| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
Plant Physiology 145:1090-1099 (2007) © 2007 American Society of Plant Biologists The Analysis of Protein-Protein Interactions in Plants by Bimolecular Fluorescence ComplementationDepartment of Plant Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel
Following the complete genome sequencing of different plant species such as Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Physcomitrella (Physcomitrella patens), as well as advances toward deciphering entire proteomes, the need for a reliable way to identify protein-protein interactions is becoming a major task for the future. Bimolecular fluorescent complementation (BiFC) is a noninvasive fluorescent-based technique that allows detection of protein-protein interactions in living cells, and furthermore can be used to determine subcellular localization of the interacting proteins, and if it changes over time, without requiring addition of external agents. BiFC is based upon reconstitution of split nonfluorescent GFP variants, primarily yellow fluorescent protein (YFP), to form a fluorescent fluorophore (Ghosh et al., 2000
Due to lack of space we did not discuss other fluorescent-based techniques for detection of protein-protein interactions, such as fluorescent resonance energy transfer, and refer the readers to a recent review on fluorescent resonance energy transfer and BiFC (Bhat et al., 2006
The BiFC Principle BiFC is based upon tethering split YFP or other GFP variants to form a functional fluorophore. The association of the split YFP/GFP/cyan fluorescent protein (CFP) molecule does not occur spontaneously and requires interaction between proteins or peptides that are fused to each of the fluorophore fragments (Fig. 1 ). Upon interaction of these fused proteins/peptides, the split fluorophore fragments can interact to form a fluorescent protein that has the same spectral properties as the unsplit YFP (or other GFP variants; Figs. 1 and 2 ). If the proteins that are fused to the split fluorophore fragments do not interact, reconstitution of the YFP/GFP/CFP usually does not take place and no fluorescence is detected.
Mutational studies have uncovered sites within the GFP molecule that allow insertions without perturbing fluorescence characteristics (Abedi et al., 1998  ; Baird et al., 1999). These findings led to the discovery that split GFP fragments could be reconstituted in vitro and in Escherichia coli when fused to interacting antiparallel coiled-coil peptides (Ghosh et al., 2000). The same study showed that each GFP fragment by itself is insoluble when expressed in E. coli; however, the reconstituted GFP protein complex is soluble and stable, with a low dissociation coefficient (Ghosh et al., 2000). Insertion of a peptide spacer within GFP to which a calmodulin (CaM) and a CaM target peptide M13 were fused at its N- and C-terminal ends, respectively, resulted in a chimeric protein, designated Pericam. Reconstitution of GFP fluorescence in Pericam takes place when Ca2+ binds the CaM moiety that in turn interacts with the M13 peptide. Pericam was used to monitor dynamic changes in Ca2+ levels in different cellular compartments of HeLa and cardiomyocytes (Nagai et al., 2001; Robert et al., 2001). It was further shown that when the two fragments of GFP were completely separated by deleting the spacer peptide between them, they could interact to reconstitute a fluorescing GFP in a Ca2+-dependent fashion (Nagai et al., 2001). These studies of split GFP in E. coli and Pericam in HeLa and cardiomyocytes prompted the development a split YFP system, designated BiFC, which was used for determining subcellular localization of protein complexes in mammalian cells (Hu et al., 2002). The work of Hu et al. (2002) demonstrated that BiFC is a simple, reliable, and efficient tool to examine protein-protein interactions in living cells, allowing determination of their subcellular localization. Following this work, BiFC became a widely used method. In the BiFC systems that were subsequently developed, including versions adapted for plants (Bracha-Drori et al., 2004; Walter et al., 2004), the YFP molecule was split between amino acids 154 and 155, based on the design of Hu et al. (2002) or between residues 174 and 175 (Citovsky et al., 2006).
The dynamics of the split YFP reconstitution has been investigated to elucidate the pathway for fluorescent molecule formation (Hu et al., 2002
Proteins under study can be expressed as either N-terminal or C-terminal fusions with the split YFP fragments, often referred to as YN and YC, respectively (Hu et al., 2002 ; Bracha-Drori et al., 2004; Citovsky et al., 2006; Kerppola, 2006a). The resulting combinations of interacting pairs of proteins are depicted in Figure 1D. Using different combinations of YN and YC fusion pairs (Fig. 1D) is advisable since the orientation of the fusion can greatly affect YFP complex formation (Bracha-Drori et al., 2004). It is also recommended to place a flexible spacer between the split YFP fragment and the proteins under investigation, to alleviate structural constraints that might compromise YFP complex formation (Hu et al., 2002; Kerppola, 2006a). Currently, however, there are no accurate predictions for designing ideal spacers. Furthermore, the structure of the proteins under study and the spacers used can greatly affect YFP complex formation and fluorescence emitted. Mature YFP complex formation could be detected even when the interaction surface between two given proteins is small and the interactions are weak or transient (Hu et al., 2002; Kerppola, 2006a, 2006b; Lavy et al., 2007; Morell et al., 2007). Thus, use of strong promoters should be avoided.
Protein complementation assays (PCAs) can take place between split GFP (Ghosh et al., 2000 ), YFP (Hu et al., 2002), and CFP (Hu and Kerppola, 2003) protein pairs, which have different excitation-emission spectra. In an expansion of their original BiFC system, Hu et al. (2002) have introduced multicolor BiFC, demonstrating that PCAs can be performed between the complementing fragments of YFP and CFP as well as between YFP and GFP fragments. The resulting GFP-YFP and CFP-YFP complexes have unique, resolvable emission spectra (Hu and Kerppola, 2003). Multicolor BiFC assays are useful when complex formation of a given protein with different interacting partners is examined. Careful analysis of fluorescence intensities has enabled prediction of preferable interactions (Hu and Kerppola, 2003). Multicolor BiFC assays in plants have not yet been described.
A foremost advantage of the BiFC assay is its simplicity and the ability to carry out experiments with either a regular epifluorescence microscope equipped with the relevant filter sets and a CCD camera, or with a CLSM. Background fluorescence does not usually constitute a problem because the signals are strong enough, especially when using split YFP. In cases of weak fluorescence, however, appropriate filter sets, such as the Ziess Pinkel Set 40, can be used to resolve true YFP fluorescence from autofluorescence (for more detail see Bracha-Drori et al., 2004 ). Certain CLSMs have spectrum scan capabilities that enable determination of fluorescence spectra (Fig. 2; Bracha-Drori et al., 2004). Furthermore, a fluorescent spectrophotometer can be used to determine whether the relevant spectra are emitted by the sample. Finally, examination of fluorescence resulting from nonlegitimate interaction between a candidate protein fused to either YN or YC with the reciprocal YN or YC only, or preferably with a mutated noninteracting protein, should be determined.
BiFC has several major advantages. (1) The assay is simple and does not require sophisticated dedicated equipment. (2) There is either no or low background signal because a fluorescing YFP would only form after interaction between proteins fused to split fragments. (3) BiFC enables determination of the subcellular localization of interacting protein complexes as well as the mutual affect of interacting partners on the subcellular localization of the complex. (4) BiFC is a sensitive assay, enabling detection of weak and transient interactions, primarily due to the stability of the reconstituted YFP complexes (Hu et al., 2002 ).
However, the assay suffers from several pitfalls that must be taken into account. (1) The slow maturation time of the reconstituted GFP/YFP/CFP compromises detection of dynamic changes in protein-protein interactions in real time (Ghosh et al., 2000
These disadvantages of the BiFC system require careful consideration of the following issues. Although BiFC-based systems have been used successfully for monitoring dynamic changes in Ca2+ concentration (Nagai et al., 2001
The Application of BiFC in Plants
Successful application of BiFC in plants was first described in three publications (Bracha-Drori et al., 2004
Below we describe several studies in which results obtained with BiFC have led to new insights and understandings of biological processes. Table I presents a list of protein-protein interactions that have been verified or identified by BiFC in plants.
BiFC proved to be useful for determining the mutual effect of interacting proteins on their subcellular localization (Fig. 4A ). The ROP GTPase-interacting coiled-coil scaffold protein INTERACTOR OF CONSTITUTIVE ACTIVE ROP1 (ICR1) makes cytoplasmic-localized homooligomers but it is recruited by ROPs to the plasma membrane. In turn, ICR1 interacts and recruits specific sets of proteins, including SEC3 exocyst subunit to the plasma membrane, regulating cell polarity. The BiFC experiments demonstrated how posttranslational lipid modifications of the ROPs determine the subcellular localization of the complex (Lavy et al., 2007 ; Fig. 1). In the same study, the colocalization of CFP-ROP9 together with a reconstituted YFP complex formed by the interaction between ICR1 and SEC3 showed that the BiFC assay could be used to demonstrate colocalization between three proteins using only two fluorescent dyes: CFP and the reconstituted YFP (Lavy et al., 2007). BiFC was used to demonstrate that ALY proteins, which interact and inhibit translocation of the tomato bushy stunt virus P19 protein from the nucleus to cytoplasm, compromise its antisilencing activity (Canto et al., 2006). SHOOTMERISTEMLESS (STM) is a transcriptional regulator that functions in maintenance of the shoot apical meristem and is localized by default to the cytoplasm. BiFC assays revealed that nuclear localization of STM depends on its heterodimerization with BELL1-LIKE HOMEODOMAIN (BLH) proteins (Cole et al., 2006).
Determination of subcellular localization of protein complexes by BiFC has opened new avenues toward understanding basic cellular processes in plants. The function of microRNAs (miR) in gene expression regulation has been at the forefront of research in plant and nonplant systems ever since they were first discovered. Much is still not known about the mechanisms of miR formation and processing. In a recent study, BiFC was used to demonstrate that DICER-LIKE1 (DCL1), HYPONASTIC LEAVES1 (HYL1), and SERRATE (SE), three proteins involved in miR processing or storage, are assembled and localized in nuclear dicing bodies (Fang and Spector, 2007 ). Plant scientists have used Agrobacterium tumefaciens for three decades to transform plants. Yet, many steps of T-DNA-mediated transformation are not well understood. BiFC was used to show that the bacterial F-box-containing protein VirF interacts with the plant protein VIP1 in the nucleus. This interaction likely leads to degradation of VIP1 and another bacterial proteinVirE2 by the proteasome (Tzfira et al., 2004). Extensive BiFC and yeast (Saccharomyces cerevisiae) two-hybrid assays were used to map the interactions between the Arp2/3 complex and its regulating SCAR/WAVE complex subunits, between the SCAR2 subunit and several ROPs, and between SCAR/WAVE subunits and the SPIKE1 ROPGEF (Uhrig et al., 2007). This study revealed internal interactions between subunits of each Arp2/3 and SCAR/WAVE complex as well as interactions taking place between the complexes. That the SCAR2 subunit of SCAR/WAVE interacted with several ROPs suggested that it is a ROP effector, and that SPIKE1 interacted with several SCAR/WAVE subunits indicated that it could be part of this complex. A new study exemplifies the usefulness of BiFC for determination of protein-protein interactions that depend on a third component (Fig. 4B). BiFC and yeast two-hybrid assays showed that the interaction between the soluble GA receptor GA-INSENSITIVE DWARF1 (GID1) and the DELLA repressor protein SLENDER RICE1 (SLR1) requires GA (Ueguchi-Tanaka et al., 2007).
Localization of protein complexes with BiFC has yielded surprises and new insights. Complexes of the polycomb (PcG) chromatin regulating complex proteins FERTILIZATION INDEPENDENT ENDOSPERM (FIE) and the SET domain MEDEA (MEA) were detected in both the nuclei and cytoplasm (Bracha-Drori et al., 2004
The topology of two membrane proteins were tested with BiFC. The amino (YN) and carboxy (YC) fragments of YFP were cloned at different positions along the P6 movement protein of Beet yellow virus and the TGBp2 protein of Potato mop top virus (Zamyatnin et al., 2006
BiFC assays have failed to confirm data obtained using in vitro or yeast two-hybrid assays. For example, homodimerization of APETALA1 (AP1), and heterodimerization of AP3 and PISTILLATA (PI) MADS-box floral regulators and their Antirrhinum homologs had previously been demonstrated by in vitro and yeast two-hybrid assays (Egea-Cortines et al., 1999 ; Honma and Goto, 2001). We used BiFC to examine AP1 homodimerization and heterodimerization of AP3 and PI. Surprisingly, no BiFC signals were detected with different combinations of split YFP pairs, even though protein expression was verified by immunoblotting (Fig. 3). Furthermore, efforts to immunoprecipitate YN-AP1 YC-AP1 homodimers were unsuccessful (K. Shichrur and S. Yalovsky, unpublished data). These negative results do not prove that homodimerization of AP1 and heterodimerization of AP3 and PI did not take place. Rather, the structure of the AP1 and AP3-PI homo- and heterodimers may have prevented successful reconstitution of YFP. Alternatively, the interaction between these MADS-box proteins in plant cells may require components that were not present in leaf epidermal cells, the tissue in which the BiFC assays were carried out, or certain components present in these cells may have interfered with the complex formation. However, the data do show that ectopic expression of split YFP fragments that accumulated in the same subcellular compartment (the nucleus in the case of AP1, AP3, and PI) does not necessarily lead to reconstitution of an active fluorophore, as was previously claimed (Zamyatnin et al., 2006).
The use of the BiFC assays could be expanded in several directions. These include determination of protein-protein interactions in time and space by using endogenous promoters of genes of interest. In addition, high-throughput screens for interacting proteins in plant cells and determination of gene expression patterns could be facilitated using BiFC.
Using endogenous promoters for gene expression in BiFC could solve several problems, as specified below. Avoiding overexpression should alleviate nonspecific interactions and will enable testing interaction at physiologically relevant conditions. Furthermore, in some cases interaction between two proteins is indirect, requiring additional factor(s) that may only exist in certain tissues or cells (Fig. 4B). Some interactions may only occur following certain stimuli, whose effects could be obscured by overexpression. Under physiologically more relevant conditions, quantification of fluorescence signals should be more readily achievable and relevant. The use of specific promoters would be invaluable for developmental studies (Fig. 4D). It is of concern that the low expression levels often observed with native promoters could potentially compromise detection of YFP signal above background fluorescence. However, the proven sensitivity of the BiFC assay together with the newer and more sensitive YFP variant, Venus, previously adapted for BiFC (Shyu et al., 2006
The simplicity and sensitivity of BiFC makes it an attractive system for high-throughput protein-protein interaction screens in plants. A split GFP system has been used for screening interacting partners of the protein kinase PKB/Akt in COS cells (Remy and Michnick, 2004 Background fluorescence should be quantified to select between specific and nonspecific interacting partners. What is the preferred experimental system? Should it be, for example, tissue culture cells, pollen, seedlings, or mature plants? Unlike mammalian cells, plant cells do not adhere to the bottom of culture plates, making it much more difficult to screen efficiently for individual cells/colonies. This problem may be alleviated by using FACS if the fluorescence is strong enough. It would be necessary to devise an efficient and reliable screening method including high transformation efficiency, reduce transgene silencing, and develop reliable methods for plasmid recovery. Finally, BiFC should be compared to other PCA systems.
A successful application of a split luciferase in plants has been described (Fujikawa and Kato, 2007 The popularity and usage of BiFC in plants systems is rapidly expanding and undoubtedly, new protein-protein interactions will be revealed. The quality and reliability of these data will depend on carrying out all the necessary controls, reducing the expression levels, and primarily using BiFC where its greatest capability lies—for the detection of subcellular localization.
The results shown in Figure 1 were produced by Meirav Lavy in the S.Y. laboratory. Keren Bracha-Drori constructed several vectors in the S.Y. laboratory. We thank Sheila McCormick and Itzhak Ohad for comments. Current research in the laboratory of S.Y. is supported by grants from the Israel Science Foundation (ISF), the German-Israeli Foundation (GIF), the Deutschland-Israeli Program (DIP), and the United States-Israel Binational Agricultural Research and Development Fund (BARD). Current research in the laboratory of N.O. is supported by grants from ISF, GIF, DIP, and BARD. Development of BiFC in our laboratories was supported by the ISF BIKURA grant. Received August 14, 2007; accepted September 5, 2007; published December 6, 2007.
1 These authors contributed equally to the article.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Shaul Yalovsky (shauly{at}tauex.tau.ac.il). www.plantphysiol.org/cgi/doi/10.1104/pp.107.107284 * Corresponding author; e-mail shauly{at}tauex.tau.ac.il.
Abe M, Kobayashi Y, Yamamoto S, Daimon Y, Yamaguchi A, Ikeda Y, Ichinoki H, Notaguchi M, Goto K, Araki T (2005) FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex. Science 309: 1052–1056 Abedi MR, Caponigro G, Kamb A (1998) Green fluorescent protein as a scaffold for intracellular presentation of peptides. Nucleic Acids Res 26: 623–630 Aparicio F, Sanchez-Navarro JA, Pallas V (2006) In vitro and in vivo mapping of the Prunus necrotic ringspot virus coat protein C-terminal dimerization domain by bimolecular fluorescence complementation. J Gen Virol 87: 1745–1750 Baird GS, Zacharias DA, Tsien RY (1999) Circular permutation and receptor insertion within green fluorescent proteins. Proc Natl Acad Sci USA 96: 11241–11246 Bazzini AA, Hopp HE, Beachy RN, Asurmendi S (2007) Infection and coaccumulation of tobacco mosaic virus proteins alter microRNA levels, correlating with symptom and plant development. Proc Natl Acad Sci USA 104: 12157–12162 Beauchemin C, Boutet N, Laliberte JF (2007) Visualization of the interaction between the precursors of VPg, the viral protein linked to the genome of turnip mosaic virus, and the translation eukaryotic initiation factor iso 4E in planta. J Virol 81: 775–782 Bedard J, Kubis S, Bimanadham S, Jarvis P (2007) Functional similarity between the chloroplast translocon component, Tic40, and the human co-chaperone, Hsp70-interacting protein (Hip). J Biol Chem 282: 21404–21414 Bhat RA, Lahaye T, Panstruga R (2006) The visible touch: in planta visualization of protein-protein interactions by fluorophore-based methods. Plant Methods 2: 12[CrossRef][Medline] Bracha-Drori K, Shichrur K, Katz A, Oliva M, Angelovici R, Yalovsky S, Ohad N (2004) Detection of protein-protein interactions in plants using bimolecular fluorescence complementation. Plant J 40: 419–427[CrossRef][ISI][Medline] Canto T, Uhrig JF, Swanson M, Wright KM, MacFarlane SA (2006) Translocation of tomato bushy stunt virus P19 protein into the nucleus by ALY proteins compromises its silencing suppressor activity. J Virol 80: 9064–9072 Chandler JW, Cole M, Flier A, Grewe B, Werr W (2007) The AP2 transcription factors DORNROSCHEN and DORNROSCHEN-LIKE redundantly control Arabidopsis embryo patterning via interaction with PHAVOLUTA. Development 134: 1653–1662 Citovsky V, Lee LY, Vyas S, Glick E, Chen MH, Vainstein A, Gafni Y, Gelvin SB, Tzfira T (2006) Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta. J Mol Biol 362: 1120–1131[CrossRef][ISI][Medline] Cole M, Nolte C, Werr W (2006) Nuclear import of the transcription factor SHOOT MERISTEMLESS depends on heterodimerization with BLH proteins expressed in discrete sub-domains of the shoot apical meristem of Arabidopsis thaliana. Nucleic Acids Res 34: 1281–1292 Diaz I, Martinez M, Isabel-LaMoneda I, Rubio-Somoza I, Carbonero P (2005) The DOF protein, SAD, interacts with GAMYB in plant nuclei and activates transcription of endosperm-specific genes during barley seed development. Plant J 42: 652–662[CrossRef][ISI][Medline] Dong G, Ni Z, Yao Y, Nie X, Sun Q (2007) Wheat Dof transcription factor WPBF interacts with TaQM and activates transcription of an alpha-gliadin gene during wheat seed development. Plant Mol Biol 63: 73–84[ISI][Medline] Egea-Cortines M, Saedler H, Sommer H (1999) Ternary complex formation between the MADS-box proteins SQUAMOSA, DEFICIENS and GLOBOSA is involved in the control of floral architecture in Antirrhinum majus. EMBO J 18: 5370–5379[CrossRef][ISI][Medline] Fang D, Kerppola TK (2004) Ubiquitin-mediated fluorescence complementation reveals that Jun ubiquitinated by Itch/AIP4 is localized to lysosomes. Proc Natl Acad Sci USA 101: 14782–14787 Fang Y, Spector DL (2007) Identification of nuclear dicing bodies containing proteins for microRNA biogenesis in living Arabidopsis plants. Curr Biol 17: 818–823[CrossRef][ISI][Medline] Fujikawa Y, Kato N (2007) Split luciferase complementation assay to study protein-protein interactions in Arabidopsis protoplasts. Plant J 52: 185–195[CrossRef][ISI][Medline] Ghosh I, Hamilton AD, Regan L (2000) Antiparallel leucine zipper-directed protein reassembly: application to the green fluorescent protein. J Am Chem Soc 122: 5658–5659[CrossRef][ISI] Gissot L, Polge C, Jossier M, Girin T, Bouly JP, Kreis M, Thomas M (2006) AKINbetagamma contributes to SnRK1 heterotrimeric complexes and interacts with two proteins implicated in plant pathogen resistance through its KIS/GBD sequence. Plant Physiol 142: 931–944 Hackbusch J, Richter K, Muller J, Salamini F, Uhrig JF (2005) A central role of Arabidopsis thaliana ovate family proteins in networking and subcellular localization of 3-aa loop extension homeodomain proteins. Proc Natl Acad Sci USA 102: 4908–4912 Honma T, Goto K (2001) Complexes of MADS-box proteins are sufficient to convert leaves into floral organs. Nature 409: 525–529[CrossRef][Medline] Hu CD, Chinenov Y, Kerppola TK (2002) Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell 9: 789–798[CrossRef][ISI][Medline] Hu CD, Kerppola TK (2003) Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nat Biotechnol 21: 539–545[CrossRef][ISI][Medline] Ihara-Ohori Y, Nagano M, Muto S, Uchimiya H, Kawai-Yamada M (2007) Cell death suppressor Arabidopsis bax inhibitor-1 is associated with calmodulin binding and ion homeostasis. Plant Physiol 143: 650–660 Jach G, Pesch M, Richter K, Frings S, Uhrig JF (2006) An improved mRFP1 adds red to bimolecular fluorescence complementation. Nat Methods 3: 597–600[CrossRef][ISI][Medline] Jelesko JG, Harper R, Furuya M, Gruissem W (1999) Rare germinal unequal crossing-over leading to recombinant gene formation and gene duplication in Arabidopsis thaliana. Proc Natl Acad Sci USA 96: 10302–10307 Kerppola TK (2006a) Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells. Nat Protocols 1: 1278–1286 Kerppola TK (2006b) Visualization of molecular interactions by fluorescence complementation. Nat Rev Mol Cell Biol 7: 449–456[CrossRef][ISI][Medline] Lavy M, Bloch D, Hazak O, Gutman I, Poraty L, Sorek N, Sternberg H, Yalovsky S (2007) A novel ROP/RAC effector links cell polarity, root-meristem maintenance, and vesicle trafficking. Curr Biol 17: 947–952[CrossRef][ISI][Medline] Miyawaki A, Nagai T, Mizuno H (2003) Mechanisms of protein fluorophore formation and engineering. Curr Opin Chem Biol 7: 557–562[CrossRef][ISI][Medline] Miyawaki A, Nagai T, Mizuno H (2005) Engineering fluorescent proteins. Adv Biochem Eng Biotechnol 95: 1–15[ISI][Medline] Morell M, Espargaro A, Aviles FX, Ventura S (2007) Detection of transient protein-protein interactions by bimolecular fluorescence complementation: the Abl-SH3 case. Proteomics 7: 1023–1036[CrossRef][ISI][Medline] Nagai T, Sawano A, Park ES, Miyawaki A (2001) Circularly permuted green fluorescent proteins engineered to sense Ca2+. Proc Natl Acad Sci USA 98: 3197–3202 Remy I, Michnick SW (2004) A cDNA library functional screening strategy based on fluorescent protein complementation assays to identify novel components of signaling pathways. Methods 32: 381–388[CrossRef][ISI][Medline] Remy I, Michnick SW (2007) Application of protein-fragment complementation assays in cell biology. Biotechniques 42: 137–145[ISI][Medline] Robert V, Gurlini P, Tosello V, Nagai T, Miyawaki A, Di Lisa F, Pozzan T (2001) Beat-to-beat oscillations of mitochondrial [Ca2+] in cardiac cells. EMBO J 20: 4998–5007[CrossRef][ISI][Medline] Shyu YJ, Liu H, Deng X, Hu CD (2006) Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions. Biotechniques 40: 61–66[ISI][Medline] Stolpe T, Susslin C, Marrocco K, Nick P, Kretsch T, Kircher S (2005) In planta analysis of protein-protein interactions related to light signaling by bimolecular fluorescence complementation. Protoplasma 226: 137–146[CrossRef][ISI][Medline] Tzfira T, Vaidya M, Citovsky V (2004) Involvement of targeted proteolysis in plant genetic transformation by Agrobacterium. Nature 431: 87–92[CrossRef][Medline] Ueguchi-Tanaka M, Nakajima M, Katoh E, Ohmiya H, Asano K, Saji S, Hongyu X, Ashikari M, Kitano H, Yamaguchi I, et al (2007) Molecular interactions of a soluble gibberellin receptor, GID1, with a rice DELLA protein, SLR1, and gibberellin. Plant Cell 19: 2140–2155 Uhrig JF, Mutondo M, Zimmermann I, Deeks MJ, Machesky LM, Thomas P, Uhrig S, Rambke C, Hussey PJ, Hulskamp M (2007) The role of Arabidopsis SCAR genes in ARP2-ARP3-dependent cell morphogenesis. Development 134: 967–977 Voelker C, Schmidt D, Mueller-Roeber B, Czempinski K (2006) Members of the Arabidopsis AtTPK/KCO family form homomeric vacuolar channels in planta. Plant J 48: 296–306[CrossRef][ISI][Medline] Walter M, Chaban C, Schutze K, Batistic O, Weckermann K, Nake C, Blazevic D, Grefen C, Schumacher K, Oecking C, et al (2004) Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J 40: 428–438[CrossRef][ISI][Medline] Weltmeier F, Ehlert A, Mayer CS, Dietrich K, Wang X, Schutze K, Alonso R, Harter K, Vicente-Carbajosa J, Droge-Laser W (2006) Combinatorial control of Arabidopsis proline dehydrogenase transcription by specific heterodimerisation of bZIP transcription factors. EMBO J 25: 3133–3143[CrossRef][ISI][Medline] Xie Z, Zhang ZL, Zou X, Yang G, Komatsu S, Shen QJ (2006) Interactions of two abscisic-acid induced WRKY genes in repressing gibberellin signaling in aleurone cells. Plant J 46: 231–242[CrossRef][ISI][Medline] Yano A, Kodama Y, Koike A, Shinya T, Kim HJ, Matsumoto M, Ogita S, Wada Y, Ohad N, Sano H (2006) Interaction between methyl CpG-binding protein and ran GTPase during cell division in tobacco cultured cells. Ann Bot (Lond) 98: 1179–1187 Zamyatnin AA Jr, Solovyev AG, Bozhkov PV, Valkonen JP, Morozov SY, Savenkov EI (2006) Assessment of the integral membrane protein topology in living cells. Plant J 46: 145–154[CrossRef][ISI][Medline] Zhang S, Ma C, Chalfie M (2004) Combinatorial marking of cells and organelles with reconstituted fluorescent proteins. Cell 119: 137–144[CrossRef][ISI][Medline] Zhang Y, Cheng YT, Qu N, Zhao Q, Bi D, Li X (2006) Negative regulation of defense responses in Arabidopsis by two NPR1 paralogs. Plant J 48: 647–656[CrossRef][ISI][Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TOP
THE PRINCIPLES AND DEVELOPMENT...
THE DEVELOPMENT OF THE...
- and β-subunits of the enzyme farnesyltransferase had different intensities depending on the orientation of the fusion of each of the subunits to the YFP fragments (Bracha-Drori et al., 2004