Green Fluorescent Protein Fusion Constructs Research Articles

Development of Genetically Encoded Fluorescent KSR1-Based Probes to Track Ceramides during Phagocytosis.

Ceramides regulate phagocytosis; however, their exact function remains poorly understood. Here, we sought (1) to develop genetically encoded fluorescent tools for imaging ceramides, and (2) to use them to examine ceramide dynamics during phagocytosis. Fourteen enhanced green fluorescent protein (EGFP) fusion constructs based on four known ceramide-binding domains were generated and screened. While most constructs localized to the nucleus or cytosol, three based on the CA3 ceramide-binding domain of kinase suppressor of ras 1 (KSR1) localized to the plasma membrane or autolysosomes. C-terminally tagged CA3 with a vector-based (C-KSR) or glycine-serine linker (C-KSR-GS) responded sensitively and similarly to ceramide depletion and accumulation using a panel of ceramide modifying drugs, whereas N-terminally tagged CA3 (N-KSR) responded differently to a subset of treatments. Lipidomic and liposome microarray analysis suggested that, instead, N-KSR may preferentially bind glucosyl-ceramide. Additionally, the three probes showed distinct dynamics during phagocytosis. Despite partial autolysosomal degradation, C-KSR and C-KSR-GS accumulated at the plasma membrane during phagocytosis, whereas N-KSR did not. Moreover, the weak recruitment of C-KSR-GS to the endoplasmic reticulum and phagosomes was enhanced through overexpression of the endoplasmic reticulum proteins stromal interaction molecule 1 (STIM1) and Sec22b, and was more salient in dendritic cells. The data suggest these novel probes can be used to analyze sphingolipid dynamics and function in living cells.

The role of Bombyx mori Bmtutl-519 protein in the infection of BmN cells by Nosema bombycis

Bmtutl-519 is an isoform of the Bombyx Turtle protein and a member of the immunoglobulin superfamily (IgSF). The relative expression level of Bmtutl-519 was significantly upregulated when BmN cells were infected by Nosema bombycis. The subcellular localization of Bmtutl-519 was studied using an indirect immunoinfluscent assay (IFA), Co-localization assay, Western blotting, and enhanced green fluorescent protein (EGFP) fusion constructs expressed in BmN cells transfected with a Bmtutl-519 expression plasmid. The results indicate that Bmtutl-519 is distributed in both the cytoplasm and the cell membrane of BmN cells. Bmtutl-519 may be involved in the infection process of N. bombycis as a cell surface receptor or regulatory factor. Interaction analysis of Bmtutl-519 with NbSWP26, a spore wall protein of N. bombycis involved in host cell adherence and infection, showed that the C-terminal heparin-binding motif (HBM) of NbSWP26 mediates the interaction between these two proteins. Mutation of the NbSWP26 HBM at K208G, K209G, K210G, and K213G led to a loss of the ability to bind the Bmtutl-519 protein. Spore adherence and infection assays showed that Bmtutl-519 enhances the binding ability of N. bombycis to the host cell surface, but this did not enhance host cell infection by N. bombycis. In contrast, the sustained high expression of Bmtutl-519 in BmN cells inhibited the proliferation of N. bombycis spores.

Nictaba Homologs from Arabidopsis thaliana Are Involved in Plant Stress Responses.

Plants are constantly exposed to a wide range of environmental stresses, but evolved complicated adaptive and defense mechanisms which allow them to survive in unfavorable conditions. These mechanisms protect and defend plants by using different immune receptors located either at the cell surface or in the cytoplasmic compartment. Lectins or carbohydrate-binding proteins are widespread in the plant kingdom and constitute an important part of these immune receptors. In the past years, lectin research has focused on the stress-inducible lectins. The Nicotiana tabacum agglutinin, abbreviated as Nictaba, served as a model for one family of stress-related lectins. Here we focus on three non-chimeric Nictaba homologs from Arabidopsis thaliana, referred to as AN3, AN4, and AN5. Confocal microscopy of ArathNictaba enhanced green fluorescent protein (EGFP) fusion constructs transiently expressed in N. benthamiana or stably expressed in A. thaliana yielded fluorescence for AN4 and AN5 in the nucleus and the cytoplasm of the plant cell, while fluorescence for AN3 was only detected in the cytoplasm. RT-qPCR analysis revealed low expression for all three ArathNictabas in different tissues throughout plant development. Stress application altered the expression levels, but all three ArathNictabas showed a different expression pattern. Pseudomonas syringae infection experiments with AN4 and AN5 overexpression lines demonstrated a significantly higher tolerance of several transgenic lines to P. syringae compared to wild type plants. Finally, AN4 was shown to interact with two enzymes involved in plant defense, namely TGG1 and BGLU23. Taken together, our data suggest that the ArathNictabas represent stress-regulated proteins with a possible role in plant stress responses. On the long term this research can contribute to the development of more stress-resistant plants.

Combination of the Endogenous lhcsr1 Promoter and Codon Usage Optimization Boosts Protein Expression in the Moss Physcomitrella patens.

The moss Physcomitrella patens is used both as an evo-devo model and biotechnological production system for metabolites and pharmaceuticals. Strong in vivo expression of genes of interest is important for production of recombinant proteins, e.g., selectable markers, fluorescent proteins, or enzymes. In this regard, the choice of the promoter sequence as well as codon usage optimization are two important inside factors to consider in order to obtain optimum protein accumulation level. To reliably quantify fluorescence, we transfected protoplasts with promoter:GFP fusion constructs and measured fluorescence intensity of living protoplasts in a plate reader system. We used the red fluorescent protein mCherry under 2x 35S promoter control as second reporter to normalize for different transfection efficiencies. We derived a novel endogenous promoter and compared deletion variants with exogenous promoters. We used different codon-adapted green fluorescent protein (GFP) genes to evaluate the influence of promoter choice and codon optimization on protein accumulation in P. patens, and show that the promoter of the gene of P. patens chlorophyll a/b binding protein lhcsr1 drives expression of GFP in protoplasts significantly (more than twofold) better than the commonly used 2x 35S promoter or the rice actin1 promoter. We identified a shortened 677 bp version of the lhcsr1 promoter that retains full activity in protoplasts. The codon optimized GFP yields significantly (more than twofold) stronger fluorescence signals and thus demonstrates that adjusting codon usage in P. patens can increase expression strength. In combination, new promotor and codon optimized GFP conveyed sixfold increased fluorescence signal.

Evidence for the role of BmNPV Bm65 protein in the repair of ultraviolet-induced DNA damage.

It is unclear how, or to what extent, baculovirus DNA that has been damaged by ultraviolet (UV) light is repaired during infection and replication. In our previous study, expression of Bombyx mori nucleopolyhedrovirus (BmNPV) ORF Bm65, a homolog of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac79, correlated with decreased inactivation of virus by UV irradiation. In the current study, we accumulated more evidence pointing to a role for Bm65 in repair of UV-induced DNA damage. The localization of Bm65 was studied using enhanced green fluorescent protein (EGFP) fusion constructs expressed in BmN cells transfected with a Bm65 expression plasmid. The results indicate that Bm65-EGFP accumulates in the nucleus. A host cell reactivation assay showed that Bm65 significantly increased the expression of UV-damaged mCherry reporter gene. An assay measuring cyclobutane pyrimidine dimers (CPDs) in UV-irradiated BmN cells found that CPD quantity was decreased in cells transfected with a Bm65 expression plasmid. We also showed that after UVC treatment, the viability of Bm65-transfected cells was higher than that of egfp-transfected cells. These results suggest that Bm65 may be involved in the repair of baculovirus DNA that has been damaged by UV light.

Divergent roles of the Drosophila melanogaster globins

In contrast to long-held assumptions, the gene repertoire of most insects includes hemoglobins. Analyses of the genome of the fruitfly Drosophila melanogaster identified three distinct hemoglobin genes (glob1, glob2, and glob3). While glob1 is predominantly associated with the tracheal system and fat body, glob2 and glob3 are almost exclusively expressed in the testis. The physiological role of globins in Drosophila is uncertain. Here, we studied the functions of the three globins in a cell culture system. Drosophila Schneider 2 (S2) cells were stably transfected with each of the three globins and the empty vector as control. Under hypoxia (1% atmospheric O2), only glob1 overexpression enhanced the activity of mitochondrial oxidases and the ATP content. However, the positive effect of glob1 expression disappeared after 24h hypoxia, suggesting metabolic adaptations of the S2 cells. glob2 and glob3 had no positive effect on hypoxia-survival. After application of oxidative stress by H2O2, glob2 dramatically enhanced the viability of S2 cells. Evaluation of the intracellular localization of the globins using specific antibodies and green fluorescent protein-fusion constructs suggested that glob1 and glob2 most likely reside in the cytoplasm, while glob3 is associated with structures that may represent parts of the intracellular transport machinery. In silico analyses of public RNA-Seq data from different developmental stages provided that glob1 is co-expressed with genes of the aerobic energy metabolism, while glob2 and glob3 expression can be related to spermatogenesis and reproduction. Together, the results indicate divergent functions of the Drosophila globins: glob1 may play a role in the O2-dependent metabolism while glob2 may protect spermatogenesis from reactive oxygen species.

Functional characterization of plasma membrane-localized organic acid transporter (CsALMT1) involved in aluminum tolerance in Camelina sativa L.

Aluminum (Al) toxicity is a major limiting factor that inhibits root elongation and decreases crop production in acidic soils. The symptoms of inhibited root growth include a reduced uptake of nutrients because the roots become stubby and brittle. The release of organic anions from roots can protect a plant from Al toxicity. The mechanism relies on the efflux of organic anions, such as malate or citrate, which protect roots by chelating the Al3+. In this study, homologs of TaALMT1, a Camelina gene that encodes an aluminum-activated malate transporter, were investigated. The expression of this gene was induced by Al in the root, but not in the shoots. Using green fluorescent protein (GFP) fusion constructs and Western-blot analysis, we observed that CsALMT1 was localized in the plasma membrane. Also, to determine the degree to which Al tolerance was affected by malate secretion in Camelina root, we generated CsALMT1 overexpressing plants. CsALMT1 overexpressing transgenic plants showed a higher root elongation rate than the wild-type plant. Damaged cell staining analysis by hematoxylin under 25 µM Al treatment for 2, 4, and 6 h showed a pattern of less damage in CsALMT1 transgenic plants than in wild-type plant, especially in the root elongation zone. Furthermore, the rate of increase of secretion of organic acid in overexpressed plants after Al treatment was higher than that in the wild-type plant. In addition, in the Al-specific dye morin staining on root protoplast under 50 µM Al treatment, less Al accumulation was observed in the CsALMT1 transgenic plants than in the wild-type plant. The Al contents in the roots of the transgenic plants were at a lower level than those in the wild-type plant. These results show that the overexpression of CsALMT1 improves Al tolerance by increasing the release of malate from the root to the soil and, thereby, detoxifies the Al3+.

Overexpression of the receptor-like kinase gene OsNRRB enhances drought-stress tolerance in rice

Receptor-like kinases (RLKs) play essential roles in plant growth, development and responses to environmental stresses. Here, we report that a putative RLK gene OsNRRB (Os10g40100.2), which encodes OsNRRB protein with a kinase domain and a U-box domain, is cloned and characterized in rice (Oryza sativa L.). Tissue-specific expression of OsNRRB tested by qRT-PCR and promoter-GUS assays shows that OsNRRB is widely expressed in rice, including internodes, roots, leaves and developing seeds. Expression profile analysis showed that OsNRRB had different transcriptional responses to drought, salt and oxidative (H2O2) stresses, as well as ABA, SA and GA3 treatments. Furthermore, OsNRRB protein is located at the surface of protoplast cells by transient expressing OsNRRB-GFP (green fluorescent protein) fusion construct in rice protoplasts. Moreover, the transgenic seedlings with overexpressed OsNRRB show better tolerance to drought stress compared to wild-type seedlings. On the contrary, the RNAi transgenic seedlings are more sensitive to drought stress. OsNRRB may positively regulate drought stress tolerance through upregulating these stress-responsive genes in rice. Indeed, the expression of stress-responsive genes of OsbZIP23, OsDREB2A, OsP5CS and OsLea3 are upregulated by overexpression of OsNRRB, which in turn increasing drought tolerance in rice.

Identification and functional analysis of the ICK gene family in maize

Inhibitors of cyclin-dependent kinases (ICKs) are key regulators of cyclin-dependent kinase activities and cell division. Herein, we identified eight ICKs in maize, which we named Zeama;ICKs (ZmICKs). Primary sequencing and phylogenetic analyses were used to divide the ZmICK family into two classes: group B and group C. Subcellular localization analysis of ZmICK:enhanced green fluorescent protein (eGFP) fusion constructs in tobacco leaf cells indicated that ZmICKs are principally nuclear. Co-localization analysis of the ZmICKs and maize A-type cyclin-dependent kinase (ZmCDKA) was also performed using enhanced green fluorescent protein (eGFP) and red fluorescent protein (RFP) fusion constructs. The ZmICKs and ZmCDKA co-localized in the nucleus. Semi-quantitative RT-PCR analysis of the ZmICKs showed that they were expressed at different levels in all tissues examined and shared similar expression patterns with cell cycle-related genes. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that ZmICK1, ZmICK2, ZmICK3, and ZmICK4 interact with ZmCDKA1 and ZmCDKA3. Interestingly, ZmICK7 interacts with D-type cyclins. Transformed and expressed ZmCDKA1 and ZmICKs together in fission yeast revealed that ZmICK1, ZmICK3, and ZmICK4 can affect ZmCDKA1 function. Moreover, the C-group of ZmICKs could interact with ZmCDKA1 directly and affect ZmCDKA1 function, suggesting that C-group ZmICKs are important for cell division regulation.

Loss of the Chloroplast Transit Peptide from an Ancestral C3 Carbonic Anhydrase Is Associated with C4 Evolution in the Grass Genus Neurachne.

Neurachne is the only known grass lineage containing closely related C3, C3-C4 intermediate, and C4 species, making it an ideal taxon with which to study the evolution of C4 photosynthesis in the grasses. To begin dissecting the molecular changes that led to the evolution of C4 photosynthesis in this group, the complementary DNAs encoding four distinct β-carbonic anhydrase (CA) isoforms were characterized from leaf tissue of Neurachne munroi (C4), Neurachne minor (C3-C4), and Neurachne alopecuroidea (C3). Two genes (CA1 and CA2) each encode two different isoforms: CA1a/CA1b and CA2a/CA2b. Transcript analyses found that CA1 messenger RNAs were significantly more abundant than transcripts from the CA2 gene in the leaves of each species examined, constituting ∼99% of all β-CA transcripts measured. Localization experiments using green fluorescent protein fusion constructs showed that, while CA1b is a cytosolic CA in all three species, the CA1a proteins are differentially localized. The N. alopecuroidea and N. minor CA1a isoforms were imported into chloroplasts of Nicotiana benthamiana leaf cells, whereas N. munroi CA1a localized to the cytosol. Sequence analysis indicated an 11-amino acid deletion in the amino terminus of N. munroi CA1a relative to the C3 and C3-C4 proteins, suggesting that chloroplast targeting of CA1a is the ancestral state and that loss of a functional chloroplast transit peptide in N. munroi CA1a is associated with the evolution of C4 photosynthesis in Neurachne spp. Remarkably, this mechanism is homoplastic with the evolution of the C4-associated CA in the dicotyledonous genus Flaveria, although the actual mutations in the two lineages differ.

Heterologous expression of the Streptococcus pneumoniae yoeB and pezT toxin genes is lethal in Chlorella vulgaris

Chlorella vulgaris is a eukaryotic microalga with potential for the production of biofuels. However, its thick and rigid cell wall is an impediment to cost-effective, large-scale harvesting of biofuels from these cells. Bacterial toxin-antitoxin (TA) systems, comprising of a stable proteic toxin and its labile cognate antitoxin, have no known homologs in eukaryotic cells. Several bacterial TA toxins have been found to be lethal when expressed in eukaryotes such as yeasts, animal and human cell lines. In this study, the functionality of the yoeBSpn and pezT toxin genes from the Gram-positive bacterium Streptococcus pneumoniae in C. vulgaris was investigated using a two-component inducible expression system. The yoeBSpn and pezT toxin genes were each cloned as green fluorescent protein (GFP) fusion constructs and introduced into C. vulgaris by Agrobacterium tumefaciens-mediated co-transformation with recombinant activator and responder vectors. Following induction for the expression of the toxin-GFP fusion transgenes, GFP fluorescence was observed in the transformed C. vulgaris cells which also showed signs of cellular damage and lysis. This is the first report of the lethal expression of bacterial TA toxins in eukaryotic microalgae, which can form the basis of a novel method for harvesting of microalgal cellular contents.

Immunological and biochemical characterisation of 7ap, a short protein translated from an alternative frame of ORF7 of PRRSV.

Sequence analysis revealed a short alternative open reading frame (ORF) named ORF7a within the nucleocapsid gene of genetically divergent porcine reproductive and respiratory syndrome virus (PRRSV) genomes. Alignment of the corresponding protein sequences (named 7ap) revealed substantial heterogeneity among 7aps of different genotypes, though all of them are predicted to be positively charged. Green fluorescent protein and FLAG fusion constructs of ORF7a of the HU-14432/2011 PRRSV demonstrated that 7ap is expressed. 7ap of HU- 14432/2011 (Hu7ap) was synthesised chemically, and ELISA experiments revealed that Hu7ap binds strongly to mammalian IgGs. Protein-protein gel retardation assays and complement fixation inhibition suggest that 7aps bind to the CH2 domain of the IgG(Fc) fragment. Cellular localisation and immunological characteristics of PRRSV 7ap may indicate multiple functions including nuclear and cytoplasmic over-tuning of normal cellular processes and immunosuppression.

Identification of Ourmiavirus 30K movement protein amino acid residues involved in symptomatology, viral movement, subcellular localization and tubule formation.

Several plant viruses encode movement proteins (MPs) classified in the 30K superfamily. Despite a great functional diversity, alignment analysis of MP sequences belonging to the 30K superfamily revealed the presence of a central core region, including amino acids potentially critical for MP structure and functionality. We performed alanine-scanning mutagenesis of the Ourmia melon virus (OuMV) MP, and studied the effects of amino acid substitutions on MP properties and virus infection. We identified five OuMV mutants that were impaired in systemic infection in Nicotiana benthamiana and Arabidopsis thaliana, and two mutants showing necrosis and pronounced mosaic symptoms, respectively, in N. benthamiana. Green fluorescent protein fusion constructs (GFP:MP) of movement-defective MP alleles failed to localize in distinct foci at the cell wall, whereas a GFP fusion with wild-type MP (GFP:MPwt) mainly co-localized with plasmodesmata and accumulated at the periphery of epidermal cells. The movement-defective mutants also failed to produce tubular protrusions in protoplasts isolated from infected leaves, suggesting a link between tubule formation and the ability of OuMV to move. In addition to providing data to support the importance of specific amino acids for OuMV MP functionality, we predict that these conserved residues might be critical for the correct folding and/or function of the MP of other viral species in the 30K superfamily.

The involvement of hexokinase in the coordinated regulation of glucose and gibberellin on cell wall invertase and sucrose synthesis in grape berry.

In plants, hexokinase (HXK, EC 2.7.1.1), an enzyme normally involved in hexose phosphorylation, plays an important role in sugar sensing and signaling. The hexokinase activity of grape HXKs was confirmed by functional complementation of the hexokinase-deficient yeast strain YSH7.4-3C (hxk1, hxk2, glk1). HXK1 and HXK2 were able to complement this mutant. The subcellular localization of HXK1 and HXK2, observed with green fluorescent protein fusion constructs, indicated that HXK1 localized to the cytosol while HXK2 was a nuclear-targeted hexokinase. Gibberellin (GA3) control various processes across plant life and has been involved in sugar accumulation. The coordinated regulation of exogenous GA3 with Glc on CWINV, SuSy1, or SuSy2 expressions indicated that GA3 can relieve the repression of Glc on CWINV or SuSy1 expression, and the repression of GA3 on SuSy2 expression overrides the Glc-inductive effect, resulting in the down-regulation of SuSy2 expression. It was concluded that GA3 negatively interfere with Glc signal transduction depending on hexokinase phosphorylation. GA3 might regulate CWINV, SuSy1 or SuSy2 expression to in order to maintain an intracellular sugar levels and normal cell metabolism. Our results provide new insights into the crosstalk mechanism of GA3 and Glc signaling depending on hexokinase in grape berry sugar accumulation.

High-throughput analytical gel filtration screening of integral membrane proteins for structural studies

BackgroundStructural studies of integral membrane proteins (IMPs) are often hampered by difficulties in producing stable homogenous samples for crystallization. To overcome this hurdle it has become common practice to screen large numbers of target proteins to find suitable candidates for crystallization. For such an approach to be effective, an efficient screening strategy is imperative. To this end, strategies have been developed that involve the use of green fluorescent protein (GFP) fusion constructs. However, these approaches suffer from two drawbacks; proteins with a translocated C-terminus cannot be tested and scale-up from analytical to preparative purification is often non-trivial and may require re-cloning. MethodsHere we present a screening approach that prioritizes IMP targets based on three criteria: expression level, detergent solubilization yield and homogeneity as determined by high-throughput small-scale immobilized metal affinity chromatography (IMAC) and automated size-exclusion chromatography (SEC). ResultsTo validate the strategy, we screened 48 prokaryotic IMPs in two different vectors and two Escherichia coli strains. A set of 11 proteins passed all preset quality control checkpoints and was subjected to crystallization trials. Four of these crystallized directly in initial sparse matrix screens, highlighting the robustness of the strategy. ConclusionsWe have developed a rapid and cost efficient screening strategy that can be used for all IMPs regardless of topology. The analytical steps have been designed to be a good mimic of preparative purification, which greatly facilitates scale-up. General significanceThe screening approach presented here is intended and expected to help drive forward structural biology of membrane proteins.

Phosphatidylinositol 4,5-Bisphosphate (PtdIns(4,5)P2) Specifically Induces Membrane Penetration and Deformation by Bin/Amphiphysin/Rvs (BAR) Domains

Cellular proteins containing Bin/amphiphysin/Rvs (BAR) domains play a key role in clathrin-mediated endocytosis. Despite extensive structural and functional studies of BAR domains, it is still unknown how exactly these domains interact with the plasma membrane containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) and whether they function by a universal mechanism or by different mechanisms. Here we report that PtdIns(4,5)P(2) specifically induces partial membrane penetration of the N-terminal amphiphilic α-helix (H(0)) of two representative N-BAR domains from Drosophila amphiphysin (dAmp-BAR) and rat endophilin A1 (EndoA1-BAR). Our quantitative fluorescence imaging analysis shows that PtdIns(4,5)P(2)-dependent membrane penetration of H(0) is important for self-association of membrane-bound dAmp-BAR and EndoA1-BAR and their membrane deformation activity. EndoA1-BAR behaves differently from dAmp-BAR because the former has an additional amphiphilic α-helix that penetrates the membrane in a PtdIns(4,5)P(2)-independent manner. Depletion of PtdIns(4,5)P(2) from the plasma membrane of HEK293 cells abrogated the membrane deforming activity of EndoA1-BAR and dAmp-BAR. Collectively, these studies suggest that the local PtdIns(4,5)P(2) concentration in the plasma membrane may regulate the membrane interaction and deformation by N-BAR domain-containing proteins during clathrin-mediated endocytosis.

Strategies for strain improvement in Fusarium fujikuroi: overexpression and localization of key enzymes of the isoprenoid pathway and their impact on gibberellin biosynthesis

The rice pathogen Fusarium fujikuroi is known to produce a wide range of secondary metabolites, such as the pigments bikaverin and fusarubins, the mycotoxins fusarins and fusaric acid, and the phytohormones gibberellic acids (GAs), which are applied as plant growth regulators in agri- and horticulture. The development of high-producing strains is a prerequisite for the efficient biotechnological production of GAs. In this work, we used different molecular approaches for strain improvement to directly affect expression of early isoprenoid genes as well as GA biosynthetic genes. Overexpression of the first GA pathway gene ggs2, encoding geranylgeranyl diphosphate synthase 2, or additional integration of ggs2 and cps/ks, the latter encoding the bifunctional ent-copalyldiphosphate synthase/ent-kaurene synthase, revealed an enhanced production level of 150%. However, overexpression of hmgR and fppS, encoding the key enzymes of the mevalonate pathway, hydroxymethylglutaryl coenzyme A reductase, and farnesyldiphosphate synthase, resulted in a reduced production level probably due to a negative feedback regulation of HmgR. Subsequent deletion of the transmembrane domains of HmgR and overexpression of the remaining catalytic domain led to an increased GA content (250%). Using green fluorescent protein and mCherry fusion constructs, we localized Cps/Ks in the cytosol, Ggs2 in small point-like structures, which are not the peroxisomes, and HmgR at the endoplasmatic reticulum. In summary, it was shown for the first time that amplification or truncation of key enzymes of the isoprenoid and GA pathway results in elevated production levels (2.5-fold). Fluorescence microscopy revealed localization of the key enzymes in different compartments.

Nuclear-to-cytoplasmic Relocalization of the Proliferating Cell Nuclear Antigen (PCNA) during Differentiation Involves a Chromosome Region Maintenance 1 (CRM1)-dependent Export and Is a Prerequisite for PCNA Antiapoptotic Activity in Mature Neutrophils

Neutrophils are deprived of proliferative capacity and have a tightly controlled lifespan to avoid their persistence at the site of injury. We have recently described that the proliferating cell nuclear antigen (PCNA), a nuclear factor involved in DNA replication and repair of proliferating cells, is a key regulator of neutrophil survival. In neutrophils, PCNA was localized exclusively in the cytoplasm due to its nuclear-to-cytoplasmic relocalization during granulocytic differentiation. We showed here that leptomycin B, an inhibitor of the chromosome region maintenance 1 (CRM1) exportin, inhibited PCNA relocalization during granulocytic differentiation of HL-60 and NB4 promyelocytic cell lines and of human CD34(+) primary cells. Using enhanced green fluorescent protein fusion constructs, we have demonstrated that PCNA relocalization involved a nuclear export signal (NES) located from Ile-11 to Ile-23 in the PCNA sequence. However, this NES, located at the inner face of the PCNA trimer, was not functional in wild-type PCNA, but instead, was fully active and leptomycin B-sensitive in the monomeric PCNAY114A mutant. To test whether a defect in PCNA cytoplasmic relocalization would affect its antiapoptotic activity in mature neutrophils, a chimeric PCNA fused with the SV40 nuclear localization sequence (NLS) was generated to preclude its cytoplasmic localization. As expected, neutrophil-differentiated PLB985 cells expressing ectopic SV40NLS-PCNA had an increased nuclear PCNA as compared with cells expressing wild-type PCNA. Accordingly, the nuclear PCNA mutant did not show any antiapoptotic activity as compared with wild-type PCNA. Nuclear-to-cytoplasmic relocalization that occurred during myeloid differentiation is essential for PCNA antiapoptotic activity in mature neutrophils and is dependent on the newly identified monomerization-dependent PCNA NES.

Ciliary and non-ciliary expression and function of PACRGduring vertebrate development

BackgroundPark2-co-regulated gene (PACRG) is evolutionarily highly conserved from green algae to mammals. In Chlamydomonas and trypanosomes, the PACRG protein associates with flagella. Loss of PACRG results in shortened or absent flagella. In mouse the PACRG protein is required for spermatogenesis. The purpose of the present study was to analyze (1) the expression patterns of PACRG during vertebrate embryogenesis, and (2) whether the PACRG protein was required for left-right (LR) axis specification through cilia-driven leftward flow in Xenopus laevis.MethodsPACRG cDNAs were cloned and expression was analyzed during early embryonic development of Xenopus, mouse, rabbit and zebrafish. Antisense morpholino oligonucleotide (MO) mediated gene knockdown was applied in Xenopus to investigate LR development at the level of tissue morphology, leftward flow and asymmetric marker gene expression, using timelapse videography, scanning electron microscopy (SEM) and whole-mount in situ hybridization. Results were statistically evaluated using Wilcoxon paired and χ2 tests.ResultsPACRG mRNA expression was found in cells and tissues harboring cilia throughout the vertebrates. Highly localized expression was also detected in the brain. During early development, PACRG was specifically localized to epithelia where leftward flow arises, that is, the gastrocoel roof plate (GRP) in Xenopus, the posterior notochord (PNC) in mammals and Kupffer’s vesicle (KV) in zebrafish. Besides its association with ciliary axonemes, subcellular localization of PACRG protein was found around the nucleus and in a spotty pattern in the cytoplasm. A green fluorescent protein (GFP) fusion construct preferentially labeled cilia, rendering PACRG a versatile marker for live imaging. Loss-of-function in the frog resulted dose dependently in LR, neural tube closure and gastrulation defects, representing ciliary and non-ciliary functions of PACRG.ConclusionsThe PACRG protein is a novel essential factor of cilia in Xenopus.

Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea(L.) and their relevance for perfume manufacture

BackgroundSclareol is a diterpene natural product of high value for the fragrance industry. Its labdane carbon skeleton and its two hydroxyl groups also make it a valued starting material for semisynthesis of numerous commercial substances, including production of Ambrox® and related ambergris substitutes used in the formulation of high end perfumes. Most of the commercially-produced sclareol is derived from cultivated clary sage (Salvia sclarea) and extraction of the plant material. In clary sage, sclareol mainly accumulates in essential oil-producing trichomes that densely cover flower calices. Manool also is a minor diterpene of this species and the main diterpene of related Salvia species.ResultsBased on previous general knowledge of diterpene biosynthesis in angiosperms, and based on mining of our recently published transcriptome database obtained by deep 454-sequencing of cDNA from clary sage calices, we cloned and functionally characterized two new diterpene synthase (diTPS) enzymes for the complete biosynthesis of sclareol in clary sage. A class II diTPS (SsLPPS) produced labda-13-en-8-ol diphosphate as major product from geranylgeranyl diphosphate (GGPP) with some minor quantities of its non-hydroxylated analogue, (9 S, 10 S)-copalyl diphosphate. A class I diTPS (SsSS) then transformed these intermediates into sclareol and manool, respectively. The production of sclareol was reconstructed in vitro by combining the two recombinant diTPS enzymes with the GGPP starting substrate and in vivo by co-expression of the two proteins in yeast (Saccharomyces cerevisiae). Tobacco-based transient expression assays of green fluorescent protein-fusion constructs revealed that both enzymes possess an N-terminal signal sequence that actively targets SsLPPS and SsSS to the chloroplast, a major site of GGPP and diterpene production in plants.ConclusionsSsLPPS and SsSS are two monofunctional diTPSs which, together, produce the diterpenoid specialized metabolite sclareol in a two-step process. They represent two of the first characterized hydroxylating diTPSs in angiosperms and generate the dihydroxylated labdane sclareol without requirement for additional enzymatic oxidation by activities such as cytochrome P450 monoxygenases. Yeast-based production of sclareol by co-expresssion of SsLPPS and SsSS was efficient enough to warrant the development and use of such technology for the biotechnological production of scareol and other oxygenated diterpenes.