Search results for "Green fluorescent protein"

Sorting Motifs Involved in the Trafficking and Localization of the PIN1 Auxin Efflux Carrier

2016

In contrast with the wealth of recent reports about the function of μ-adaptins and clathrin adaptor protein (AP) complexes, there is very little information about the motifs that determine the sorting of membrane proteins within clathrin-coated vesicles in plants. Here, we investigated putative sorting signals in the large cytosolic loop of the Arabidopsis (Arabidopsis thaliana) PIN-FORMED1 (PIN1) auxin transporter, which are involved in binding μ-adaptins and thus in PIN1 trafficking and localization. We found that Phe-165 and Tyr-280, Tyr-328, and Tyr-394 are involved in the binding of different μ-adaptins in vitro. However, only Phe-165, which binds μA(μ2)- and μD(μ3)-adaptin, was found …

Enhancing heterologous protein expression and secretion in HEK293 cells by means of combination of CMV promoter and IFNα2 signal peptide

2016

Efficient production and secretion of recombinant proteins in mammalian cell lines relies in a combination of genetic, metabolic and culture strategy factors. The present work assesses the influence of two key genetic components of expression vectors (promoter and signal peptide) on protein production and secretion effciency in HEK293 cells expressing eGFP as a reporter protein. Firstly, the strength of 3 different promoters was evaluated using transient expression methods. Flow cytometry analysis revealed that the highest level of intracellular protein expression was found when eGFP was under the control of CMV promoter, being 3-times higher in comparison to the rest of the promoters teste…

Thioredoxin (Trxo1) interacts with proliferating cell nuclear antigen (PCNA) and its overexpression affects the growth of tobacco cell culture.

2017

Thioredoxins (Trxs), key components of cellular redox regulation, act by controlling the redox status of many target proteins, and have been shown to play an essential role in cell survival and growth. The presence of a Trx system in the nucleus has received little attention in plants, and the nuclear targets of plant Trxs have not been conclusively identified. Thus, very little is known about the function of Trxs in this cellular compartment. Previously, we studied the intracellular localization of PsTrxo1 and confirmed its presence in mitochondria and, interestingly, in the nucleus under standard growth conditions. In investigating the nuclear function of PsTrxo1 we identified proliferati…

2014

The Vipp1 protein is essential in cyanobacteria and chloroplasts for the maintenance of photosynthetic function and thylakoid membrane architecture. To investigate its mode of action we generated strains of the cyanobacteria Synechocystis sp. PCC6803 and Synechococcus sp. PCC7942 in which Vipp1 was tagged with green fluorescent protein at the C-terminus and expressed from the native chromosomal locus. There was little perturbation of function. Live-cell fluorescence imaging shows dramatic relocalisation of Vipp1 under high light. Under low light, Vipp1 is predominantly dispersed in the cytoplasm with occasional concentrations at the outer periphery of the thylakoid membranes. High light ind…

AtCCS is a functional homolog of the yeast copper chaperone Ccs1/Lys7

2005

AbstractIn plant chloroplasts two superoxide dismutase (SOD) activities occur, FeSOD and Cu/ZnSOD, with reciprocal regulation in response to copper availability. This system presents a unique model to study the regulation of metal-cofactor delivery to an organelle. The Arabidopsis thaliana gene AtCCS encodes a functional homolog to yeast Ccs1p/Lys7p, a copper chaperone for SOD. The AtCCS protein was localized to chloroplasts where it may supply copper to the stromal Cu/ZnSOD. AtCCS mRNA expression levels are upregulated in response to Cu-feeding and senescence. We propose that AtCCS expression is regulated to allow the most optimal use of Cu for photosynthesis.

Visualizing fungicide action: anin vivotool for rapid validation of fungicides with target location HOG pathway

2018

BACKGROUND The mitogen-activated protein kinase MoHog1p was fused with a green fluorescent protein (GFP) in the filamentous fungus Magnaporthe oryzae. The MoHOG1::GFP mutant was found to be an excellent tool visualizing in vivo fungicide-dependent translocation of MoHog1p into the nucleus. Validation of pathway specificity was achieved by generating fluorescence-labelled MoHog1p in the ΔMohik1 'loss of function' mutant strain. RESULTS GFP-labelled MoHog1p expressed in the wildtype and in ΔMohik1 demonstrates that fludioxonil is acting on the HOG pathway and even more precisely that fungicide action is dependent on the group III histidine kinase MoHik1p. GFP-tagged MoHog1p translocated into …

No inbreeding depression in sperm storage ability or offspring viability in Drosophila melanogaster females.

2013

Mating between relatives usually decreases genetic quality of progeny as deleterious recessive alleles are expressed in inbred individuals. Inbreeding degrades sperm traits but its effects on sperm storage and fate within females are currently unknown. We quantified the relationship between the degrees of inbreeding relevant to natural populations (f=0, 0.25 and 0.50) and the number of sperm inseminated and stored, sperm swimming speed, long-term sperm viability while in storage, pattern of sperm precedence, mating latency, and offspring viability of female Drosophila melanogaster. The use of transgenic flies that have either red or green fluorescent sperm heads allowed us to distinguish tw…

Postcopulatory sexual selection generates speciation phenotypes in Drosophila.

2013

Background: Identifying traits that reproductively isolate species and the selective forces underlying their divergence is a central goal of evolutionary biology and speciation research. There is growing recognition that postcopulatory sexual selection which can drive rapid diversification of interacting ejaculate and female reproductive tract traits that mediate sperm competition may be an engine of speciation. Conspecific sperm precedence (CSP) is a taxonomically widespread form of reproductive isolation but the selective causes and divergent traits responsible for CSP are poorly understood. Results: To test the hypothesis that postcopulatory sexual selection can generate reproductive iso…

In vivoanalysis of the lumenal binding protein (BiP) reveals multiple functions of its ATPase domain

2007

International audience; The endoplasmic reticulum (ER) chaperone binding protein (BiP) binds exposed hydrophobic regions of misfolded proteins. Cycles of ATP hydrolysis and nucleotide exchange on the ATPase domain were shown to regulate the function of the ligand-binding domain in vitro. Here we show that ATPase mutants of BiP with defective ATP-hydrolysis (T46G) or ATP-binding (G235D) caused permanent association with a model ligand, but also interfered with the production of secretory, but not cytosolic, proteins in vivo. Furthermore, the negative effect of BiP(T46G) on secretory protein synthesis was rescued by increased levels of wild-type BiP, whereas the G235D mutation was dominant. U…

2019

Abstract Tyrosine nitration is a post-translational protein modification relevant to various pathophysiological processes. Chemical nitration procedures have been used to generate and study nitrated proteins, but these methods regularly lead to modifications at other amino acid residues. A novel strategy employs a genetic code modification that allows incorporation of 3-nitrotyrosine (3-NT) during ribosomal protein synthesis to generate a recombinant protein with defined 3-NT-sites, in the absence of other post-translational modifications. This approach was applied to study the generation and stability of the 3-NT moiety in recombinant proteins produced in E.coli. Nitrated alpha-synuclein (…