediates red/far-red light reversible, low fluence responses (LFR). phyA mediates both very-low-fluence responses (VLFR), which saturate with single or infrequent light pulses of very low fluence, and high irradiance responses (HIR), which require sustained activation with far-red light. We investigated whether VLFR, LFR, and HIR are genetically coregulated. The Arabidopsis enhanced very-low-fluence response1 mutant, obtained in a novel screening under hourly far-red light pulses, showed enhanced VLFR of hypocotyl growth inhibition, cotyledon unfolding, blocking of greening, and anthocyanin synthesis. However, eve1 showed reduced LFR and HIR. eve1 was found allelic to the brassinosteroid biosynthesis mutant dim/dwarf1. The analysis of both the brassinosteroid mutant det2 in the Columbia background (where VLFR are repressed) and the phyA eve1 double mutant indicates that the negative effect of brassinosteroid mutations on LFR requires phyA signaling in the VLFR mode but not the expression of the VLFR. Under sunlight, hypocotyl growth of eve1 showed little difference with the wild type but failed to respond to canopy shadelight. We propose that the opposite regulation of VLFR versus LFR and HIR could be part of a context-dependent mechanism of adjustment of sensitivity to light signals.     

PC-1在前列腺癌细胞系LNCaP中的定位

目的:研究PC-1分子在前列腺癌细胞系LNCaP中的亚细胞定位。方法:利用常规PCR和重叠PCR技术在pEGFP-C1-PC-1上分别扩增PC-1的不同截短体及缺失体基因,PCR产物经酶切后克隆到真核表达载体pEGFP-C1中;经测序确定构建成功的载体在LNCaP细胞中瞬时高表达,在荧光显微镜下观察这些载体表达产物在LNCaP细胞中的定位情况,并通过Western印迹验证这些载体在LNCaP细胞中的表达。结果:构建了多个用于PC-1亚细胞定位研究的、能在LNCaP细胞中表达的载体;同时,还找到了一段对PC-1定位有重要影响的氨基酸序列。结论:为进一步研究PC-1的亚细胞定位及其发挥功能的方式提供了基础。     

Protein Subcellular Localization in Bacteria

Like their eukaryotic counterparts, bacterial cells have a highly organized internal architecture. Here, we address the question of how proteins localize to particular sites in the cell and how they do so in a dynamic manner. We consider the underlying mechanisms that govern the positioning of proteins and protein complexes in the examples of the divisome, polar assemblies, cytoplasmic clusters, cytoskeletal elements, and organelles. We argue that geometric cues, self-assembly, and restricted sites of assembly are all exploited by the cell to specifically localize particular proteins that we refer to as anchor proteins. These anchor proteins in turn govern the localization of a whole host of additional proteins. Looking ahead, we speculate on the existence of additional mechanisms that contribute to the organization of bacterial cells, such as the nucleoid, membrane microdomains enriched in specific lipids, and RNAs with positional information.Our view of the organization of the bacterial cell has changed radically over the past two decades. Once seen as an amorphous vessel harboring a homogeneous solution of proteins, these primitive organisms are now known to have an intricate subcellular architecture in which individual proteins localize to particular sites in the cell, often in a dynamic manner. Of course, bacteria frequently show conspicuous morphological features, such as division septa, flagella, pili, and stalks, which implied a nonuniform, underlying distribution of proteins. But it was not until the early 1990s that it became clear that proteins can, and often do, have distinctive subcellular addresses. Among the earliest discoveries were: (1) the formation of a ringlike structure at the mid cell position by the cytokinetic protein FtsZ (Bi and Lutkenhaus 1991), (2) the clustering of chemotaxis proteins at the poles of cells (Alley et al. 1992), (3) the compartment-specific production of sporulation proteins and their assembly into shell-like structures (Driks and Losick 1991), and (4) the asymmetric distribution of proteins involved in actin polymerization along the cell surface (Goldberg et al. 1993; Kocks et al. 1993). These discoveries were initially made by immunoelectron and immunofluorescence microscopy with fixed cells, but the discovery of green fluorescent protein (GFP) and the demonstration that proteins could retain their proper subcellular localization as GFP fusions opened the way to visualizing proteins and their dynamic behavior in living cells, including, importantly, in bacteria (Arigoni et al. 1995).Knowing where proteins are in the cell is often critical to understanding their function. Thus, the position of the aforementioned FtsZ ring (the Z-ring) dictates where cytokinesis will take place (Margolin 2005). The clustering of chemotaxis proteins plays an important role in the extraordinary gain in the responsiveness of chemotatic behavior to small changes in attractants (Ames and Parkinson 2006). Where sporulation proteins are produced and the way in which they assemble governs spore morphogenesis (Stragier and Losick 1996; Errington 2003). The asymmetric distribution of actin-polymerization proteins on the cell surface explains how certain pathogens harness host cytoskeletal proteins for their own motility (Smith et al. 1995). From these and other examples emerge a view of the bacterial cell as a dynamic, three-dimensional system in which protein localization and changes in protein localization over time orchestrate growth, the cell cycle, behavior, and differentiation.Here, after an initial discussion of general principles governing the positioning of proteins within the cell, we consider five broad categories of subcellular localization: the divisome, polar assemblies, cytoplasmic clusters, cytoskeletal elements, and organelles. We end by looking ahead to exciting new aspects of bacterial cytology just emerging from current research. Our goal is not to be comprehensive but rather to focus on examples that are illustrative of general principles of protein localization. Comprehensive treatment of individual topics can be found in other articles on this topic.     

A BIN2-GLK1 Signaling Module Integrates Brassinosteroid and Light Signaling to Repress Chloroplast Development in the Dark

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Interdependency of Brassinosteroid and Auxin Signaling in Arabidopsis

How growth regulators provoke context-specific signals is a fundamental question in developmental biology. In plants, both auxin and brassinosteroids (BRs) promote cell expansion, and it was thought that they activated this process through independent mechanisms. In this work, we describe a shared auxin:BR pathway required for seedling growth. Genetic, physiological, and genomic analyses demonstrate that response from one pathway requires the function of the other, and that this interdependence does not act at the level of hormone biosynthetic control. Increased auxin levels saturate the BR-stimulated growth response and greatly reduce BR effects on gene expression. Integration of these two pathways is downstream from BES1 and Aux/IAA proteins, the last known regulatory factors acting downstream of each hormone, and is likely to occur directly on the promoters of auxin:BR target genes. We have developed a new approach to identify potential regulatory elements acting in each hormone pathway, as well as in the shared auxin:BR pathway. We show that one element highly overrepresented in the promoters of auxin- and BR-induced genes is responsive to both hormones and requires BR biosynthesis for normal expression. This work fundamentally alters our view of BR and auxin signaling and describes a powerful new approach to identify regulatory elements required for response to specific stimuli.     

Molecular Cloning, Subcellular Localization and Functional Analysis of ThCLC-a from Thellungiella halophila

Often, nitrate is the major source of available nitrogen for plants. Nitrate can accumulate in central vacuoles via tonoplast transporters. In the present study, a gene termed ThCLC-a that encodes a chloride channel protein was isolated from Thellungiella halophila. Deduced amino acid sequence analysis revealed high identity with AtCLC-a. RT-PCR analysis showed that the ThCLC-a gene was expressed ubiquitously in all major organs and its expression was induced by nitrate treatment. Confocal microscopy using green fluorescent fusion proteins revealed that ThCLC-a was localized specifically to the tonoplast membrane. Furthermore, an RNAi construct expressing a ThCLC-a cDNA fragment was used to silence the endogenous ThCLC-a in T. halophila. HPLC analysis showed that the nitrate content in shoots or roots of silenced plants was 19–36 % lower than in wild-type plants. Transgenic Arabidopsis plants ectopically expressing the ThCLC-a gene could accumulate 15–21 % more nitrate content than wild type plants under limited nitrogen conditions. Finally, our results suggest ThCLC-a may play an important role in the transport of nitrate via the vacuolar membrane.     

Functional Expression and Subcellular Localization of the Aflatoxin Pathway Enzyme Ver-1 Fused to Enhanced Green Fluorescent Protein

Aflatoxin, a mycotoxin synthesized by Aspergillus spp., is among the most potent naturally occurring carcinogens known. Little is known about the subcellular organization of aflatoxin synthesis. Previously, we used transmission electron microscopy and immunogold labeling to demonstrate that the late aflatoxin enzyme OmtA localizes primarily to vacuoles in fungal cells on the substrate surface of colonies. In the present work, we monitored subcellular localization of Ver-1 in real time in living cells. Aspergillus parasiticus strain CS10-N2 was transformed with plasmid constructs that express enhanced green fluorescent protein (EGFP) fused to Ver-1. Analysis of transformants demonstrated that EGFP fused to Ver-1 at either the N or C terminus functionally complemented nonfunctional Ver-1 in recipient cells. Western blot analysis detected predominantly full-length Ver-1 fusion proteins in transformants. Confocal laser scanning microscopy demonstrated that Ver-1 fusion proteins localized in the cytoplasm and in the lumen of up to 80% of the vacuoles in hyphae grown for 48 h on solid media. Control EGFP (no Ver-1) expressed in transformants was observed in only 13% of the vacuoles at this time. These data support a model in which middle and late aflatoxin enzymes are synthesized in the cytoplasm and transported to vacuoles, where they participate in aflatoxin synthesis.     

香蕉MuMADS1基因表达产物的亚细胞定位

MuMADS1是从香蕉果实cDNA文库中筛选分离到的一个MADS—box基因．通过生物信息学分析表明,该基因编码的蛋白可能作为转录因子定位于细胞核中,而且芯片分析表明：该基因在果实成熟早期表达上调．是乙烯的上游调控因子,可能与花的发育、果实发育及成熟相关．为进一步深入研究该基因功能。构建了以绿色荧光蛋白（Green fluorescent protein．GFP）为报告基因的融合植物表达载体pCAMBIA1304 MuMADS1．利用基因枪转化法将重组载体转入洋葱表皮细胞瞬时表达．荧光显微镜检测结果表明。该基因表达产物定位于细胞核中．符合转录因子特性．     

香蕉Maasr1基因表达产物的亚细胞定位

利用SSH分离香蕉果实采后差异表达基因,获得香蕉的ASR基因,并将其命名为Maasr1。对该基因与香蕉采后成熟衰老进行相关性研究,发现其在果实采后早期表达上调。通过对Maasr1基因进行生物信息学分析表明,Maasr1基因编码的蛋白可能作为转录因子定位于细胞核或细胞质中。为进一步深入研究该基因功能,构建了香蕉Maasr1基因与绿色荧光蛋白基因融合的植物表达载体pCAMBIA1304-Maasr1。利用基因枪转化法将重组载体转入洋葱表皮细胞瞬时表达,荧光显微镜检测结果表明,Maasr1基因表达产物定位在细胞核中,符合转录因子特性。     

香蕉MaROP1基因表达产物的亚细胞定位

植物类Rho相关G蛋白(Rho-related GTPases from plants,ROP)属于小G蛋白超家族,是高等植物体内广泛存在的一类重要信号分子,在植物生长发育过程中起着关键的调控作用.本实验室从香蕉果实采后抑制差减杂交文库中获得一个香蕉ROP基因,命名为MaROP1.半定量RT-PCR表明该基因在香蕉的根、球茎、叶、花和果实中的表达存在差异,其中在球茎中的表达量最高且与其它器官的表达差异显著.为进一步研究该基因的功能,构建了以绿色荧光蛋白(green fluorescent protein,GFP)为报告基因的融合植物表达载体pCAMBIA1302-MaROP1,并利用基因枪转化法将重组载体转入洋葱表皮细胞瞬时表达,荧光显微镜检测表明该基因表达产物定位在细胞膜上.