Dimerization of N-methyltransferases involved in caffeine biosynthesis

Kodama, Yutaka; Shinya, Tomotaka; Sano, Hiroshi

doi:10.1016/j.biochi.2007.10.001

Cited by 18 publications

(21 citation statements)

References 22 publications

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“…cDNA fragments of CaMXMT1 (Uefuji et al 2003) were subcloned into the BsrGI/NotI sites of the above respective vectors, and CN155-MXMT, CC155-MXMT, GN155-MXMT, GC155-MXMT, RN168-MXMT and RC169-MXMT fusion genes driven by the CaMV35S promoter were constructed. The pSY736-CaMXMT1 encoding YN154-MXMT and pSY735-CaMXMT1 encoding YC155-MXMT were used as YFP-based BiFC vectors (Kodama et al 2008) ( Table 1). cDNA fragments of CaDXMT1 (Uefuji et al 2003) were subcloned into the BsrGI site of pBRN168 and pBRC169 vectors by the in-fusion method (Clontech), constructing the pBRN168-DXMT and the pBRC169-DXMT vectors, respectively (Table 1).…”

Section: Methodsmentioning

confidence: 99%

Simultaneous visualization of two protein complexes in a single plant cell using multicolor fluorescence complementation analysis

Kodama

Wada

2009

Plant Mol Biol

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Bimolecular fluorescence complementation (BiFC) is an approach used to analyze protein-protein interaction in vivo, in which non-fluorescent N-terminal and C-terminal fragments of a fluorescent protein are reconstituted to emit fluorescence only when they are brought together by interaction of two proteins to fuse both fragments. A method for simultaneous visualization of two protein complexes by multicolor BiFC with fragments from green fluorescent protein (GFP) and its variants such as cyan and yellow fluorescent proteins (CFP and YFP) was recently reported in animal cells. In this paper we describe a new strategy for simultaneous visualization of two protein complexes in plant cells using the multicolor BiFC with fragments from CFP, GFP, YFP and a red fluorescent protein variant (DsRed-Monomer). We identified nine different BiFC complexes using fragments of CFP, GFP and YFP, and one BiFC complex using fragments of DsRed-Monomer. Fluorescence complementation did not occur by combinations between fragments of GFP variants and DsRed-Monomer. Based on these findings, we achieved simultaneous visualization of two protein complexes in a single plant cell using two colored fluorescent complementation pairs (cyan/red, green/red or yellow/red).

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Section: Methodsmentioning

confidence: 99%

Simultaneous visualization of two protein complexes in a single plant cell using multicolor fluorescence complementation analysis

Kodama

Wada

2009

Plant Mol Biol

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“…The details of the BiFC fragments used in this study are listed (Table 1). Because the 7-methylxanthine methyltransferase (MXMT) protein from Coffea arabica (Uefuji et al 2003) forms a homodimer that can be visualized by the BiFC assay (Kodama et al 2008;Kodama and Wada 2009), it was fused with either GN, GC or CC (Table 2). Combinations of appropriate BiFC plasmids (1 mg each) were co-bombarded with gold particles into onion (Allium cepa) epidermal cell layers.…”

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confidence: 99%

A bright green-colored bimolecular fluorescence complementation assay in living plant cells

Kodama

2011

Plant Biotechnology

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“…Since the 7-methylxanthine methyltransferase (MXMT) protein from Coffea Arabica forms a homodimer that can be visualized using the BiFC assay (Kodama et al 2008(Kodama et al , 2009, it was fused with either the N-or C-terminal fragments of each GFP variant (abbreviated as GN and GC, respectively). Each GFP variant was split between A155 and D156: psGN, psGC, eGN, eGC, frGN, frGC, sfGN and sfGC (Figures 1, 3A).…”

Section: Bifc With Gfp Variants In Plant Cellsmentioning

confidence: 99%

In planta comparative analysis of improved green fluorescent proteins with reference to fluorescence intensity and bimolecular fluorescence complementation ability

Fujii

Kodama

2015

Plant Biotechnology

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Green fluorescent protein (GFP) was discovered from the jellyfish Aequorea victoria, and several improvements have been carried out to change its physicochemical properties. The resulting improved GFP variants have been used as reporter proteins for bioimaging techniques in various research fields including plant science. Almost all GFP variants were developed using Escherichia coli to improve fluorescence properties in mammalian cells, but the impact in other organisms such as plant cells remains to be determined. In this study, we performed comparative analysis of four improved GFP variants, GFP-S65T, eGFP, frGFP and sfGFP, with reference to the fluorescence intensity in Arabidopsis protoplasts, and found that sfGFP is the brightest. Using non-fluorescent fragments from the GFP variants, we also conducted bimolecular fluorescence complementation (BiFC) assays to find appropriate fragment pairs of GFP-based BiFC for visualization of protein-protein interactions in living plant cells. Our observations revealed that the brightest is the sfGFP-based BiFC. Further, as an evaluation method for the sfGFP-based BiFC, a BiFC competition assay was successfully completed for the first time in planta. The present study provides useful information for selection and improvement of the GFP molecule and its application to BiFC technology in plants.Key words: Arabidopsis, BiFC, BiFC competition, GFP, protein-protein interaction.Since the first attempt to employ wild-type green fluorescent protein (wtGFP) as a reporter protein, it has been developed for bioimaging techniques in various research fields including plant science (Chalfie et al. 1994;Heim et al. 1995). wtGFP is a typical β-barrel structure harboring a chromophore spontaneously formed by the three residues (S65-T66-G67), and thereby exhibits green fluorescence without any cofactor (Tsien 1998). To date, wtGFP has been improved by targeted or random mutations to increase its fluorescence intensity, and almost all of the improvements were performed using Escherichia coli for suitable expression in mammalian cells (Shaner et al. 2005). The first improvement was a single mutation of serine to threonine residue at position 65 (S65T) for the chromophore (Figure 1); the mutation shifted the excitation peak to 490 nm from 395 nm and 470 nm of wtGFP (Heim et al. 1995). Compared with wtGFP, this peak-shifted GFP (psGFP) gives 6-fold brighter Abbreviations: BiFC, bimolecular fluorescence complementation; eGC, the C-terminal fragment of eGFP; eGFP, enhanced GFP; eGN, the N-terminal fragment of eGFP; EYFP, enhanced yellow fluorescent protein; frGC, the C-terminal fragment of frGFP; frGFP, folding reporter GFP; frGN, the N-terminal fragment of frGFP; GFP, green fluorescent protein; MXMT, 7-methylxanthine methyltransferase; psGC, the C-terminal fragment of psGFP; psGFP, peak-shifted GFP; psGN, the N-terminal fragment of psGFP; PEG, polyethylene glycol; RC, the C-terminal fragment of DsRED monomer; sfGC, the C-terminal fragment of sfGFP; sfGFP, superfolder GFP; sfGN, the N-...

show abstract

Dimerization of N-methyltransferases involved in caffeine biosynthesis

Cited by 18 publications

References 22 publications

Simultaneous visualization of two protein complexes in a single plant cell using multicolor fluorescence complementation analysis

Simultaneous visualization of two protein complexes in a single plant cell using multicolor fluorescence complementation analysis

A bright green-colored bimolecular fluorescence complementation assay in living plant cells

In planta comparative analysis of improved green fluorescent proteins with reference to fluorescence intensity and bimolecular fluorescence complementation ability

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