Subcellular Localization Determines the Stability and Axon Protective Capacity of Axon Survival Factor Nmnat2

Axons require a constant supply of the labile axon survival factor Nmnat2 from their cell bodies to avoid spontaneous axon degeneration. Here we investigate the mechanism of fast axonal transport of Nmnat2 and its site of action for axon maintenance. Using dual-colour live-cell imaging of axonal transport in SCG primary culture neurons, we find that Nmnat2 is bidirectionally trafficked in axons together with markers of the trans-Golgi network and synaptic vesicles. In contrast, there is little co-migration with mitochondria, lysosomes, and active zone precursor vesicles. Residues encoded by the small, centrally located exon 6 are necessary and sufficient for stable membrane association and vesicular axonal transport of Nmnat2. Within this sequence, a double cysteine palmitoylation motif shared with GAP43 and surrounding basic residues are all required for efficient palmitoylation and stable association with axonal transport vesicles. Interestingly, however, disrupting this membrane association increases the ability of axonally localized Nmnat2 to preserve transected neurites in primary culture, while re-targeting the strongly protective cytosolic mutants back to membranes abolishes this increase. Larger deletions within the central domain including exon 6 further enhance Nmnat2 axon protective capacity to levels that exceed that of the slow Wallerian degeneration protein, Wld^S. The mechanism underlying the increase in axon protection appears to involve an increased half-life of the cytosolic forms, suggesting a role for palmitoylation and membrane attachment in Nmnat2 turnover. We conclude that Nmnat2 activity supports axon survival through a site of action distinct from Nmnat2 transport vesicles and that protein stability, a key determinant of axon protection, is enhanced by mutations that disrupt palmitoylation and dissociate Nmnat2 from these vesicles.     

Arabidopsis thaliana PGR7 Encodes a Conserved Chloroplast Protein That Is Necessary for Efficient Photosynthetic Electron Transport

A significant fraction of a plant''s nuclear genome encodes chloroplast-targeted proteins, many of which are devoted to the assembly and function of the photosynthetic apparatus. Using digital video imaging of chlorophyll fluorescence, we isolated proton gradient regulation 7 (pgr7) as an Arabidopsis thaliana mutant with low nonphotochemical quenching of chlorophyll fluorescence (NPQ). In pgr7, the xanthophyll cycle and the PSBS gene product, previously identified NPQ factors, were still functional, but the efficiency of photosynthetic electron transport was lower than in the wild type. The pgr7 mutant was also smaller in size and had lower chlorophyll content than the wild type in optimal growth conditions. Positional cloning located the pgr7 mutation in the At3g21200 (PGR7) gene, which was predicted to encode a chloroplast protein of unknown function. Chloroplast targeting of PGR7 was confirmed by transient expression of a GFP fusion protein and by stable expression and subcellular localization of an epitope-tagged version of PGR7. Bioinformatic analyses revealed that the PGR7 protein has two domains that are conserved in plants, algae, and bacteria, and the N-terminal domain is predicted to bind a cofactor such as FMN. Thus, we identified PGR7 as a novel, conserved nuclear gene that is necessary for efficient photosynthetic electron transport in chloroplasts of Arabidopsis.     

Identification of an Apically-Located Antigen That Is Conserved in Sporozoan Parasites

ABSTRACT. Sporozoan parasites of the phylum Apicomplexa all possess common apical structures. The current study used a monoclonal antibody (mAb-E12) to identify a conserved antigen in the apical region of merozoites of seven species of Plasmodium (including rodent, primate and human pathogens), tachyzoites of Toxoplasma gondii , bradyzoites of Sarcocystis bovis , and sporozoites and merozoites of Eimeria tenella and E. acervulina. The antigen was also present in sporozoites of haemosporinid parasites. Immunofluorescence studies showed that the antigen was restricted to the apical 3rd of these invasive stages. Using immunoelectron microscopy, labeling was demonstrated in the region of the polar ring, below the paired inner membranes of the parasite pellicle, and near the subpellicular microtubules radiating from the polar ring of merozoites and sporozoites of E. tenella . The majority of the antigen could be extracted with 1% Triton-X 100, but a portion remained associated with the cytoskeletal elements. The molecule has a relative rate of migration (M_r) of 47,000 in Plasmodium spp. and 43–46,000 in coccidian species. Since the epitope recognized by mAb-El 2 is highly conserved, restricted to motile stages, and appears to be associated with microtubules, this antigen could be involved in cellular motility and cellular invasion.     

Messenger RNA 5′ NAD+ Capping Is a Dynamic Regulatory Epitranscriptome Mark That Is Required for Proper Response to Abscisic Acid in Arabidopsis

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Evidence for a Shared Nuclear Pore Complex Architecture That Is Conserved from the Last Common Eukaryotic Ancestor

The nuclear pore complex (NPC) is a macromolecular assembly embedded within the nuclear envelope that mediates bidirectional exchange of material between the nucleus and cytoplasm. Our recent work on the yeast NPC has revealed a simple modularity in its architecture and suggested a common evolutionary origin of the NPC and vesicle coating complexes in a progenitor protocoatomer. However, detailed compositional and structural information is currently only available for vertebrate and yeast NPCs, which are evolutionarily closely related. Hence our understanding of NPC composition in a full evolutionary context is sparse. Moreover despite the ubiquitous nature of the NPC, sequence searches in distant taxa have identified surprisingly few NPC components, suggesting that much of the NPC may not be conserved. Thus, to gain a broad perspective on the origins and evolution of the NPC, we performed proteomics analyses of NPC-containing fractions from a divergent eukaryote (Trypanosoma brucei) and obtained a comprehensive inventory of its nucleoporins. Strikingly trypanosome nucleoporins clearly share with metazoa and yeast their fold type, domain organization, composition, and modularity. Overall these data provide conclusive evidence that the majority of NPC architecture is indeed conserved throughout the Eukaryota and was already established in the last common eukaryotic ancestor. These findings strongly support the hypothesis that NPCs share a common ancestry with vesicle coating complexes and that both were established very early in eukaryotic evolution.Nearly all eukaryotic cells possess an extensive endomembrane system that is principally responsible for protein targeting and modification (1). The nucleus, the defining eukaryotic feature, is separated from the cytoplasm by a double bilayered nuclear envelope (NE)¹ that is contiguous with the rest of this endomembrane system via connections to the endoplasmic reticulum. Nuclear pore complexes (NPCs) fenestrate the NE, serving as the exclusive sites mediating exchange between the nucleoplasmic and cytoplasmic compartments. Macromolecules are chaperoned through the NPC by numerous transport factors. It has been proposed that the endomembrane system and nucleus have an autogenous origin (i.e. evolving from invaginations of an ancestral plasma membrane) and were established early in eukaryotic evolution (2).The composition of the NPC has been cataloged at ∼30 distinct nucleoporins (Nups) (3) for the yeast Saccharomyces cerevisiae (4) and vertebrates (5), two members of the Opisthokonta (animals, fungi, and closely related protists). Ultrastructural studies have identified objects morphologically similar (at a first approximation) to opisthokont NPCs in the other major eukaryote supergroups (6–8). However, very few data are available concerning the detailed NPC molecular composition and architecture for nearly all eukaryotic lineages, leaving a relatively narrow view of the “typical” NPC and its origins. A few examples of potential Nup orthologs beyond the opisthokonts have been reported, leading to the suggestion that substantial portions of the NPC may have an ancient, pre-last common eukaryotic ancestor (LCEA) origin (9). However, a more extensive study has concluded that LCEA possessed a primitive ancestral NPC that passed few components to its modern descendants (10).In yeast and vertebrates, the NPC consists of an eight-spoked core surrounding a central tube that serves as the conduit for macromolecular exchange. Each spoke can be divided into two similar nucleoplasmic and cytoplasmic halves. The eight spokes connect to form several coaxial rings: the membrane rings, the two outer rings at the nucleoplasmic and cytoplasmic periphery, and the two adjacent inner rings (11). Groups of Nups that we term “linker Nups” are attached between both sets of outer and inner rings. Another group of related proteins, collectively termed phenylalanine-glycine (FG) Nups, are largely exposed on the inner surface of the spokes and anchored either to the inner rings or to the linker Nups (11).Opisthokont Nups can be grouped into three structural classes (11, 12). The first class comprises membrane-bound proteins that anchor the NPC into the NE. The second class is the core scaffold Nups; these proteins constitute the bulk of the NPC mass, form the central tube, and provide the scaffold for the deployment of the third class of Nups across both faces of the NPC. The core scaffold Nups are remarkably restricted at the structural level and contain only three distinct arrangements of 2-fold types: proteins dominated by an α-solenoid fold (also termed a helix-turn-helix repeat domain), proteins consisting of a β-propeller fold, and finally proteins composed of an amino-terminal β-propeller fold followed by a carboxyl-terminal α-solenoid fold (which we here term a β-α structure) (12). FG Nups comprise the third class. These Nups carry multiply repeated degenerate “Phe-Gly” motifs (FG repeats) separated by hydrophilic or charged residues that form large unstructured domains. Each FG Nup also contains a small structured domain (often a coiled coil motif) that serves as the anchor site for interaction with the remainder of the NPC.Many transport factors belong to a structurally related protein family collectively termed karyopherins (Kaps) (13, 14). Transport across the NPC depends on the interactions between Kaps, cargo molecules, and the disordered repeat domains of FG Nups; the latter are thought to form the selective barrier for nucleocytoplasmic transport, guiding the Kap·cargo complexes (and other transport factors) through the central tube while excluding other macromolecules (for reviews, see Refs. 3 and 15–22).Significantly we have previously noted that the fold composition and arrangement of many of the core scaffold Nups are shared with proteins that form coating structures that participate in the generation and transport of vesicles between different endomembrane compartments; significantly many vesicle coating complex proteins and NPC scaffold Nups share an α-solenoid fold, β-propeller fold, or β-α structure (12, 23–28). These similarities gave rise to the “protocoatomer hypothesis,” which suggests a common ancestry for the NPC and these vesicle coat complexes. However, it is unclear how many, if any, of these particular core scaffold Nups are widely conserved, and hence it is unclear how general this potential relationship is throughout the Eukaryota. Thus, two scenarios are possible. The first is that the coatomer-like proteins are only found in a subset of the eukaryotes (including the opisthokonts), indicating that they are a relatively recent acquisition of only some eukaryotes and are not a general feature of all NPCs. The second is that the coatomer-like proteins are conserved in all eukaryotes, providing strong support to the protocoatomer hypothesis. To directly address this issue we characterized the NPC of Trypanosoma brucei, a highly divergent but experimentally tractable organism, using proteomics. The resulting data indicate an ancient origin for the majority of the NPC components and shed light on the origin of LCEA itself.     

Genome-Wide Mutagenesis Reveals That ORF7 Is a Novel VZV Skin-Tropic Factor

The Varicella Zoster Virus (VZV) is a ubiquitous human alpha-herpesvirus that is the causative agent of chicken pox and shingles. Although an attenuated VZV vaccine (v-Oka) has been widely used in children in the United States, chicken pox outbreaks are still seen, and the shingles vaccine only reduces the risk of shingles by 50%. Therefore, VZV still remains an important public health concern. Knowledge of VZV replication and pathogenesis remains limited due to its highly cell-associated nature in cultured cells, the difficulty of generating recombinant viruses, and VZV''s almost exclusive tropism for human cells and tissues. In order to circumvent these hurdles, we cloned the entire VZV (p-Oka) genome into a bacterial artificial chromosome that included a dual-reporter system (GFP and luciferase reporter genes). We used PCR-based mutagenesis and the homologous recombination system in the E. coli to individually delete each of the genome''s 70 unique ORFs. The collection of viral mutants obtained was systematically examined both in MeWo cells and in cultured human fetal skin organ samples. We use our genome-wide deletion library to provide novel functional annotations to 51% of the VZV proteome. We found 44 out of 70 VZV ORFs to be essential for viral replication. Among the 26 non-essential ORF deletion mutants, eight have discernable growth defects in MeWo. Interestingly, four ORFs were found to be required for viral replication in skin organ cultures, but not in MeWo cells, suggesting their potential roles as skin tropism factors. One of the genes (ORF7) has never been described as a skin tropic factor. The global profiling of the VZV genome gives further insights into the replication and pathogenesis of this virus, which can lead to improved prevention and therapy of chicken pox and shingles.     

盐爪爪Na^＋/H^＋逆向转运蛋白和焦磷酸酶的亚细胞定位

将盐爪爪Na+/H+逆向转运蛋白基因(KfNHX1)和焦磷酸酶基因(KfVP1)分别构建至植物表达载体,利用基因枪介导的方法转化洋葱表皮细胞,通过荧光显微镜观察研究其亚细胞定位.结果表明,转化了KfNHX1(或KfVP1)-GFP融合蛋白的洋葱表皮细胞仅膜系统散发荧光,而对照组即未转入KfNHX1(或KfVP1)基因的细胞则整体均匀发出荧光.说明KfNHX1和KfVP1可能定位于细胞的膜系统,作为跨膜转运蛋白在离子的调控运输中发挥重要作用.     

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.     

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。     

BRD7的亚细胞定位及其假定核输出信号序列的分离与鉴

BRD7被鉴定为一个鼻咽癌密切相关新基因和潜在的核转录调节因子．通过绿色荧光蛋白(GFP)介导的亚细胞定位方法,系统研究BRD7在非洲绿猴肾COS7细胞、人宫颈癌HeLa细胞以及人鼻咽癌HNE1细胞中的亚细胞定位,发现BRD7主要定位在细胞核,呈细点状或条梭状分布,3株细胞中没有明显的细胞类型差异．通过对BRD7编码蛋白氨基酸序列进行比对分析,发现了1个具有亮氨酸富集特征的假定核输出信号序列pNES,该区域具有类似核输出信号特征序列“ L-x(2,3)-［LIVFM］-x(2,3)-L-x-［LI］ "（X代表任意氨基酸）的结构;通过功能分析,发现它不具有介导异源蛋白GFP胞浆定位的功能,且其亚细胞定位或胞浆/胞核分布比例不受细霉素B(leptomycin B)干预的影响,说明这个pNES不具核输出信号结构域的功能,不是BRD7的核输出信号．     

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。