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.     

人肝脏辅酶Ⅱ依赖性视黄醇脱氢/还原酶的表达与细胞内定位

应用杆状病毒蛋白表达系统在Sf9细胞中对人肝脏辅酶Ⅱ依赖性视黄醇脱氢／还原酶(NADP-dependent retinol dehydrogenase／reductase,NRDR)进行表达。以纯化的重组蛋白质为材料,分析NRDR的催化活性和酶促反应性质。结果证实,人肝脏NRDR具有视黄醛还原酶活性,在辅酶Ⅱ存在条件下其催化视黄醛还原成视黄醇的Km值和Vmax值分别(2.8±0.24)μM和(0.468±0.036)μmol／(min·mg)。构建删除和不删除羧基端过氧化物酶体定位信号SRL序列的NRDR绿色荧光蛋白报告基因表达载体。转染HeLa细胞后,应用免疫荧光双染色的方法分析NRDR细胞内定位。结果表明NRDR定位在细胞内过氧化物酶体。羧基端SRL序列对NRDR定位到过氧化物酶体是必需的,删除SRL序列的NRDR分布在细胞质中。因此,人肝脏NRDR是存在于过氧化物酶体中的辅酶Ⅱ依赖性视黄醛还原酶。     

Molecular Characterization and Subcellular Localization of Arabidopsis Class VIII Myosin,ATM1

Land plants possess myosin classes VIII and XI. Although some information is available on the molecular properties of class XI myosins, class VIII myosins are not characterized. Here, we report the first analysis of the enzymatic properties of class VIII myosin. The motor domain of Arabidopsis class VIII myosin, ATM1 (ATM1-MD), and the motor domain plus one IQ motif (ATM1-1IQ) were expressed in a baculovirus system and characterized. ATM1-MD and ATM1-1IQ had low actin-activated Mg²⁺-ATPase activity (V_max = 4 s⁻¹), although their affinities for actin were high (K_actin = 4 μm). The actin-sliding velocities of ATM1-MD and ATM1-1IQ were 0.02 and 0.089 μm/s, respectively, from which the value for full-length ATM1 is calculated to be ∼0.2 μm/s. The results of actin co-sedimentation assay showed that the duty ratio of ATM1 was ∼90%. ADP dissociation from the actin·ATM1 complex (acto-ATM1) was extremely slow, which accounts for the low actin-sliding velocity, low actin-activated ATPase activity, and high duty ratio. The rate of ADP dissociation from acto-ATM1 was markedly biphasic with fast and slow phase rates (5.1 and 0.41 s⁻¹, respectively). Physiological concentrations of free Mg²⁺ modulated actin-sliding velocity and actin-activated ATPase activity by changing the rate of ADP dissociation from acto-ATM1. GFP-fused full-length ATM1 expressed in Arabidopsis was localized to plasmodesmata, plastids, newly formed cell walls, and actin filaments at the cell cortex. Our results suggest that ATM1 functions as a tension sensor/generator at the cell cortex and other structures in Arabidopsis.     

Characterization of RanBPM Molecular Determinants that Control Its Subcellular Localization

RanBPM/RanBP9 is a ubiquitous, nucleocytoplasmic protein that is part of an evolutionary conserved E3 ubiquitin ligase complex whose function and targets in mammals are still unknown. RanBPM itself has been implicated in various cellular processes that involve both nuclear and cytoplasmic functions. However, to date, little is known about how RanBPM subcellular localization is regulated. We have conducted a systematic analysis of RanBPM regions that control its subcellular localization using RanBPM shRNA cells to examine ectopic RanBPM mutant subcellular localization without interference from the endogenously expressed protein. We show that several domains and motifs regulate RanBPM nuclear and cytoplasmic localization. In particular, RanBPM comprises two motifs that can confer nuclear localization, one proline/glutamine-rich motif in the extreme N-terminus which has a dominant effect on RanBPM localization, and a second motif in the C-terminus which minimally contributes to RanBPM nuclear targeting. We also identified a nuclear export signal (NES) which mutation prevented RanBPM accumulation in the cytoplasm. Likewise, deletion of the central RanBPM conserved domains (SPRY and LisH/CTLH) resulted in the relocalization of RanBPM to the nucleus, suggesting that RanBPM cytoplasmic localization is also conferred by protein-protein interactions that promote its cytoplasmic retention. Indeed we found that in the cytoplasm, RanBPM partially colocalizes with microtubules and associates with α-tubulin. Finally, in the nucleus, a significant fraction of RanBPM is associated with chromatin. Altogether, these analyses reveal that RanBPM subcellular localization results from the combined effects of several elements that either confer direct transport through the nucleocytoplasmic transport machinery or regulate it indirectly, likely through interactions with other proteins and by intramolecular folding.     

Molecular Cloning, Expression and Subcellular Localization of a BiP Homolog from Rice Endosperm Tissue

The ER luminal binding protein, BiP, has been linked to prolamineprotein body formation in rice. To obtain further informationon the possible role of this chaperone in protein body formationwe have cloned and sequenced a BiP cDNA homolog from rice endosperm.The rice sequence is very similar to the maize BiP exhibiting92% nu-cleotide identity and 96% deduced amino acid sequenceidentity in the coding region. Substantial amino acid sequencehomology exists between rice BiP and BiP homo-logs from severalother plant and animal species including long stretches of conservationthrough the amino-terminal ATPase domain. Considerable variation,however, is observed within the putative carboxy-terminal peptide-bind-ingdomain between the plant and nonplant BiP sequences. A singleband of approximately 2.4 kb was visible when RNA gel blotsof total RNA purified from seed tissue were probed with radiolabeledrice BiP cDNA. This band increased in intensity during seeddevelopment up to 10 days after flowering, and then decreasedgradually until seed maturity. Protein gel blots indicated thatBiP polypeptide accumulation parallels that of the prolaminepolypeptides throughout seed development. Immunocytochemicalanalysis demonstrated that BiP is localized in a non-stochasticfashion in the endoplasmic reticulum membrane complex of developingendosperm cells. It is abundant on the periphery of the proteininclusion body but not in the central portion of the proteinbody or in the cisternal ER membranes connecting the proteinbodies. These data support a model which proposes that BiP associateswith the newly synthesized prolamine polypeptide to facilitateits folding and assembly into a protein inclusion body, andis then recycled. (Received October 21, 1996; Accepted January 20, 1997)     

日本七鳃鳗钙调蛋白分子克隆、组织定位及功能分析

钙调蛋白（calmodulin,CaM）是高度保守的钙离子结合蛋白质,可形成Ca ²⁺-CaM复合体,从而调节细胞代谢以及靶酶的功能。日本七鳃鳗（Lampetra japonica）作为原始的无颌类脊椎动物,对研究脊椎动物分子起源进化及器官发育分化具有重要的研究价值。通过提取日本七鳃鳗髓组织总RNA,利用RT-PCR方法获得日本七鳃鳗CaM（简称Lj-CaM）基因并进行生物信息学分析。将Lj-CaM基因分别构建到原核表达载体pColdⅠ和真核表达载体pEGFP-N1中,利用亲和层析技术纯化得到Lj-CaM蛋白。圆二色谱分析结果表明,Lj-CaM属于典型的α-螺旋结构型蛋白质。免疫印迹和免疫组化结果表明,CaM主要存在于日本七鳃鳗的肠、鳃、髓、肾组织中,在心和肝组织中几乎不表达。细胞免疫荧光结果显示,CaM定位于细胞核中。qPCR和免疫印迹方法检测发现,当293T细胞中Lj-CaM过表达时,对下游靶基因CaMKⅡ作用不明显,但促进PLA2G2A表达。本研究报道了日本七鳃鳗CaM结构、细胞组织定位分布以及基因调控研究,对其结构、分子起源与进化、分子调控及功能方面的研究奠定了基础。     

Nodule Nitrate Reductase as a Source of Reduced Nitrogen in Soybean, Glycine max

Hydroponically grown soybeans were fed ¹⁵N-enriched NaNO₃ at nine reproductive stages of development. The stem exudates contained excess ¹⁵N in the fully reduced nitrogen fraction. The soybean nodules had high nitrate reductase activity, whereas the roots had no detectable nitrate reductase activity. Based on these results, we concluded that the nodule nitrate reductase system has the potential of contributing significantly to the nitrogen economy of the plant.     

Molecular Cloning, Chromosomal Localization, and Cell Cycle-Dependent Subcellular Distribution of the A-Kinase Anchoring Protein, AKAP95

The cyclic AMP-dependent protein kinase (PKA) type II is directed to different subcellular loci through interaction of the RII subunits with A-kinase anchoring proteins (AKAPs). A full-length human clone encoding AKAP95 was identified and sequenced, and revealed a 692-amino acid open reading frame that was 89% homologous to the rat AKAP95 (V. M. Coghlan, L. K. Langeberg, A. Fernandez, N. J. Lamb, and J. D. Scott (1994)J. Biol. Chem.269, 7658–7665). The gene encoding AKAP95 was mapped to human chromosome 19p13.1-q12 using somatic cell hybrids and PCR. A fragment covering amino acids 414–692 of human AKAP95 was expressed inEscherichia coliand shown to bind RIIα. Competition with a peptide covering the RII-binding domain of AKAP Ht31 abolished RIIα binding to AKAP95. Immunofluorescence studies in quiescent human Hs-68 fibroblasts showed a nuclear localization of AKAP95, whereas RIIα was excluded from the nucleus. In contrast, during mitosis AKAP95 staining was markedly changed and appeared to be excluded from the condensed chromatin and localized outside the metaphase plate. Furthermore, the subcellular localizations of AKAP95 and RIIα overlapped in metaphase but started to segregate in anaphase and were again separated as AKAP95 reentered the nucleus in telophase. Finally, RIIα was coimmunoprecipitated with AKAP95 from HeLa cells arrested in mitosis, but not from interphase HeLa cells, demonstrating a physical association between these two molecules during mitosis. The results show a distinct redistribution of AKAP95 during mitosis, suggesting that the interaction between AKAP95 and RIIα may be cell cycle-dependent.     

Accumulation and Subcellular Localization of alpha-Galactosidase-Hemagglutinin in Developing Soybean Cotyledons

We have investigated the accumulation and intracellular localization of soybean (Glycine max [L.] Merr. cv Forrest) α-galactosidase-hemagglutinin during seed development. Cotyledon tissue was embedded in Lowicryl K4M and immunocytochemical localization was accomplished through treating thin sections with α-galactosidase antisera followed by indirect labeling with protein A coupled to colloidal gold. Gold particles were localized on the Golgi apparatus and protein bodies. We interpret this to indicate that α-galactosidase-hemagglutinin is transferred to and transported through the Golgi apparatus and finally deposited within the protein body by a Golgi apparatus-mediated process.     

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。     

Molecular Cloning and Functional Characterization of Oxidosqualene Cyclases from Panax vietnamensis

Panax vietnamensis is a valuable medicinal resource with promising preclinical applications. Ginsenosides, which are triterpenoids, are the primary active components in P. vietnamensis. Oxidosqualene cyclases (OSCs) catalyze the formation of the basic skeleton of triterpenes from 2,3-oxidosqualene, which is a crucial step in the biosynthesis of triterpenoids. The OSCs involved in triterpenoid biosynthesis in P. vietnamensis have not yet been characterized. Four OSC genes (PvOSC1–4) were cloned from P. vietnamensis and functionally characterized via heterologous expression in yeast. Transgenic yeast expressing PvOSC1, PvOSC3, and PvOSC4 produced the corresponding products β-amyrin, cycloartenol, and dammarenediol-II, respectively. PvOSC1, PvOSC3, and PvOSC4 are monofunctional OSCs. In this study, we characterized three PvOSC genes, providing a better understanding of the biosynthesis of triterpenoids in P. vietnamensis and the multiple choices of plant OSCs for metabolic engineering in yeast and other hosts.     

Subcellular Localization and Characterization of Neuronal Nitric Oxide Synthase

Abstract: In contrast to the predominantly participate, Ca²⁺/calmodulin-dependent nitric oxide (NO) synthase in endothelial cells, the corresponding neuronal isoenzyme is considered to be mainly soluble, presumably owing to the lack of a posttranslational myristoylation. However, preliminary findings from this and other laboratories suggest that a substantial portion of the neuronal NO synthase activity may in fact be membrane bound. We have therefore investigated the distribution of this enzyme among subcellular fractions of the rat and rabbit cerebellum in more detail. Up to 60% of the total NO synthase activity was found in the particulate fraction and, according to density gradient ultracentrifugation, associated mainly with the endoplasmic reticulum fraction. There was no apparent difference between the soluble and particulate enzymes with respect to their specific activity, Ca²⁺ and pH dependency, inhibitor sensitivity, or immunoreactivity, suggesting that both rat and rabbit cerebella contain a single Ca²⁺/calmodulin-dependent NO synthase. The inhibition by the cytochrome P450 inhibitor SKF-525A of the NO synthase activity in these subcellular fractions (IC₅₀= 90 μ M ) and the fact that mammalian cytochrome P450 enzymes are endoplasmic reticulum-bound proteins support the notion that the cerebellar NO synthase is a cytochrome P450-type hemoprotein. Moreover, the aforementioned findings suggest that posttranslational myristoylation may not be the only factor determining the intracellular localization of NO synthase.     

Leaf Nitrate Reductase, d-Ribulose-1,5-bisphosphate Carboxylase, and Root Nodule Development of Genetic Male-Sterile and Fertile Soybean Isolines

The objectives of this study were to determine the effect of pod and seed development on leaf chlorophyll concentration, and on activities of leaf ribulose bisphosphate carboxylase, leaf nitrate reductase, and root nodule acetylene reduction in field-grown soybean (Glycine max [L.] Merr.). Two genetic male-sterile lines and their fertile counterparts (Williams and Clark 63) were compared in both 1978 and 1979. Two additional lines (Wells × Beeson and Wells × Corsoy) were compared in 1979.

The expression of male-sterile character was nearly complete as very little outcrossing due to insect pollinators was observed. Male-sterile plants showed a delayed late season decline in leaf chlorophyll content and ribulose bisphosphate carboxylase activity when compared with fertile plants. A slight delay in the loss of in vivo leaf nitrate reductase activity was also observed for male-sterile plants. Root nodule fresh weight and acetylene reduction activity declined slightly more rapidly for fertile lines than for male-sterile lines in both years with differences significant on the last two to three sampling dates as leaf loss occurred in the control plants.

Seed development was found to increase slightly, the rate of decline of metabolic activity in fertile lines compared with that of male-sterile lines. However, pod development was not an a priori requirement for leaf and root nodule senescence. Male-sterile plants also lost photosynthetic and nitrogen metabolic competence, but at a slower rate. These results support the concept that pod and seed development does not signal monocarpic senescence per se but rather affects the rate at which senescence occurs after flowering.

     

Multiple Molecular Forms of Acetylcholine Receptors in Cultured Skeletal Muscle Cells: Subcellular Localization and Characterization

Abstract: Skeletal muscle cells of newborn rats, cultured in the absence of neuronal influence, were found to contain two types of cell surface acetylcholine receptors as demonstrated by isoelectric focusing. The isoelectric points of the two types of receptors were indistinguishable from those of junctional and extrajunctional types of receptors in mature animals. The cultured cells had two classes of intracellular α-bungarotoxin (αBT) binding components; one had the same sedimentation coefficient as that of surface receptors (9S), and the other had much smaller apparent molecular weights. Only a single major component was detected by isoelectric focusing analysis of the 9s intracellular aBT binding component, with a PI value close to that of the extra junctional receptor. These results suggest that the junctional and extrajunctional types of receptors may be synthesized through a common precursor.     

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.     

大青杨PubZIP1基因的克隆及亚细胞定位与抗旱表达特性分析

从大青杨中克隆得到PubZIP1基因,通过基因测序结果可知,PubZIP1基因全长1 083 bp,编码360个氨基酸。分析得出PubZIP1蛋白含有BZIP和DOG1两个结构域,其二级结构包括α-螺旋（62.50%）、无规卷曲（29.72%）、延伸链（5.56%）、β-折叠（2.22%）。通过亚细胞定位试验表明PubZIP1基因位于细胞核。通过qRT-PCR分析表明,在模拟干旱的不同7% PEG6000胁迫时间下,分析结果表明胁迫后PubZIP1基因在大青杨根中的表达量呈下降趋势。而在大青杨茎和叶片中的表达量呈上升趋势,尤其是在叶片中明显被诱导表达。预测该基因可能主要在叶片中表达并行使功能。     

Molecular Cloning and Functional Characterization of the Receptor for Clostridium perfringens Enterotoxin

A cDNA encoding the Clostridium perfringens enterotoxin receptor gene (CPE-R) was cloned from an expression library of enterotoxin-sensitive Vero cells. The nucleotide sequence of CPE-R showed that the enterotoxin receptor consists of 209 amino acids with a calculated molecular mass of 22,029 D. This receptor is highly hydrophobic, contains four putative transmembrane segments, and has significant similarity to the rat androgen withdrawal apoptosis protein RVP1 and the mouse oligodendrocyte specific protein, the functions of which are unknown. The expression of CPE-R was detected in the enterotoxin-sensitive Vero, Hep3B, and Intestine 407 cell lines, but not in the enterotoxin-insensitive K562 and JY cell lines. The CPE-R gene product expressed in enterotoxin-resistant L929 cells bound to enterotoxin specifically and directly and with high affinity and rendered the cells sensitive to the toxin, indicating that the cloned receptor is functional. Results showed that enterotoxin could not assemble into a complex with a defined structure unless it interacted with the receptor. From these results, it is proposed that the enterotoxin receptor is required for both target cell recognition and poreformation in the cell membrane.