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Vectors for multi-color bimolecular fluorescence complementation to investigate protein-protein interactions in living plant cells.

Lee LY, Fang MJ, Kuang LY, Gelvin SB - Plant Methods (2008)

Bottom Line: Additional expression of mCherry indicates transfected cells and sub-cellular structures.Using this system, we have determined in both tobacco BY-2 protoplasts and in onion epidermal cells that Agrobacterium VirE2 protein interacts with the Arabidopsis nuclear transport adapter protein importin alpha-1 in the cytoplasm, whereas interaction of VirE2 with a different importin alpha isoform, importin alpha-4, occurs predominantly in the nucleus.The vectors we have constructed and tested will facilitate the study of protein-protein interactions in many different plant systems.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA. zofangmj@gate.sinica.edu.tw.

ABSTRACT

Background: The investigation of protein-protein interactions is important for characterizing protein function. Bimolecular fluorescence complementation (BiFC) has recently gained interest as a relatively easy and inexpensive method to visualize protein-protein interactions in living cells. BiFC uses "split YFP" tags on proteins to detect interactions: If the tagged proteins interact, they may bring the two split fluorophore components together such that they can fold and reconstitute fluorescence. The sites of interaction can be monitored using epifluorescence or confocal microscopy. However, "conventional" BiFC can investigate interactions only between two proteins at a time. There are instances when one may wish to offer a particular "bait" protein to several "prey" proteins simultaneously. Preferential interaction of the bait protein with one of the prey proteins, or different sites of interaction between the bait protein and multiple prey proteins, may thus be observed.

Results: We have constructed a series of gene expression vectors, based upon the pSAT series of vectors, to facilitate the practice of multi-color BiFC. The bait protein is tagged with the C-terminal portion of CFP (cCFP), and prey proteins are tagged with the N-terminal portions of either Venus (nVenus) or Cerulean (nCerulean). Interaction of cCFP-tagged proteins with nVenus-tagged proteins generates yellow fluorescence, whereas interaction of cCFP-tagged proteins with nCerulean-tagged proteins generates blue fluorescence. Additional expression of mCherry indicates transfected cells and sub-cellular structures. Using this system, we have determined in both tobacco BY-2 protoplasts and in onion epidermal cells that Agrobacterium VirE2 protein interacts with the Arabidopsis nuclear transport adapter protein importin alpha-1 in the cytoplasm, whereas interaction of VirE2 with a different importin alpha isoform, importin alpha-4, occurs predominantly in the nucleus.

Conclusion: Multi-color BiFC is a useful technique to determine interactions simultaneously between a given" bait" protein and multiple "prey" proteins in living plant cells. The vectors we have constructed and tested will facilitate the study of protein-protein interactions in many different plant systems.

No MeSH data available.


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Schematic diagrams of the multi-color BiFC vectors. A, Vectors for tagging proteins at their N-termini with autofluorescent protein fragments; B and C, Vectors for tagging proteins at their C-termini with autofluorescent protein fragments. The pSAT-NA series described in Panel C have the upstream NcoI site deleted. Note that an ORF fragment tailored to maintain the open reading frame with the autofluorescent protein fragment of the pSAT-C vectors will be out of frame if ligated into pSAT-N vectors.
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Figure 1: Schematic diagrams of the multi-color BiFC vectors. A, Vectors for tagging proteins at their N-termini with autofluorescent protein fragments; B and C, Vectors for tagging proteins at their C-termini with autofluorescent protein fragments. The pSAT-NA series described in Panel C have the upstream NcoI site deleted. Note that an ORF fragment tailored to maintain the open reading frame with the autofluorescent protein fragment of the pSAT-C vectors will be out of frame if ligated into pSAT-N vectors.

Mentions: To facilitate the use of multi-color BiFC in plants, we adapted a previously-described series of pSAT vectors [8]. The pSAT vectors [11] are built in modular fashion, with rare-cutting restriction endonuclease or homing endonuclease sites surrounding an "expression cassette". Each "set" of pSAT vectors is flanked by different rare-cutting sites, and includes a double Cauliflower Mosaic Virus (CaMV) promoter, a Tobacco Etch Virus (TEV) translational leader, a multi-cloning site either preceding or following an autofluorescent protein N- or C-terminal fragment, and a CaMV polyA addition signal. Shyu et al. [12] showed that pairing the C-terminal fragment of cyan fluorescent protein (cCFP) with either the N-terminal fragment of Cerulean (nCerulean) or the N-terminal fragment of Venus (nVenus) results in more intense blue or yellow fluorescence, respectively, than using other GFP-derived autofluorescent protein fragments. "Overlapping" the N- and C-terminal autofluorescent protein fragments additionally increased signal intensity. We therefore used nVenus or nCerulean fragments from amino acids 1–173, and cCFP from amino acids 155–238. Figures 1A–C show the final constructions. Table 1 lists the currently available multi-color BiFC vectors that we have constructed.


Vectors for multi-color bimolecular fluorescence complementation to investigate protein-protein interactions in living plant cells.

Lee LY, Fang MJ, Kuang LY, Gelvin SB - Plant Methods (2008)

Schematic diagrams of the multi-color BiFC vectors. A, Vectors for tagging proteins at their N-termini with autofluorescent protein fragments; B and C, Vectors for tagging proteins at their C-termini with autofluorescent protein fragments. The pSAT-NA series described in Panel C have the upstream NcoI site deleted. Note that an ORF fragment tailored to maintain the open reading frame with the autofluorescent protein fragment of the pSAT-C vectors will be out of frame if ligated into pSAT-N vectors.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2572157&req=5

Figure 1: Schematic diagrams of the multi-color BiFC vectors. A, Vectors for tagging proteins at their N-termini with autofluorescent protein fragments; B and C, Vectors for tagging proteins at their C-termini with autofluorescent protein fragments. The pSAT-NA series described in Panel C have the upstream NcoI site deleted. Note that an ORF fragment tailored to maintain the open reading frame with the autofluorescent protein fragment of the pSAT-C vectors will be out of frame if ligated into pSAT-N vectors.
Mentions: To facilitate the use of multi-color BiFC in plants, we adapted a previously-described series of pSAT vectors [8]. The pSAT vectors [11] are built in modular fashion, with rare-cutting restriction endonuclease or homing endonuclease sites surrounding an "expression cassette". Each "set" of pSAT vectors is flanked by different rare-cutting sites, and includes a double Cauliflower Mosaic Virus (CaMV) promoter, a Tobacco Etch Virus (TEV) translational leader, a multi-cloning site either preceding or following an autofluorescent protein N- or C-terminal fragment, and a CaMV polyA addition signal. Shyu et al. [12] showed that pairing the C-terminal fragment of cyan fluorescent protein (cCFP) with either the N-terminal fragment of Cerulean (nCerulean) or the N-terminal fragment of Venus (nVenus) results in more intense blue or yellow fluorescence, respectively, than using other GFP-derived autofluorescent protein fragments. "Overlapping" the N- and C-terminal autofluorescent protein fragments additionally increased signal intensity. We therefore used nVenus or nCerulean fragments from amino acids 1–173, and cCFP from amino acids 155–238. Figures 1A–C show the final constructions. Table 1 lists the currently available multi-color BiFC vectors that we have constructed.

Bottom Line: Additional expression of mCherry indicates transfected cells and sub-cellular structures.Using this system, we have determined in both tobacco BY-2 protoplasts and in onion epidermal cells that Agrobacterium VirE2 protein interacts with the Arabidopsis nuclear transport adapter protein importin alpha-1 in the cytoplasm, whereas interaction of VirE2 with a different importin alpha isoform, importin alpha-4, occurs predominantly in the nucleus.The vectors we have constructed and tested will facilitate the study of protein-protein interactions in many different plant systems.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA. zofangmj@gate.sinica.edu.tw.

ABSTRACT

Background: The investigation of protein-protein interactions is important for characterizing protein function. Bimolecular fluorescence complementation (BiFC) has recently gained interest as a relatively easy and inexpensive method to visualize protein-protein interactions in living cells. BiFC uses "split YFP" tags on proteins to detect interactions: If the tagged proteins interact, they may bring the two split fluorophore components together such that they can fold and reconstitute fluorescence. The sites of interaction can be monitored using epifluorescence or confocal microscopy. However, "conventional" BiFC can investigate interactions only between two proteins at a time. There are instances when one may wish to offer a particular "bait" protein to several "prey" proteins simultaneously. Preferential interaction of the bait protein with one of the prey proteins, or different sites of interaction between the bait protein and multiple prey proteins, may thus be observed.

Results: We have constructed a series of gene expression vectors, based upon the pSAT series of vectors, to facilitate the practice of multi-color BiFC. The bait protein is tagged with the C-terminal portion of CFP (cCFP), and prey proteins are tagged with the N-terminal portions of either Venus (nVenus) or Cerulean (nCerulean). Interaction of cCFP-tagged proteins with nVenus-tagged proteins generates yellow fluorescence, whereas interaction of cCFP-tagged proteins with nCerulean-tagged proteins generates blue fluorescence. Additional expression of mCherry indicates transfected cells and sub-cellular structures. Using this system, we have determined in both tobacco BY-2 protoplasts and in onion epidermal cells that Agrobacterium VirE2 protein interacts with the Arabidopsis nuclear transport adapter protein importin alpha-1 in the cytoplasm, whereas interaction of VirE2 with a different importin alpha isoform, importin alpha-4, occurs predominantly in the nucleus.

Conclusion: Multi-color BiFC is a useful technique to determine interactions simultaneously between a given" bait" protein and multiple "prey" proteins in living plant cells. The vectors we have constructed and tested will facilitate the study of protein-protein interactions in many different plant systems.

No MeSH data available.


Related in: MedlinePlus