Visualization of Nuclear Localization of Transcription Factors with Cyan and Green Fluorescent Proteins in the Red Alga Porphyra yezoensis

Uji, Toshiki; Takahashi, Megumu; Saga, Naotsune; Mikami, Koji

doi:10.1007/s10126-009-9210-5

Cited by 32 publications

(20 citation statements)

References 40 publications

(44 reference statements)

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“…The full-length cDNAs for the other isoforms were fused to the cDNA encoding ZsGreen1 and protein expression was imaged in living cells. ZsGreen1 fluorescent protein is derived from Zoanthus species [12] and can be fused to a different protein without interfering with the normal localization [13]. ZsGreen1 has high solubility, extremely bright emission, and rapid chromophore maturation in mammalian cells [14], [15].…”

Section: Resultsmentioning

confidence: 99%

Compartmentalization of Mammalian Pantothenate Kinases

et al. 2012

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The pantothenate kinases (PanK) catalyze the first and the rate-limiting step in coenzyme A (CoA) biosynthesis and regulate the amount of CoA in tissues by differential isoform expression and allosteric interaction with metabolic ligands. The four human and mouse PanK proteins share a homologous carboxy-terminal catalytic domain, but differ in their amino-termini. These unique termini direct the isoforms to different subcellular compartments. PanK1α isoforms were exclusively nuclear, with preferential association with the granular component of the nucleolus during interphase. PanK1α also associated with the perichromosomal region in condensing chromosomes during mitosis. The PanK1β and PanK3 isoforms were cytosolic, with a portion of PanK1β associated with clathrin-associated vesicles and recycling endosomes. Human PanK2, known to associate with mitochondria, was specifically localized to the intermembrane space. Human PanK2 was also detected in the nucleus, and functional nuclear localization and export signals were identified and experimentally confirmed. Nuclear PanK2 trafficked from the nucleus to the mitochondria, but not in the other direction, and was absent from the nucleus during G2 phase of the cell cycle. The localization of human PanK2 in these two compartments was in sharp contrast to mouse PanK2, which was exclusively cytosolic. These data demonstrate that PanK isoforms are differentially compartmentalized allowing them to sense CoA homeostasis in different cellular compartments and enable interaction with regulatory ligands produced in these same locations.

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Section: Resultsmentioning

confidence: 99%

Compartmentalization of Mammalian Pantothenate Kinases

et al. 2012

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“…RT-PCR using the PyAly-specific primers (PA-F1 and PA-R2, Table 1 ) and PrimeSTAR HS DNA polymerase (Takara) was performed as follows: initial incubation at 94 o C for 1 min, and 30 cyles of 94 o C for 30 sec, 55 o C for 30 sec, and 72 o C for 1 min then final incubation at 72 o C for 7 min. As an internal control, RT-PCR was performed with a specific primer pair PyElf1-F and PyElf1-R (Table 1 ) that can amplify a transcription elongation factor, PyElf1, of P. yezoensis [43]. …”

Section: Methodsmentioning

confidence: 99%

Characterization of an eukaryotic PL-7 Alginate Lyase in the Marine Red Alga Pyropia Yezoensis

Inoue

Mashino

Uji

et al. 2015

CBIOT

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Background: Alginate lyases belonging to polysaccharide lyase family-7 (PL-7) are the most well studied on their structures and functions among whole alginate lyases. However, all characterized PL-7 alginate lyases are from prokaryotic bacteria cells. Here we report the first identification of eukaryotic PL-7 alginate lyase from marine red alga Pyropia yezoensis.Methods: The cDNA encoding an alginate lyase PyAly was cloned and was used for the construction of recombinant PyAly (rPyAly) expression system in Escherichia coli. Purified rPyAly was assayed to identify its enzymatic properties. Its expression pattern in P. yessoensis was also investigated.Results: PyAly is likely a secreted protein consisting of an N-terminal signal peptide of 25 residues and a catalytic domain of 216 residues. The amino-acid sequence of the catalytic domain showed 19-29% identities to those of bacterial characterized alginate lyases classified into family PL-7. Recombinant PyAly protein, rPyAly, which was produced with E. coli BL21(DE3) by cold-inducible expression system, drastically decreased the viscosity of alginate solution in the early stage of reaction. The most preferable substrate for rPyAly was the poly(M) of alginate with an optimal temperature and pH at 35oC and 8.0, respectively. After reaction, unsaturated tri- and tetra-saccharides were produced from poly(M) as major end products. These enzymatic properties indicated that PyAly is an endolytic alginate lyase belonging to PL-7. Moreover, we found that the PyAly gene is split into 4 exons with 3 introns. PyAly was also specifically expressed in the gametophytic haplopid stage.Conclusion: This study demonstrates that PyAly in marine red alga P. yezoensis is a novel PL-7 alginate lyase with an endolytic manner. PyAly is a gametophyte-specifically expressed protein and its structural gene is composed of four exons and three introns. Thus, PyAly is the first enzymatically characterized eukaryotic PL-7 alginate lyase.

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“…1 and 2), indicating the enhancement of the expression level of the GUS reporter gene. Optimization of the codon usage of the reporter gene is therefore one of the important factors for successful expression in P. yezoensis cells (Fukuda et al, 2008;Mikami et al, 2009;Takahashi et al, 2010;Uji et al, 2010;Hirata et al, 2011).…”

Section: Codon-optimization Of the Reporter Genementioning

confidence: 99%