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Koji Mikami Toshiki Uji Lin Li Megumu Takahashi Hajime Yasui Naotsune Saga 《Marine biotechnology (New York, N.Y.)》2009,11(5):563-569
Phosphoinositides (PIs) play important roles in signal transduction pathways and the regulation of cytoskeleton and membrane
functions in eukaryotes. Subcellular localization of individual PI derivative is successfully visualized in yeast, animal,
and green plant cells using PI derivative-specific pleckstrin homology (PH) domains fused with a variety of fluorescent proteins;
however, expression of fluorescent proteins has not yet been reported in any red algal cells. In the present study, we developed
the system to visualize these PIs using human PH domains fused with a humanized cyan fluorescent protein (AmCFP) in the red
alga Porphyra yezoensis. Plasma membrane localization of AmCFP fused with the PH domain from phospholipase Cδ1 and Akt1, but not Bruton’s tyrosine
kinase, was observed in cell wall-free monospores, demonstrating the presence of phosphatidylinositol-4,5-bisphosphate and
phosphatidylinositol-3,4-bisphosphate in P. yezoensis cells. This is the first report of the successful expression of fluorescent protein and the monitoring of PI derivatives
in red algal cells. Our system, based on transient expression of AmCFP, could be applicable for the analysis of subcellular
localization of other proteins in P. yezoensis and other red algal cells. 相似文献
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Nieva C Gwóźdź T Dutko-Gwóźdź J Wiedenmann J Spindler-Barth M Wieczorek E Dobrucki J Duś D Henrich V Ozyhar A Spindler KD 《Biological chemistry》2005,386(5):463-470
The heterodimer consisting of ecdysteroid receptor (EcR) and ultraspiracle (USP), both of which are members of the nuclear receptor superfamily, is considered to be the functional ecdysteroid receptor. Here we analyzed the subcellular distribution of EcR and USP fused to fluorescent proteins. The experiments were carried out in mammalian COS-7, CHO-K1 and HeLa cells to facilitate investigation of the subcellular trafficking of EcR and USP in the absence of endogenous expression of these two receptors. The distribution of USP tagged with a yellow fluorescent protein (YFP-USP) was almost exclusively nuclear in all cell types analyzed. The nuclear localization remained constant for at least 1 day after the first visible signs of expression. In contrast, the intracellular distribution of EcR tagged with a yellow fluorescent protein (YFP-EcR) varied and was dependent on time and cell type, although YFP-EcR alone was also able to partially translocate into the nuclear compartment. Coexpression of YFP-EcR with USP tagged with a cyan fluorescent protein (CFP-USP) resulted in exclusively nuclear localization of both proteins in all cell types analyzed. The USP-induced nuclear localization of YFP-EcR was stable for at least 20 hours. These experiments suggest that USP has a profound effect on the subcellular distribution of EcR. 相似文献
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To a great extent, the cellular compartmentalization and molecular interactions are indicative of the function of a protein.
The development of simple and efficient tools for testing the subcellular location of proteins is indispensable to elucidate
the function of genes in plants. In this report, we assessed the feasibility ofAgrobacterium-mediated transformation of hydroponically grown roots to follow intracellular targeting of proteins fused to green fluorescent
protein (GFP). We developed a simple in planta assay for subcellular localization of proteins inArabidopsis roots via transient transformation and tested this method by expressing a GFP fusion of a known nuclear protein, IQD1. Visualization
of transiently expressed GFP fusion proteins in roots by means of confocal microscopy is superior to the analysis of green
tissues because the roots are virtually transparent and free of chlorophyll autofluorescence. 相似文献
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The Arabidopsis genome contains seven cyclin-dependent kinase (CDK) inhibitors (ICK for inhibitor/interactor with cyclin-dependent kinase)
which share a small conserved C-terminal domain responsible for the CDK-inhibition activity by these proteins. Different ICK/KRPs
have been shown to have unique expression patterns within tissues, organs and during the cell cycle. Previous studies have
shown that overexpressing one of the ICK/KRPs inhibits CDK activity, cell division, and profoundly affects plant growth and
development. In this study, we investigated the subcellular localization of the seven Arabidopsis ICK proteins and domains responsible for this localization. Using transgenic expression in Arabidopsis plants and transient expression in tobacco leaf cells, all ICK/KRPs fused to green fluorescent protein (GFP) were localized
to the nucleus, suggesting that the nucleus is the cellular compartment for the plant CDK inhibitors to function. While ICK2/KRP2,
ICK4/KRP6, and ICK5/KRP7 were localized to the nucleoplasm in a homogeneous manner, ICK1/KRP1, ICK3/KRP5, ICK6/KRP3, and ICK7/KRP4
showed a punctate pattern of localization. A small motif conserved amongst the latter group of ICK/KRPs is required to confer
this subcellular pattern as deletion of this motif from ICK7/KRP4 resulted in a shift from a punctate to a homogeneous pattern
of localization. While a single nuclear localization signal (NLS) is responsible for the nuclear localization of ICK2/KRP2,
multiple mechanisms for nuclear localization are suggested to exist for the other six ICK/KRPs since deletion mutants lacking
predicted NLS motifs and the conserved C-terminal domain are still localized in the nucleus. 相似文献
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Both binding and activation of p38 mitogen-activated protein kinase (MAPK) play essential roles in regulation of the nucleocytoplasmic distribution of MAPK-activated protein kinase 5 by cellular stress 总被引:10,自引:0,他引:10
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Seternes OM Johansen B Hegge B Johannessen M Keyse SM Moens U 《Molecular and cellular biology》2002,22(20):6931-6945
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Plastid proteome prediction for diatoms and other algae with secondary plastids of the red lineage
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Peter G. Kroth E. Virginia Armbrust Thomas Mock 《The Plant journal : for cell and molecular biology》2015,81(3):519-528
The plastids of ecologically and economically important algae from phyla such as stramenopiles, dinoflagellates and cryptophytes were acquired via a secondary endosymbiosis and are surrounded by three or four membranes. Nuclear‐encoded plastid‐localized proteins contain N‐terminal bipartite targeting peptides with the conserved amino acid sequence motif ‘ASAFAP’. Here we identify the plastid proteomes of two diatoms, Thalassiosira pseudonana and Phaeodactylum tricornutum, using a customized prediction tool (ASAFind) that identifies nuclear‐encoded plastid proteins in algae with secondary plastids of the red lineage based on the output of SignalP and the identification of conserved ‘ASAFAP’ motifs and transit peptides. We tested ASAFind against a large reference dataset of diatom proteins with experimentally confirmed subcellular localization and found that the tool accurately identified plastid‐localized proteins with both high sensitivity and high specificity. To identify nucleus‐encoded plastid proteins of T. pseudonana and P. tricornutum we generated optimized sets of gene models for both whole genomes, to increase the percentage of full‐length proteins compared with previous assembly model sets. ASAFind applied to these optimized sets revealed that about 8% of the proteins encoded in their nuclear genomes were predicted to be plastid localized and therefore represent the putative plastid proteomes of these algae. 相似文献