Toward an Interaction Map of the Two-Component Signaling Pathway

Toward an Interaction Map of the Two-Component Signaling Pathway of Arabidopsis thaliana Hakan Dortay,† Nijuscha Gruhn, Andreas Pfeifer, Mareike Schwerdtner, Thomas Schmu ¨ lling, and Alexander Heyl* Institute of Biology/Applied Genetics, Free University of Berlin, Albrecht-Thaer-Weg 6, 14195 Berlin, Germany Received June 19, 2007

Among the signal transduction pathways in higher eukaryotes, the two-component system (TCS) is unique to plants. In the model plant Arabidopsis thaliana, it consists of more than 30 proteins, including eight receptors, five phosphotransmitters and 23 response regulators. One of its important functions is to perceive and transduce the signal of the plant hormone cytokinin. The basic signal flow within the TCS is well-understood, but it is unclear how this pathway is integrated with the remainder of the proteome. Thus, knowledge about the interactions of TCS proteins should contribute to the understanding of their mode of action. Therefore, we conducted medium-scale yeast two-hybrid screens focusing on those members of the TCS, which are thought to be involved in cytokinin signaling. In total, more than 6.3 × 107 transformants were screened resulting in the identification of 160 different interactions, of which 136 were novel. Most of the interacting proteins belong to the functional categories of signal transduction and protein metabolism. TCS proteins and their interactors localized to the same subcellular compartment in many cases, a prerequisite to being of biological relevance. The resulting interaction network map revealed large differences in the connectivity. Cytokinin receptors (AHK2, CRE1/AHK4) showed the highest numbers of different interaction partners. This study is the first systematic protein-protein interaction experiment for a plant signal system and provides numerous starting points for further analysis of the molecular mechanisms used to convert the signal carried by the TCS into biological processes. Keywords: Two-component signaling • yeast two-hybrid system • protein-protein interaction • cytokinin • interactome

Introduction In recent years, tremendous progress has been made toward the elucidation and characterization of the signal transduction pathways of various plant hormones.1 This is also true for the phytohormone cytokinin. The basic scheme of its signaling pathway, a His-to-Asp, multistep phosphorelay system, resembles the classical two-component system (TCS), the major signal transduction system among bacteria (reviewed in refs 2 and 3). The cytokinin signaling pathway is best characterized in the model plant Arabidopsis thaliana, where the binding of cytokinins to membrane bound hybrid histidine kinase receptors (AHK) is predicted to cause an autophosphorylation of the receptors. After an intramolecular phosphorelay to a conserved aspartate residue at the C-terminus of the receptors, the phosphate is transferred to histidine phosphotransfer proteins (AHP). These then translocate into the nucleus, where they activate via phosphorylation the B-type response regulators (Btype ARR), which form a subclass of the Myb-domain contain* To whom correspondence should be addressed. Alexander Heyl, phone, ++49 (0)30 838 56550; fax, ++49 (0)30 838 54345, e-mail, [email protected]. † Current address: Institute of Biochemistry and Biology/Molecular Biology and Plant Physiology, University of Potsdam, Karl-Liebknecht-Str. 2425, 14476 Golm, Germany. 10.1021/pr0703831 CCC: $40.75

 2008 American Chemical Society

ing transcription factors. Among their target genes are the A-type response regulators (A-type ARR), which have been shown to participate in a negative feedback regulation of the cytokinin signaling pathway.4 This model is supported by genetic data involving multiple mutants in the different protein classes5–10 and is consistent with the results of targeted interaction studies using yeast two-hybrid and phosphorylation experiments involving most of the more than 30 members of the TCS.4,11–18 Genetic and biochemical studies have shown that a major function of the plant TCS is to perceive and transmit cytokinin signals.4–10 While many of the physiological and developmental activities of cytokinin are well-studied, little is known about how the signal information is specified downstream of the TCS. Some studies have identified cytokinin response genes, which could contribute to signal specification. Another possibility of signal specification could be achieved through interaction of TCS proteins with other Arabidopsis proteins. One known example is the interaction between the A-type response regulator ARR4 and the light receptor PhyB.19 As most cellular processes are mediated by protein interactions, a broader knowledge of interactions of TCS proteins is expected to lead to the discovery of molecular mechanisms, which convert the hormone signal into physiological and developmental responses. Journal of Proteome Research 2008, 7, 3649–3660 3649 Published on Web 07/22/2008

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Table 1. Summary of the Results of the Yeast Two-Hybrid Screens Using Arabidopsis Proteins of the TCS as Baits bait

transformants

primary positives

total interactions (secondary positives)

different interactions

no. of total interactions with TCS proteins

no. of interactions with different TCS proteins

proportion of interactions with TCS proteins

AHK2 AHK3 AHK4 AHP5 ARR3 ARR4 ARR5 ARR6 ARR7 ARR8 ARR9 ARR15 ARR16 ARR1 ARR2 ARR10 ARR14

∼1.5 × 107 ∼6.9 × 106 ∼8.4 × 106 ∼2.5 × 105 ∼9 × 105 ∼1.7 × 105 ∼3.7 × 105 ∼2.8 × 105 ∼2 × 107 ∼8.6 × 105 ∼2.2 × 105 ∼2.2 × 106 ∼1.7 × 106 ∼7 × 105 ∼7.9 × 105 ∼1.5 × 106 ∼1.7 × 106

>200 >200 >200 12 181 101 >200 110 >200 >200 >200 >200 >200 112 150 >200 >200

65 3 73 5 32 2 40 5 66 14 14 12 2 25 37 4 25

42 1 26 3 6 2 19 4 11 2 4 4 2 8 6 4 16

6 0 5 4 28 0 20 0 45 14 12 0 0 17 32 0 2

2 0 1 2 3 0 2 0 3 2 2 0 0 3 3 0 1

9% 0% 7% 80% 88% 0% 50% 0% 68% 100% 86% 0% 0% 68% 86% 0% 8%

The yeast two-hybrid system (Y2H) is the most widely used method for the detection of such interactions (for review, see refs 20–22). In fact, most interaction data of species-wide interactome studies to date have been generated using this method.23–29 However, so far these studies have been conducted only in phages, bacteria, yeast, animals and humans. In plants, small to medium-scale protein interaction studies have been done mostly to analyze the interaction patterns within a chosen pathway or protein family 11,30–32 and reviewed in.20 While those studies are very informative about regulatory patterns and interaction patterns within the respective group, they are less useful in revealing connections to other protein families and pathways. To better understand how the two-component system interacts with other pathways of the A. thaliana proteome, we conducted a systematic screen for interaction partners using the yeast two-hybrid assay. While the detection of previously known interactions validated the approach used, many new interactions were identified from a variety of functional categories and signaling pathways.

Experimental Procedures Cloning of cDNAs Encoding TCS Signaling Components. The full-length cDNAs encoding the TCS proteins were amplified either by PCR using a cDNA library from A. thaliana C2433 or by RT-PCR using total RNA extract from A. thaliana Col-0 primary leaves as described previously.11 In the case of the receptors, we cloned the part encoding the cytoplasmic domains, as transmembrane domains are known to lead to misfolding in the Y2H. For the sequence of primers used in the different amplifications see Supplemental Table 1. Plasmid Construction and Yeast Two-Hybrid Screens. To screen for protein-protein interaction, a LexA DNA binding domain-based bait vector (pBTM116-D9, kind gift of E. Wanker, MDC Berlin, Germany)34 and a Gal4 activation domain encoding prey vector (pACT2, Clontech, Mountain View, CA), adapted to the GATEWAY system, were used in the Saccharomyces cerevisiae, strain L40ccua (MATa his3∆200 trp1-901 leu2-3,112 LYS2::(lexAop)4-HIS3,URA3::(lexAop)8-lacZ,ADE2::(lexAop)8-URA3, GAL4 gal80 can1 cyh2).11 Of the six cDNA libraries used in the screens, four were derived from total RNA obtained from seedlings, hormone-treated seedlings, flowers and seeds, re3650

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spectively. These libraries were generated in the pENTR1A vector (Invitrogen, Carlsbad, CA) and shuttled into the prey vector by in vitro recombination.35 The remaining two cDNA libraries were derived from root cell culture (courtesy of C. Koncz, MPI Cologne, Germany) and from seedlings (TAIR stock CD4-22, kindly provided by A. Theologis, Berkeley, CA) and were already cloned in the pACT2 vector (Clontech, Mountain View, CA). The Y2H transformations were conducted based on the LiAc method as described before35 and the selection scheme was performed as shown in Supplemental Figure 1. Expression of Fusion Proteins and in Vitro Interaction Assay. For the pull-down assay, the cDNAs were cloned by in vitro recombination using the GATEWAY system into the pDEST15 vector (Invitrogen, Carlsbad, CA), for the expression as GST-tag fusion proteins. For the expression as His-tag fusion proteins, cDNAs were cloned either by in vitro recombination or by PCR-amplification, restriction and subsequent ligation into the pDEST17 vector (Invitrogen, Carlsbad, CA). For the ligation-mediated cloning, the cDNA-inserts of the interacting prey clones were amplified using the following primers: sense, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCT3′; and antisense, 5′-GGGGACCACTTTGTACAAGAAAGCTGGGT3′. Subsequently, the PCR products were digested using Bsp1407I (Fermentas, St. Leon Rot, Germany) and ligated into the pDEST17 using T4-DNA-Ligase (Fermentas, St. Leon Rot, Germany). Protein expression, purification and in vitro interaction assays were performed using the MagneGST pull-down system (Promega, Madison, WI) as described previously.11 Subcellular Localization Using GFP-Fusion Proteins. Subcellular localization experiments were done by expressing the respective cDNAs as a GFP-fusion protein in onion epidermal cells under control of the constitutive CaMV 35S promoter (pB7WGF2).36 The constructs were transformed into onion epidermal cells by particle bombardment using a biolistic PDS1000/He system (Bio-Rad, Hercules, CA). Gold particles (1.6 µm) were coated with the respective plasmid and a helium pressure of 9 MPa (rupture disks, 1350 psi) was employed. The distance between target stop screen and onion cells was set to 9 cm. After bombardment, onion epidermis cells were incubated in the dark from 24 to 48 h at 26 °C and subsequently examined under a fluorescence microscope (Zeiss, Go¨ttingen, Germany).

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Interaction Map for Arabidopsis Two-Component Signaling Proteins

Table 2. Most Common Interactors of TCS Proteins, Their Localization and Functional Annotation According to the Arabidopsis Database (www.arabidopsis.org) interacting prey AGI ID

no.

description

subcellular localization/methodd

biological process

AHK AHK2 AT3G29350a AT4G12060 AT5G62740b

5×1c 3×1c 2×1,4c

Hpt (AHP2) Clp Band 7

Cytosol/GFP Plastid/MS Plasma membrane/GFP + MS

AT5G08790

3×1c

NAM (ANAC081)

-

AT5G42080

5×1,5c

Dynamin (ADL1A)

AT1G26270 AHK3 AT5G43560 AHK4 AT3G29350a AT5G16070

4×1c

-

Cytoskeleton, plasma membrane, plastid/GFP; Plasma membrane, Vacuole/MS -

3×

MATH

-

5×1c 4×1c

Hpt (AHP2) Chaperonin

Cytosol/GFP -

AT1G79530

14×1c

-

AT1G13440

3×1c

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH1) Glyceraldehyde 3-phosphate dehydrogenase (GAPDH2)

AT5G28490a AHP AHP5 AT1G10470

3×1c

LSH1

3×1c

Response regulator receiver (ARR4)

Nucleus/SwissProt

AT3G16857

1×1c

Nucleus/SwissProt

AT3G02065 A-type ARRs ARR3 AT5G39340a AT3G29350a AT3G21510a AT5G53880 ARR4 AT3G57670a

1×1c

Response regulator receiver, Myb-like DNA binding domain (ARR1) DEAD-box helicase

-

Unkown biological processes

16×1c 7×1c 5×1c 2×1c

Hpt (AHP3) Hpt (AHP2) Hpt (AHP1) -

Cytosol/GFP Cytosol/GFP Cytosol/GFP Mitochondria/MS

Signal transduction Signal transduction Signal transduction Unkown biological processes

1×1c

Zn-finger (WIP2)

-

1×1c

Protein phosphatase

-

13×1c 7×1c 2×1c

Hpt (AHP3) Hpt (AHP2) Cytochrome P450 (ATC4H)

Cytosol/GFP Cytosol/GFP ER, extracellular/MS

2×1c

-

Nucleus/MS

Other metabolic processes, other cellular processes, developmental processes, transcription Unknown biological processes Signal transduction Signal transduction Other metabolic processes, other cellular processes, response to stimulus, response to stress Unknown biological processes

2×1c

Jacalin-related lectin

-

AT3G12620 ARR5 AT5G39340a AT3G29350a AT2G30490

AT1G68790a AT1G52070 ARR6 a

1c

1c

Nucleus/GFP; mitochondrium, nucleus, plasma membrane, plastid/ MS -

AT5G43170

2×

Zn-finger (AZF3)

Nucleus/GFP

AT5G63460

1×1c

DNA-binding SAP

-

AT5G65630

1×1c

Bromo

-

Signal transduction Protein metabolism Other metabolic processes, other cellular processes, protein metabolism Developmental processes, other biological processes, response to stimulus, response to stress Other cellular processes, developmental processes, cell organization Unknown biological processes Unknown biological processes Signal transduction Other metabolic processes, other cellular processes, protein metabolism Other metabolic processes, other cellular processes Other metabolic processes, other cellular processes, response to stress Unknown biological processes Developmental processes, other biological processes, response to stimulus, response to stress, signal transduction Other biological processes, signal transduction

Unknown biological processes Response to stimulus, response to stress Unknown biological processes Other biological processes

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Table 2. Continued interacting prey AGI ID a

no. 1c

description


biological process

AT5G28490

1×

LSH1

-

Unknown biological processes

ARR7 AT5G39340a,b AT3G29350a AT3G21510a AT5G43170a

27×1,3c 12×1c 6×3c 2×2c

Hpt (AHP3) Hpt (AHP2) Hpt (AHP1) Zn-finger (AZF3)

Cytosol/GFP Cytosol/GFP Cytosol/GFP Nucleus/GFP

11×1,4c

-

Nucleus/MS

13×1c 1×1c

Hpt (AHP3) Hpt (AHP1)

Cytosol/GFP Cytosol/GFP

Signal transduction Signal transduction Signal transduction Response to stimulus, response to stress Unknown biological processes Signal transduction Signal transduction

9×1c 3×1c 1×1c

Cytosol/GFP Cytosol/GFP Cytosol, nucleus/GFP

AT1G68790a,b ARR8 AT5G39340a AT3G21510a ARR9 AT5G39340a AT3G29350a AT1G16970 AT5G08720a ARR15 AT1G29920 AT5G43170a

1×1c

Hpt (AHP3) Hpt (AHP2) DNA-binding SAP, DNA helicase (KU70) -

1×5c 2×2c

Chlorophyll a/b binding Zn-finger (AZF3)

Plastid/MS Nucleus/GFP

AT5G08720a,b AT1G04850 ARR16

8×2,3,5c

-

Mitochondria/MS

1×3c

Zn-finger

-

AT3G57670a

1×1c

Zn-finger (WIP2)

-

AT5G08720a B-type ARRs ARR1 AT5G39340a AT1G53170

1×1c

-

Mitochondria/MS

14×1c 3×1c

Hpt (AHP3) -

Cytosol/GFP -

AT3G29350a AT3G21510a AT4G26000 ARR2 AT5G39340a AT3G29350a AT3G21510a AT3G25900

2×1c 1×1c 2×1c

Hpt (AHP2) Hpt (AHP1) KH

Cytosol/GFP Cytosol/GFP -

20×1c 7×1c 5×1c 2×1c

Cytosol/GFP Cytosol/GFP Cytosol/GFP -

Signal transduction Signal transduction Signal transduction Other metabolic processes, other cellular processes

AT1G69930

2×1c

Hpt (AHP3) Hpt (AHP2) Hpt (AHP1) Homocysteine S-methyltransferase (HMT-1) Glutathion S-transferase (GSTU11)

-

Other metabolic processes, other cellular processes

ARR10 AT1G01300

1×1c

Aspartic protease A1

Extracellular/MS

1×1c

Copia-like retrotransposon protein

-

Other metabolic processes, other cellular processes, protein metabolism Unknown biological processes

1×1c

-

-

1×1c

-

-

2×1c 1×1c

Hpt (AHP2) DnaJ

Cytosol/GFP -

AT3G42433 AT5G11090 AT5G03230 ARR14

AT3G29350a AT1G16680

3652


Mitochondria/MS

Signal transduction Signal transduction Cell organization, response to stress, DNA metabolism Unknown biological processes Other metabolic processes Response to stimulus, response to stress Unknown biological processes Unknown biological processes Other metabolic processes, other cellular processes, developmental processes, transcription Unknown biological processes Signal transduction Other metabolic processes, other cellular processes, transcription, signal transduction Signal transduction Signal transduction Developmental processes

Unknown biological processes Unknown biological processes Signal transduction Other metabolic processes, other cellular processes, protein metabolism

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Interaction Map for Arabidopsis Two-Component Signaling Proteins Table 2. Continued interacting prey AGI ID

no. 1c

description


AT2G47110

1×

Ubiquitin (UBQ6)

-

AT1G20100

8×1c

-

-

AT5G49710

2×1c

-

-

biological process

Other metabolic processes, other cellular processes, protein metabolism Unknown biological processes Unknown biological processes

a Interacting prey identified with different baits. b Interacting prey identified with the same bait from different libraries. c cDNA-library derived from A. thaliana Col-0 (1) root cell culture, (2) flowers, (3) 5-7 days old hormone treated seedlings, (4) seeds, (5) 5-7 days old seedlings and (6) 3 days old seedlings. d ER, endoplasmatic reticulum; GFP, green fluorescent protein; MS, mass spectrometry.

Results The Protein Interaction Screens Identified Numerous New Interactions. To begin to elucidate the interaction network of the TCS of Arabidopsis, we screened 17 of the TCS proteins against at least one of the six cDNA libraries derived from different tissues (Supplemental Figure 1). In total, more than 40 screens were performed using the cytoplasmic part of the three cytokinin receptors (AHK2, AHK3 and CRE1/AHK4), one phosphotransmitter (AHP5), nine A-type response regulators (ARR3-ARR9, ARR15 and ARR16) and four B-type response regulators (ARR1, ARR2, ARR10 and ARR14) as bait. These screens resulted in more than 6.3 × 107 transformants (Table 1). The other previously cloned TCS proteins (AHP1, AHP2, AHP3, ARR11 and ARR20) could not be used as bait due to a very high level of autoactivation, which could not be suppressed by the addition of even up to 20 mM 3-amino-1,2,4-triazol in the selection medium.11 For a large number of the baits, 1.2 × 106 to over 1.5 × 107 transformants were screened, yielding in total more than 2800 primary positives of which a maximum of 96 primary positives per screen were further investigated (Table 1). This resulted finally in a total of 569 interactions (secondary positives, Supplemental Figure 1). Of these, sequence analyses identified 145 (26%) as histone encoding genes. These clones also interacted with the LexA DNA-binding domain alone and thus proved to be unspecific interactors. These clones were therefore excluded from the subsequent analysis. The remaining 424 interactions represent 160 different interaction pairs with 129 different prey proteins (Table 1 and Supplemental Table 2). Most of the different interactions (136) are novel. The other 24 interactions have been previously described (ref 11 and references therein) and were thus confirmed in this study. Of the 129 different prey proteins, a majority (83) were identified only once, while 46 were found more often, in some cases more than 15 times (e.g., ARR2-AHP3 or ARR3-AHP3; see Table 2). For some baits, like ARR3, a high number of interactions was detected (32); however, these interactions were with only six different proteins. Other baits, like AHK2 or ARR5, interacted with a high number of different proteins (Table 2 and Supplemental Table 2). Comparison with Previously Published Data and in Vitro Binding Assay Indicate High Reliability of Detected Interactions. The Y2H screens identified 136 new and 24 previously published interactions. Of the 24 published interactions, 13 were found by the yeast two-hybrid system only and the remaining 11 were additionally confirmed by other methods, for example, pull-down or phosphorelay (summarized in ref 11). The fact that a relatively high number of previously described interactions was also detected in our screens underlines the quality of the particular vector-strain-library combination used in our experiments.

To further estimate the reliability of our data set, we selected several novel interactions detected in our screens and attempted to confirm the interactions in vitro by affinity copurification. As the cytokinin receptors AHK2 and CRE1/AHK4 and the A-type ARRs ARR7 and ARR15 showed a high number of different interactors, 20 different interactions of those proteins were investigated. Using the affinity co-purification assay,11interactions,namely,AHK2withAT5G64070,AT5G62740, AT5G13120, AT5G42080, AT4g27160 and AT1G69840; AHK4 with AT3G51630, AT2G38280 and AT4G15630; ARR7 with AT1G79690 and ARR15 with AT5G43170 could be verified. For the other 9 interactions, AHK2 with AT1G26270, AT4G29040, AT5G52410, AT5G48470 and AT1G17100; AHK4 with AT2G23760 and ARR7 with AT5G48630, AT2G19480 and AT1G68790 an interaction could not been shown using this in vitro method (Figure 1). Subcellular Localization of the Proteins of the TCS Correlates with That of Most Interactors. For a detected proteinprotein interaction to be biologically meaningful, both interacting proteins have to be present in the same subcellular compartment. Thus, we examined the subcellular localization of most TCS proteins by transiently expressing them as GFP-fusion proteins in onion epidermal cells. For the AHP proteins, a localization in the cytosol as well as in the nucleus was detected (Figure 2A). Both, A- and B-type response regulator proteins were found to be localized in the nucleus only (Figure 2B,C). The only exceptions were the A-type ARRs ARR3 and ARR16, which were found to be localized to the cytosol. However, a weak signal could also be detected in the nucleus. A comparison of the published data with our findings revealed an overlap for almost all investigated TCS proteins (Table 3). In a second step, the subcellular localization of identified interactors was determined by using the SUBA database.37 This resulted in 50 candidates with known subcellular localization among the 129 different prey proteins (Supplemental Table 2). Of these interactors, three were identified as AHPs (AHP1, AHP2 and AHP3) which are localized to the cytosol according to the Arabidopsis database and our experimental data (Table 3), but have been shown to shuttle to the nucleus after induction by cytokinin.4 They interacted with almost all A- and B-type ARRs and the AHKs which were used as baits (Table 2). Furthermore, two interactors of AHP5 detected in the screens, the A-type ARR4 and the B-type ARR1, are also localized in the nucleus (Table 3). The other 45 proteins showed different localizations (e.g., cytosol, nucleus, mitochondria, plastid and plasma membrane). A comparison between the localization of the TCS proteins and their respective interaction partner showed a localization to the same subcellular compartment in most cases, thus, fulfilling an important prerequisite for in planta interaction. This Journal of Proteome Research • Vol. 7, No. 9, 2008 3653

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Dortay et al. A majority of interactors belong to the functional categories of signal transduction and protein metabolism. The interactors found in the yeast two-hybrid screens were further characterized by analyzing their respective functional annotations. For this purpose, the GO annotation of the TAIR database (TAIR7 release at www.arabidopsis.org) was used to group the interactors according to their functions. Some proteins play a role in several different processes and were therefore annotated for more than one category. Thus, in total, 304 functional annotations were assigned for the 160 interactors found in the screens. Most interactors fall into the little defined categories of “other metabolic processes” (17%) and “other cellular processes” (18%) or into the undefined category of “unknown biological processes” (18%) (Figure 3). Among those interactors having a clearly defined functional annotation, members of the categories “signal transduction” (9%) and “protein metabolism” (11%) are overrepresented, when compared to the other categories.

Discussion

Figure 1. In vitro interaction analysis of selected proteins interacting in the Y2H assay. Indicated GST-fusion proteins were immobilized on glutathione agarose beads and incubated with the respective [35S] methionine-labeled prey-protein produced by in vitro transcription/translation. The purified proteins were separated by SDS/PAGE and analyzed by autoradiography. Lane 1, 35 S-labeled prey-protein (input); lane 2, GST immobilized on glutathione agarose beads + 35S-labeled prey-protein; lane 3, GST-fusion protein immobilized on glutathione agarose beads + 35S-labeled prey-protein. In the rows marked by an asterisk (*), the lane representing the input and the other two lanes representing the GST and the GST-fusion protein, respectively, had to be run on separate gels for technical reasons.

further increased the confidence in the interactions detected in our screens (Tables 2 and 3 and Supplemental Table 2). 3654


Toward the TCS Interactome. The generation of protein interaction networks is a stepping stone toward the system biology of a given organism and thus has moved more into the focus of current research.38,39 The Y2H system has been the method of choice to conduct most protein-protein interaction studies and has contributed greatly toward the generation of in vivo interactome maps.26 One reason favoring the Y2H assay is the great flexibility of the basic concept of this method, which has allowed it to evolve from the prototype to a wide range of different modifications and applications as well as the adaptation for high-throughput usage.21 A comparison of protein-protein interaction networks, generated from high-throughput screens of different species, has revealed a high level of conservation.40 Interactions between homologous proteins, so-called interologs, are overrepresented in the interactomes of different species.41 However, as the TCS is unique to plants among higher eukaryotes, it is not possible to use data from other higher eukaryotes to extrapolate the interactome of the cytokinin signaling pathway. Furthermore, while the TCS is widespread in bacteria, their proteomes are too different for a reasonable comparison. Thus, this study represents a start toward elucidating the interaction network emanating from the TCS. We conducted medium-scale interaction screens using members from all four protein families of the TCS as baits, namely, the cytoplasmic part of the three cytokinin receptors, one AHP, nine A-type and four B-type ARRs, and screened them at least once with one or several of six cDNA libraries generated from different tissues.35 While our data set identified many new potential interactions, previously known interactions were also detected, validating our data set. Independent confirmation by affinity co-purification of randomly selected pairs was successful for more than 50% of the tested interactions, a percentage comparable to other screens.24 The subcellular localization of interactors reveals a high level of localization in the same subcellular compartment as the TCS components. For an interaction to be biologically meaningful, both interaction partners have to be localized in the same subcellular compartment. Therefore, the subcellular localization of all cloned members of the Arabidopsis TCS was determined by transient expression as GFP-fusion protein in onion epidermal cells. Five members of the TCS (ARR3, ARR8, ARR9, ARR14 and ARR20) were localized for the first time and all, with the exception of the A-type ARR3, were found to be


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Figure 2. Subcellular localization of proteins of the two-component signaling system. GFP-fusion proteins were transiently expressed in onion epidermis cells under the control of the 35S promoter. GFP alone was expressed as a control. After 24-48 h of incubation, the cells were analyzed for GFP expression with a fluorescence microscope. The same cells which showed GFP signals were also photographed using Nomarski optics. (A) Localization of GFP-AHP fusion proteins. (B) Localization of GFP-B-type ARR fusion proteins. (C) Localization of GFP-A-type ARR fusion proteins. Red frame ) GFP control; bar size ) 100 µm; arrows indicate the cell nucleus.

exclusively in the nucleus. ARR3 was clearly detected in the cytosol, but a weak signal was also found in the nucleus. The same is true for the subcellular localization of all AHPs and for ARR16. However, as the size of the respective fusion proteins

is below the size exclusion limit of the nuclear pore, the nuclear signal might be due to diffusion rather than active transport.42 Thus, for all members of the Arabidopsis TCS, previously reported localization was confirmed in this study.4,15,17,43–46 Journal of Proteome Research • Vol. 7, No. 9, 2008 3655

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Table 3. Subcellular Localization of Cytokinin Signaling Proteins protein family

protein

our data

literature

AHKs

AHK2 AHK3 AHK4 AHP1 AHP2 AHP3 AHP4 AHP5 ARR1 ARR2 ARR10 ARR11 ARR12 ARR13 ARR14 ARR18 ARR19 ARR20 ARR21 ARR3 ARR4 ARR5 ARR6 ARR7 ARR8 ARR9 ARR15 ARR16 ARR17

Cytosol, Nucleus Cytosol, Nucleus Cytosol, Nucleus Cytosol, Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Cytosol, Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Cytosol, Nucleus -

Plasma membranea Cytosol, Nucleusb Cytosol, Nucleusb Cytosolb Cytosolb Cytosol, Nucleusb Nucleusc Nucleusc Nucleusd Nucleuse Nucleusf Nucleusf Cytosolg, Nucleus Nucleusg Nucleush Nucleusi Nucleusi Cytosoli -

AHPs

Type-B ARRs

Type-A ARRs

a Kim et al.63 b Tanaka et al.15 c Sakai et al.43 d Imamura et al.17 Imamura et al.17 f Mason et al.44 g Hwang et al.45 h Hwang and Sheen4 i Kiba et al.46 e

For the majority of the interactions (61%) detected in the screens, both partners are predicted or localize to the same subcellular compartment (Tables 2 and 3 and Supplemental Table 2), fulfilling a prerequisite for a potential biological relevance of the respective interaction. However, one has to keep in mind that, even in those cases where no localization to the same subcellular compartment is predicted or could be detected, the interaction partner could translocate to the same

compartment under certain physiological conditions. In fact, it has been proposed that the regulation of the intracellular localization of proteins is a biological mechanism for diversification of protein function.47 For example, for AHK2 and CRE1/AHK4, a number of transcription factors have been identified as interactors. While interactions between membrane bound receptors and transcriptional activators seem contradictory at first glance, there are several examples known, where transcription factors are tethered to membrane proteins and translocate to the nucleus upon activation.48,49 Also for the AHPs and for some cytokinin response factors (CRFs), it has been shown that they translocate, in this case from the cytoplasm to the nucleus, upon cytokinin treatment.4,50 Thus, it might be a more widespread mechanism in this signaling pathway that proteins change their subcellular localization in a cytokinin-dependent fashion. The Majority of Interactors Belongs to Regulatory Functional Categories. As the TCS represents the starting point of the cytokinin signaling cascade, one might expect that a relatively high portion of the interactors will belong to the functional categories involving regulatory functions. Thus, it is not surprising that in our data set those interacting proteins, which are annotated as being involved in signal transduction and protein metabolism, represent the two largest fractions among proteins with a clearly defined functional category (Figure 3). Indeed, it has been shown that the interaction of proteins from both groups is necessary for the proper completion of cellular processes. Protein degradation and synthesis are important mechanisms for fine-tuning the signal strength and are a major means in feedback regulation of signaling systems.48,49,51,52 In fact, the signal transduction of three other plant hormones, namely, auxin, brassinosteroids and gibberellins, are all initiated by the degradation of a repressor (reviewed in ref 1). Interestingly, protein-protein interaction pairs (interologs) from signal transduction and protein metabolism belong to the most highly conserved across multiple species.53 Interaction Analysis Indicates a Role for Intracellular Trafficking in Receptor Function. In protein networks, those proteins which are connected to a high number of other proteins are considered to be “hub” proteins. Their high level of connectivity makes them instant candidates for being a key

Figure 3. Functional categories of the interacting proteins identified in the yeast two-hybrid screens. Interacting prey-proteins identified in the yeast two-hybrid screens were assigned to 14 functional categories according to the gene ontologies of the Arabidopsis database (TAIR7 release at www.arabidopsis.org). The pie chart represents percentage of the functional categories of all identified interacting proteins. 3656



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Figure 4. Interaction network of the members of the two-component signaling system with other Arabidopsis proteins. Protein-protein interactions of all investigated TCS signaling components in the yeast two-hybrid screens visualized in an interaction network using the program Osprey 1.2.0 (http://biodata.mshri.on.ca/osprey/servlet/Index). Proteins are shown as dots and interactions as lines. Cytokinin signaling bait proteins are shown as orange dots. Prey proteins found in the yeast two-hybrid screens interacting with one bait protein are shown as blue dots and those interacting with different bait proteins as violet dots. Previously found interactions11 are shown as black lines and interactions with other proteins as blue lines.

connector protein, essential for the respective organism.54 Thus, it is tempting to look at interaction data to identify the main players of a particular system. This might be especially informative if genetic analysis of the respective pathway is complicated by a high level of redundancy, as it is the case for the cytokinin signaling pathway. The interaction map presented here clearly shows that some proteins display a higher level of connectivity than others (Figure 4). These potential “hub” proteins include AHK2 and CRE1/AHK4 with a total of 68 different interactors. Interestingly, all three receptors interact frequently with proteins outside the TCS. This may indicate that, while the majority of the cytokinin signaling was shown to be mediated via the TCS, some responses can divert already directly at the level of the receptors, bypassing the downstream components of this signaling pathway. Additionally, interactions with pro-

teins of regulatory function might point at possible mechanism of post-translational regulation of receptor function. In this context, it is especially interesting that a relatively high number of interactors for the receptors are implicated in intracellular trafficking from and to the plasma membrane. Intracellular transport has been well-established as an important factor in many signaling and developmental processes in general.55 As mentioned before, one way to regulate a signal transduction pathway is by changing the subcellular localization of the participating proteins4,50,56 and it has been shown for other plant membrane proteins that their activity is regulated by changing the location within the cell.57,58 One of the interactors of the receptors is the ADL1A protein for which a role in membrane recycling and more generally polarized cell growth has been described.59 Adaptin, another interactor, shows high homology to a subunit of the Adaptin Journal of Proteome Research • Vol. 7, No. 9, 2008 3657

research articles complex in Drosophila and mice, which provides an interface between proteins in vesicles and the clathrin scaffold stabilizing these structures during their journey to and from the plasma membrane.60 The GNOM protein, which was found to interact with CRE1/AHK4, is a mediator of intracellular membrane trafficking and essential for hormone-dependent plant growth.61 All three proteins, ADL1A, Adaptin and GNOM, are also implicated in the vesicular transport of the auxin transporterPIN1betweenthetrans-Golgiandtheplasmamembrane.57,61 One other interactor (AT1G64330) shows a high homology to USO1. This yeast protein has also been shown to play a role in intracellular transport; it recruits vesicles transported from the ER to the Golgi.62 The identification of these interactors makes it tempting to speculate that the cytokinin receptors are not only located in the plasma membrane as predicted by the model and as was shown for AHK3,63 but also in other cellular compartments. Indeed our preliminary subcellular results obtained using an AHK3-GFP-fusion protein indicate a more diverse localization than previously reported (data not shown). Different subcellular localizations may represent an additional layer of posttranslational regulation of cytokinin signaling and the proteins identified as interactors of the receptors may be involved in regulating their trafficking within the cell. While it is tempting to interpret the data of an interaction map in such a manner, one should do it with several caveats in mind. First, not all proteins are suitable for the Y2H system, and thus, a given protein might have more interactors, that is, being more highly connected, than the data show. Such a bias might also be caused by the fact that most baits were screened with only one cDNA library. Thus, the number of interactors for all proteins is likely to be larger than detected in this study. In addition, although we employed a vector/strain combination, which has proven to be very reliable in the past,11,34 the interactions detected in this study need to be verified using different experimental set ups.

Conclusions This study and the resulting interaction network represent an important, albeit first step, toward elucidating the interactome of the TCS of Arabidopsis. The identification of several known interactions in our screens underlines the suitability of this system for the elucidation of protein interaction networks. The numerous new interactions detected in the screens point toward new routes of crosstalk with other pathways or mechanism of regulation of protein function and thus provide important stepping stones for the generation of testable hypotheses. However, further experiments are necessary to confirm the detected interactions and the next challenge will be to determine their function in planta.

Acknowledgment. We thank Prof. Erich Wanker (MDC, Berlin) for the bait plasmid and the yeast strain. This work was supported by the AFGN Program and by the SFB 449 of the DFG and by a NaFo¨G stipend to H.D. Supporting Information Available: Supplemental Figure 1, flowchart of library-screens; Supplemental Table 1, oligonucleotide sequences for the cloning of the genes used as baits in the yeast two-hybrid screens; Supplemental Table 2, interactors of TCS proteins, their localization and functional annotation according to the Arabidopsis database (TAIR7 release; www.arabidopsis.org).This material is available free of charge via the Internet at http://pubs.acs.org. 3658


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Toward an Interaction Map of the Two-Component Signaling Pathway

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