Characterization and Subcellular Localization of Chlorophyllase from Ginkgo biloba

2006 ◽  
Vol 61 (1-2) ◽  
pp. 111-117 ◽  
Author(s):  
Atsushi Okazawa ◽  
Lei Tang ◽  
Yoshiko Itoh ◽  
Ei’ichiro Fukusaki ◽  
Akio Kobayashi
Keyword(s):  
Subcellular Localization ◽  
Ginkgo Biloba ◽  
Fluorescent Protein ◽  
Initial Step ◽  
Subcellular Fractionation ◽  
Natural Phenomena ◽  
Chl A ◽  
Green Fluorescent ◽  
Common Understanding

Abstract Chlorophyllase (Chlase) catalyzes the initial step of chlorophyll (Chl)-degradation, but the physiological significance of this reaction is still ambiguous. Common understanding of its role is that Chlase is involved in de-greening processes such as fruit ripening, leaf senescence, and flowering. But there is a possibility that Chlase is also involved in turnover and homeostasis of Chls. Among the de-greening processes, autumnal coloration is one of the most striking natural phenomena, but the involvement of Chlase during autumnal coloration is not clear. Previously, it was shown that Chlase activity and expression level of the Chlase gene were not increased during autumnal coloration in Ginkgo biloba, indicating that Chlase does not work specially in the de-greening processes in G. biloba. In this study, we characterized the recombinant Chlase and analyzed its subcellular localization to understand the role of the cloned Chlase of G. biloba (GbCLH). GbCLH exhibited its highest activity at pH 7.5, 40 °C. Kinetic analysis revealed that GbCLH hydrolyzes pheophytin (Pheo) a and Chl a more rapidly than Pheo b and Chl b. Transient expression analysis of 40 N-terminus amino acids of GbCLH fused with GFP (green fluorescent protein) and subcellular fractionation showed that GbCLH localizes within chloroplasts. Together with our previous results, property of GbCLH and its location within the chloroplasts suggest that GbCLH plays a role in the turnover and homeostasis of Chls in green leaves of G. biloba

scholarly journals Rice black-streaked dwarf virus P10 suppresses protein kinase C in insect vector through changing the subcellular localization of LsRACK1

2019 ◽  
Vol 374 (1767) ◽  
pp. 20180315 ◽  
Author(s):  
Lina Lu ◽  
Qi Wang ◽  
Deqing Huang ◽  
Qiufang Xu ◽  
Xueping Zhou ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Subcellular Localization ◽  
Fluorescent Protein ◽  
Brown Planthopper ◽  
Subcellular Fractionation ◽  
Dwarf Virus ◽  
Insect Vector ◽  
Kinase C ◽  
Green Fluorescent

Rice black-streaked dwarf virus (RBSDV) was known to be transmitted by the small brown planthopper (SBPH) in a persistent, circulative and propagative manner in nature. Here, we show that RBSDV major outer capsid protein (also known as P10) suppresses the protein kinase C (PKC) activity of SBPH through interacting with the receptor for activated protein kinase C 1 (LsRACK1). The N terminal of P10 (amino acids (aa) 1–270) and C terminal of LsRACK1 (aa 268–315) were mapped as crucial for the interaction. Confocal microscopy and subcellular fractionation showed that RBSDV P10 fused to enhanced green fluorescent protein formed vesicular structures associated with endoplasmic reticulum (ER) membranes in Spodoptera frugiperda nine cells. Our results also indicated that RBSDV P10 retargeted the initial subcellular localization of LsRACK1 from cytoplasm and cell membrane to ER and affected the function of LsRACKs to activate PKC. Inhibition of RACK1 by double stranded RNA-induced gene silencing significantly promoted the replication of RBSDV in SBPH. In addition, the PKC pathway participates in the antivirus innate immune response of SBPH. This study highlights that RACK1 negatively regulates the accumulation of RBSDV in SBPH through activating the PKC signalling pathway, and RBSDV P10 changes the subcellular localization of LsRACK1 and affects its function to activate PKC. This article is part of the theme issue ‘Biotic signalling sheds light on smart pest management’.

scholarly journals The N-terminus of the serum- and glucocorticoid-inducible kinase Sgk1 specifies mitochondrial localization and rapid turnover

2006 ◽  
Vol 399 (1) ◽  
pp. 69-76 ◽  
Author(s):  
Arne Engelsberg ◽  
Franziska Kobelt ◽  
Dietmar Kuhl
Keyword(s):  
Amino Acids ◽  
Subcellular Localization ◽  
Half Life ◽  
Fluorescent Protein ◽  
Subcellular Fractionation ◽  
Mitochondrial Fraction ◽  
Mouse Tissue ◽  
N Terminus ◽  
Green Fluorescent

The serine/threonine protein kinase Sgk1 (serum- and glucocorticoid-inducible kinase 1) is characterized by a short half-life and has been implicated in the control of a large variety of functions in different subcellular compartments and tissues. Here, we analysed the influence of the N-terminus of Sgk1 on protein turnover and subcellular localization. Using green fluorescent protein-tagged Sgk1 deletion variants, we identified amino acids 17–32 to function as an anchor for the OMM (outer mitochondrial membrane). Subcellular fractionation of mouse tissue revealed a predominant localization of Sgk1 to the mitochondrial fraction. A cytosolic orientation of the kinase at the OMM was determined by in vitro import of Sgk1 and protease protection assays. Pulse–chase experiments showed that half-life and subcellular localization of Sgk1 are inseparable and determined by identical amino acids. Our results provide evidence that Sgk1 is primarily localized to the OMM and shed new light on the role of Sgk1 in the control of cellular function.

scholarly journals The Vaccinia Virus B5 Protein Requires A34 for Efficient Intracellular Trafficking from the Endoplasmic Reticulum to the Site of Wrapping and Incorporation into Progeny Virions

2007 ◽  
Vol 82 (5) ◽  
pp. 2161-2169 ◽  
Author(s):  
Amalia K. Earley ◽  
Winnie M. Chan ◽  
Brian M. Ward
Keyword(s):  
Endoplasmic Reticulum ◽  
Vaccinia Virus ◽  
Intracellular Trafficking ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Subcellular Fractionation ◽  
Gfp Fluorescence ◽  
Virion Morphogenesis ◽  
Green Fluorescent

ABSTRACT The glycoproteins encoded by the vaccinia virus A34R and B5R genes are involved in intracellular envelope virus formation and are highly conserved among orthopoxviruses. A recombinant virus that has the A34R gene deleted and the B5R gene replaced with a B5R gene fused to the enhanced green fluorescent protein (B5R-GFP) gene was created (vB5R-GFP/ΔA34R) to investigate the role of A34 during virion morphogenesis. Cells infected with vB5R-GFP/ΔA34R displayed GFP fluorescence throughout the cytoplasm, which differed markedly from that seen in cells infected with a normal B5R-GFP-expressing virus (vB5R-GFP). Immunofluorescence and subcellular fractionation demonstrated that B5-GFP localizes with the endoplasmic reticulum in the absence of A34. Expression of either full-length A34 or a construct consisting of the lumenal and transmembrane domains restored normal trafficking of B5-GFP to the site of wrapping in the juxtanuclear region. Coimmunoprecipitation studies confirmed that B5 and A34 interact through their luminal domains, and further analysis revealed that in the absence of A34, B5 is not efficiently incorporated into virions released from the cell.

scholarly journals 20-HETE-mediated cytotoxicity and apoptosis in ischemic kidney epithelial cells

2008 ◽  
Vol 294 (3) ◽  
pp. F562-F570 ◽  
Author(s):  
Vani Nilakantan ◽  
Cheryl Maenpaa ◽  
Guangfu Jia ◽  
Richard J. Roman ◽  
Frank Park
Keyword(s):  
Caspase 3 ◽  
Fluorescent Protein ◽  
Ischemia Reperfusion ◽  
Atp Depletion ◽  
Cellular Damage ◽  
Apoptotic Pathway ◽  
Injury Model ◽  
Green Fluorescent

20-HETE, a metabolite of arachidonic acid, has been implicated as a mediator of free radical formation and tissue death following ischemia-reperfusion (IR) injury in the brain and heart. The present study examined the role of this pathway in a simulated IR renal injury model in vitro. Modified self-inactivating lentiviral vectors were generated to stably overexpress murine Cyp4a12 following transduction into LLC-PK1 cells (LLC-Cyp4a12). We compared the survival of control and transduced LLC-PK1 cells following 4 h of ATP depletion and 2 h of recovery in serum-free medium. ATP depletion-recovery of LLC-Cyp4a12 cells resulted in a significantly higher LDH release ( P < 0.05) compared with LLC-enhanced green fluorescent protein (EGFP) cells. Treatment with the SOD mimetic MnTMPyP (100 μM) resulted in decreased cytotoxicity in LLC-Cyp4a12 cells. The selective 20-HETE inhibitor HET-0016 (10 μM) also inhibited cytotoxicity significantly ( P < 0.05) in LLC-Cyp4a12 cells. Dihydroethidium fluorescence showed that superoxide levels were increased to the same degree in LLC-EGFP and LLC-Cyp4a12 cells after ATP depletion-recovery compared with control cells and that this increase was inhibited by MnTMPyP. There was a significant increase ( P < 0.05) of caspase-3 cleavage, an effector protease of the apoptotic pathway, in the LLC-Cyp4a12 vs. LLC-EGFP cells ( P < 0.05). This was abolished in the presence of HET-0016 ( P < 0.05) or MnTMPyP ( P < 0.01). These results demonstrate that 20-HETE overexpression can significantly exacerbate the cellular damage that is associated with renal IR injury and that the programmed cell death is mediated by activation of caspase-3 and is partially dependent on enhanced CYP4A generation of free radicals.

Defining the Role of Arginine 96 in Green Fluorescent Protein Fluorophore Biosynthesis†,‡

Biochemistry ◽  
2005 ◽  
Vol 44 (49) ◽  
pp. 16211-16220 ◽  
Author(s):  
Timothy I. Wood ◽  
David P. Barondeau ◽  
Chiharu Hitomi ◽  
Carey J. Kassmann ◽  
John A. Tainer ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent

Removal of Magnesium by Mg-dechelatase Is a Major Step in the Chlorophyll-Degrading Pathway in Ginkgo biloba in the Process of Autumnal Tints

2000 ◽  
Vol 55 (11-12) ◽  
pp. 923-926 ◽  
Author(s):  
Lei Tang ◽  
Atsushi Okazawa ◽  
Eiichiro Fukusaki ◽  
Akio Kobayashi
Keyword(s):  
Ginkgo Biloba ◽  
Hplc Analysis ◽  
Deciduous Trees ◽  
Natural Phenomena ◽  
Major Step ◽  
Pheophytin A ◽  
Chl A ◽  
Degrading Enzymes ◽  
Green Leaves ◽  
Related Compounds

Autumnal tints are one of the most manifest and fascinating natural phenomena, but the mechanism of chlorophyll (Chl)-breakdown in deciduous trees has not been fully elucidated. In this study, we analyzed the composition of Chl-related compounds and determined the activities of initial Chl-degrading enzymes in Ginkgo leaves at various stages in the process of autumnal coloring. Only pheophytin a (Pheo a, Mg-free Chl a) was detected in yellow leaves by HPLC analysis, and the activity of Mg-dechelatase in yellow leaves was found to be higher than in green leaves. These findings showed that the removal of magnesium from Chl a occurred in advance of dephytylation in the Ginkgo.

scholarly journals Targeting of Protein Kinase C-ϵ during Fcγ Receptor-dependent Phagocytosis Requires the ϵC1B Domain and Phospholipase C-γ1

2006 ◽  
Vol 17 (2) ◽  
pp. 799-813 ◽  
Author(s):  
Keylon L. Cheeseman ◽  
Takehiko Ueyama ◽  
Tanya M. Michaud ◽  
Kaori Kashiwagi ◽  
Demin Wang ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Fcγ Receptor ◽  
Kinase C ◽  
Green Fluorescent

Protein kinase C-ϵ (PKC-ϵ) translocates to phagosomes and promotes uptake of IgG-opsonized targets. To identify the regions responsible for this concentration, green fluorescent protein (GFP)-protein kinase C-ϵ mutants were tracked during phagocytosis and in response to exogenous lipids. Deletion of the diacylglycerol (DAG)-binding ϵC1 and ϵC1B domains, or the ϵC1B point mutant ϵC259G, decreased accumulation at phagosomes and membrane translocation in response to exogenous DAG. Quantitation of GFP revealed that ϵC259G, ϵC1, and ϵC1B accumulation at phagosomes was significantly less than that of intact PKC-ϵ. Also, the DAG antagonist 1-hexadecyl-2-acetyl glycerol (EI-150) blocked PKC-ϵ translocation. Thus, DAG binding to ϵC1B is necessary for PKC-ϵ translocation. The role of phospholipase D (PLD), phosphatidylinositol-specific phospholipase C (PI-PLC)-γ1, and PI-PLC-γ2 in PKC-ϵ accumulation was assessed. Although GFP-PLD2 localized to phagosomes and enhanced phagocytosis, PLD inhibition did not alter target ingestion or PKC-ϵ localization. In contrast, the PI-PLC inhibitor U73122 decreased both phagocytosis and PKC-ϵ accumulation. Although expression of PI-PLC-γ2 is higher than that of PI-PLC-γ1, PI-PLC-γ1 but not PI-PLC-γ2 consistently concentrated at phagosomes. Macrophages from PI-PLC-γ2-/-mice were similar to wild-type macrophages in their rate and extent of phagocytosis, their accumulation of PKC-ϵ at the phagosome, and their sensitivity to U73122. This implicates PI-PLC-γ1 as the enzyme that supports PKC-ϵ localization and phagocytosis. That PI-PLC-γ1 was transiently tyrosine phosphorylated in nascent phagosomes is consistent with this conclusion. Together, these results support a model in which PI-PLC-γ1 provides DAG that binds to ϵC1B, facilitating PKC-ϵ localization to phagosomes for efficient IgG-mediated phagocytosis.

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