Characterization of Light-Regulated Protein–Protein Interactions by In Vivo Coimmunoprecipitation (Co-IP) Assays in Plants

Abstract

In light signaling pathways, protein complexes play essential roles in light perception and signal transduction. The phytochrome family of photoreceptors perceives red/far-red region of the light spectrum and then translocates into the nucleus to form protein complexes. Many phytochrome interacting proteins have been identified based on yeast two-hybrid screening and other protein-protein interaction methods. However, it is essential to demonstrate that these proteins interact with phytochromes in vivo to be functionally relevant. In this chapter, a protocol for demonstrating light dependent in vivo interactions between phytochromes and phytochrome interacting proteins is described. This protocol can be adapted for any putative phytochrome interacting protein for validation of their interaction in vivo.

1. Introduction

Light provides energy source for plant growth and development. However, light also acts as a pivotal informational signal that regulates plant adaptation to prevailing environmental conditions. Plants perceive light signal through multiple classes of photoreceptors responding to different spectrum of light. These include UVR8 (for sensing UV-B light), phototropins (phot1/2), cryptochromes (cry1/2/3) and Zeitlupe family of receptors (ZTL/LKP1/FKF) (for sensing UV-A/Blue lights), and phytochromes (for sensing blue/red/far-red lights) [1]. Among these photoreceptors, phytochromes (phys) are well-characterized light perception system in Arabidopsis and other plants [2]. Phytochromes are chromoproteins with an attached bilin chromophore that perceives light. There are two light-switchable forms of phytochromes: Pr, an inactive form that perceives light in the red region of the spectrum and Pfr, a biologically active form that perceives far-red region of the spectrum. These two forms can interconvert and exist as an equilibrium in plant cells. In darkness, the Pr form of phytochromes is present in the cytosol and is photoinactive. Upon light exposure, phytochromes undergo conformational changes to the Pfr form and translocate into the nucleus [3]. The photoactivated phytochromes in the nucleus induce a large change in protein–protein complex formation and transcriptional reprogramming that drive photomorphogenesis.

Within the nucleus, phytochromes interact with bHLH transcription factors called the phytochrome interacting factors (PIFs) [4, 5], circadian clock factors [6, 7], splicing factors [8], E3 ubiquitin ligases [9], and others. Among the best-characterized phytochrome interacting proteins are the PIFs. PIFs are negative regulators in the light signaling pathways. The interaction between phytochromes and PIFs results in rapid phosphorylation, ubiquitination, and degradation of the PIFs through the 26S proteasome system [2]. PIFs are phosphorylated by various kinases in response to light. The phosphorylated forms are recognized by various E3 ubiquitin ligases and then rapidly degraded to promote photomorphogenesis [2]. Thus, multiple dynamic and rapid changes in protein–protein interactions and protein complex formation are necessary for rapid responses to light signal. Therefore, identification and in vivo demonstration of protein–protein interaction under both dark and light conditions are necessary to understand the mechanisms by which plants respond to light signal. In this chapter, we provide a detailed protocol for demonstrating in vivo light-dependent interaction between phytochrome and PIF1 as an example. The in vivo coimmunoprecipitation (co-IP) method is suitable for detecting protein interacting partners under both dark and light conditions.

Although, the in vivo coimmunoprecipitation (co-IP) method demonstrates interaction between phytochromes and putative interaction partners, there are several advantages and disadvantages for this method. The advantages include the following: (1) this method provides robust verification of interactions in vivo where the interaction is functionally relevant, (2) proteins can be post-translationally modified in vivo if the posttranslational modification is necessary for interaction with phytochromes, and (3) the interaction between phytochromes and the putative phytochrome interacting partner might still be detected even if the interaction is indirect. The disadvantages include the following: (1) the interaction between phytochromes and the putative phytochrome interacting partner might be indirect, as a bridging protein can facilitate the interaction, (2) this method cannot be used to distinguish interactions in a cell- and/or tissue-specific manner in vivo; although, a modified version of this method can be used for this purpose.

2. Materials

2.1. Plant Material Preparation

1. 10 cm ø petri dishes.

2. 7.5 cm ø filter paper.

3. Standard dark room and light chambers (Model: E30LED, Percival Scientific, Inc.).

4. MS medium: dissolve 4.33 g Murashige and Skoog basal medium in 1 L H₂O, adjust pH to 5.7 by NaOH, add 9 g agar and autoclave.

5. Liquid MS medium: dissolve 4.33 g Murashige and Skoog basal medium in 1 L H₂O, adjust pH to 5.7 by NaOH and autoclave.

6. Bortezomib stock: make 40 mM bortezomib (Fisher Scientific, #B1408100MG) stock in DMSO; aliquot to 20 μL volume and store at −20 °C.

7. 40 μM bortezomib in liquid MS media: mix 5 μL 40 mM bortezomib stock with 5 mL liquid MS in 10 cm ø petri dish.

8. Liquid nitrogen.

9. Mortar and pestle.

10. Photometer (e.g., Beckman DU530 UV-VIS Life Science Spectrophotometer).

2.2. Coimmunoprecipitation

1. 100 mM phosphate buffer, pH 7.8: Mix 90.8 mL 1 M K₂HPO₄ and 9.2 mL 1 M KH₂PO₄, and dilute the combined 1 M stock solutions to 1 L with distilled H₂O.

2. Native extraction buffer/IP buffer: 100 mM phosphate buffer, pH 7.8, 150 mM NaCl, 0.1% NP40, 1× protease inhibitor cocktail (Sigma-Aldrich Co., St Louis, MO, Cat. No: P9599), 1 mM PMSF, 10 mM iodoacetamide, 40 μM bortezomib, 25 mM β-glycerophosphate, 10 mM sodium fluoride (NaF), and 2 mM sodium orthovanadate (Na₃VO₄).

3. IP wash buffer: 100 mM phosphate buffer, pH 7.8, 150 mM NaCl, 0.5% NP40, 1× protease inhibitor cocktail, 1 mM PMSF, 40 μM bortezomib, 25 mM β-glycerophosphate, 10 mM sodium fluoride (NaF), and 2 mM sodium orthovanadate (Na₃VO₄).

4. Dynabeads Protein A (Life Technologies Co., Carlsbad, CA, Cat. No: 10002D).

5. Magnetic rack.

6. Primary antibodies used for immunoprecipitation.

7. Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, Inc., Hercules, CA, Cat. No: 500-0006).

2.3. Western Blot

1. 6× SDS gel loading buffer: 300 mM Tris–HCl, pH 6.8, 12% SDS, 0.6% bromophenol blue, 60% glycerol, 600 mM DTT.

2. 1.5× SDS gel loading buffer: 75 mM Tris–HCl, pH 6.8, 3% SDS, 0.15% bromophenol blue, 15% glycerol, 150 mM DTT.

3. 4× lower gel buffer: 1.5 M Tris–HCl, pH 8.8, 0.4% SDS.

4. 4× upper gel buffer: 0.5 M Tris–HCl, pH 6.8, 0.4% SDS.

5. 6.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel:

   1. Lower gel (total 10 mL): Add 2.2 mL 30% acrylamide–bis solution, 2.5 mL 4× lower gel buffer, 5 μL tetramethyle-thylenediamine (TEMED), 50 μL 10% ammonium persulfate (APS), 5.25 mL H₂O.
   2. Upper gel (total 5 mL): Add 0.83 mL 30% acrylamide–bis solution, 1.25 mL 4× lower gel buffer, 5 μL TEMED, 50 μL 10% APS, 2.87 mL H₂O.

6. PVDF membrane.

7. Transfer buffer for semidry method: 48 mM Tris, 39 mM glycine, 20% methanol.

8. Whatman 3 MM filter paper.

9. Standard equipment to run SDS-PAGE gels.

10. Semidry electroblotter.

11. Primary and secondary antibodies.

12. Dry milk.

13. Bovine serum albumin (BSA).

14. 1× TBST: 20 mM Tris, 150 mM NaCl, 0.05% Tween 20; adjust pH to 7.6 by HCl.

15. LumiGLO Reserve^® Chemiluminescent Substrate Kit (SeraCare, Milford, MA, Cat. No: 54-71-00).

16. X-ray film or imaging system for detection of chemiluminescent signals (e.g., FluorChem Q, ProteinSimple, San Jose, CA, Cat. No: 92-14095-00).

3. Methods

3.1. Plant Material Preparation

3.1.1. Seed Plating and Germination

1. Sterilize 0.04 g Arabidopsis seeds for each sample, and plate onto 7.5 cm ø circular filter in a 10 cm ø MS/agar plate. Try to align the seeds on the filter paper in a line and keep ~1.5 cm space between the lines.

2. Keep the plates at 4 °C for 4 days for stratification.

3. Expose the seeds for 3 h to white light at room temperature to stimulate germination. Keep the plates open in the hood for the first half an hour to reduce water accumulation in the plates.

4. Keep the plates at 21 °C for 21 h and give 3000 μmol m⁻² far-red light total afterward to photoinactivate phytochromes. Align all the plates in the dark and wrap with aluminum foil. Place plates vertically at 21 °C for 3–4 days, so seedlings will grow vertically.

5. The seedlings will grow flat on the filter paper.

3.1.2. Bortezomib Pretreatment and Sample Collection

1. Prepare final concentration of 40 μM bortezomib with liquid MS media in 10 cm ø plates.

2. In the dark room, with very dim safe green light, carefully transfer the filter paper with seedlings grown on MS agar plates into MS liquid media with 40 μM bortezomib. Make sure the seedlings are fully soaked in the liquid but not floating around (see Notes 1 and 2).

3. Keep all the samples in MS liquid media supplemented with 40 μM bortezomib for 4 h.

4. Either keep seedlings in darkness or treat seedlings with light pulse (see Note 3). Collect individual samples and dry seedlings very well using paper towels. Samples can be wrapped with aluminum foil and stored at −80 °C or directly ground for protein extraction.

3.2. Coimmunoprecipitation

3.2.1. Binding Primary Antibodies to Dynabeads

1. Resuspend the Dynabeads by vortexing for 30 s.

2. Transfer 20 μL Dynabeads per sample to an eppendorf tube.

3. Place the tubes in a magnetic rack for 1 min and discard the supernatant.

4. Wash the Dynabeads with 1 mL IP buffer per tube by inverting the tubes multiple times to fully resuspend beads in IP buffer.

5. Place the tubes in a magnetic rack for 1 min and discard the supernatant.

6. Resuspend beads in 20 μL IP buffer per sample and add 1 μg of the desired antibody into individual tube.

7. Couple Dynabeads and antibodies at 4 °C by end-to-end rotation for 30–60 min.

8. Place the tube in a magnetic rack for 1 min and discard the supernatant.

9. Wash Dynabeads with 1 mL IP buffer by fully resuspending Dynabeads; place tubes back in a magnetic rack for 1 min and discard the supernatant.

10. Repeat washing step as described in step 9 for another two times.

11. After three times wash, add 50 μL IP buffer per tube and store on ice. The antibody precoupled Dynabeads are ready to use.

3.2.2. Preparation of Crude Extracts

1. Grind samples into powder in liquid nitrogen using a mortar and pestle; add three times liquid nitrogen for grinding to make sure samples turning into fine powder and maximize the total protein yield.

2. Add 800 μL of native extraction buffer per sample directly into the mortar and keep grinding the sample till the sample melts into liquid form (see Notes 4 and 5).

3. Collect individual samples into 1.5 mL eppendorf tubes and keep on ice.

4. Centrifuge samples at 16,000 × g in a microcentrifuge at 4 °C in the dark for 15 min.

5. Transfer 1 mL of the supernatant for each sample into new eppendorf tubes as cell lysate (see Note 6).

3.2.3. Measurement of Protein Concentration in Cell Lysate

1. Pipette samples for calibration curve for measurement of protein concentration as shown in Table 1; mix the samples well and incubate for 5 min at room temperature before measuring the absorption at 595 nm using a photometer (e.g., Beckman DU530 UV-VIS Life Science Spectrophotometer) (see Note 7); plot the protein concentration of the samples against the absorption at 595 nm.

2. Measure protein concentration in cell lysate; add 795 μL H₂O, 200 μL Bio-Rad Protein Assay Dye Reagent Concentrate and 5 μL of protein crude extracts for each sample. Mix well by pipetting up and down a few times and incubate for 5 min at room temperature before measuring the absorption at 595 nm using a photometer; calculate the protein concentration in the protein crude extracts using the calibration curve from the previous step.

Table 1.

Calibration curve for measurement of protein concentration

Protein concentration (μg/mL)	0.5 mg/mL BSA (μL)	H2O (μL)	IP buffer (μL)	Protein assay dye reagent concentrate (μL)
0	0	795	5	200
2.5	5	790	5	200
5	10	785	5	200
10	20	775	5	200
20	40	755	5	200

3.2.4. Coimmunoprecipitation Using Antibody Precoupled Dynabeads

1. For each sample add 20 μL antibody precoupled Dynabeads to equal amount (usually 1 mL) of crude protein extract (total protein); compensate for different volumes of crude protein extracts by adding IP buffer; work in the darkroom using safety green light.

2. Incubate the samples by end-to-end rotation at 4 °C in the dark for 3 h.

3. After 3 h IP in the dark, all the wash steps need to be done in darkness.

4. Place the tubes in a magnetic rack for 1 min.

5. Save 50 μL supernatant from each sample as input controls and add 10 μL 6× SDS gel loading buffer into each tube.

6. Discard the rest of supernatant and wash the Dynabeads with 1 mL wash buffer per tube by inverting the tubes multiple times to fully resuspend beads in wash buffer.

7. Place the tubes in a magnetic rack for 1 min and discard the supernatant.

8. Repeat washing step as described in steps 6 and 7 for another two times.

9. After three times wash, centrifuge tubes briefly with 500 rpm for 1 min and place tubes back in a magnetic rack for 1 min.

10. Discard any wash buffer left over and add 20 μL 1.5× SDS gel loading buffer.

11. Wrap IP sample and input control samples with aluminum foil. Freeze them together at −80 °C.

3.3. Detection of Protein–Protein Interactions

3.3.1. Electrophoresis of Protein Complex

1. Thaw input and IP samples from −80 °C quickly.

2. Mix and incubate samples at 65 °C for 10 min.

3. Load 10 μL input and 20 μL IP samples onto 6.5% SDS-PAGE gel. Using a constant voltage of 120 V, run gel until the bromophenol blue dye runs off the bottom of the gel (see Note 8).

3.3.2. Transfer Protein Complex Onto PVDF Membrane Using Semidry Method

1. Dissemble the gel cassette and cut off the upper gel.

2. Cut a piece of PVDF membrane and eight pieces of Whatman 3 MM filter paper with slightly larger size than the gel.

3. Rinse the PVDF membrane thoroughly with methanol and keep it in transfer buffer.

4. Prewet eight pieces of Whatman 3 MM filter paper with transfer buffer.

5. Clean the semidry electroblotter using 70% ethanol; dry it very well with Kimwipes.

6. Put four pieces of prewet Whatman 3 MM filter paper and get rid of any bubbles using a glass Pasteur pipette.

7. Gently lay the gel, PVDF membrane, and the remaining four pieces of prewet Whatman 3 MM filter papers sequentially. Make sure to remove any bubbles within all layers.

8. Wipe off any excess transfer buffer and carefully attach the cover of the semidry electroblotter; do not move the cover afterward.

9. Tight screws at all three positions evenly; start transfer using a constant current of 50 mA per gel; transfer at room temperature for 90 min.

3.3.3. Western Blot

1. After transfer, discard the SDS-PAGE gel and directly put the PVDF membrane in 1× TBST/5% dry milk; incubate under constant shaking at room temperature for 1 h.

2. Wash the membrane with 1× TBST multiple times to wash off residual milk.

3. Rotate the membrane with primary antibody in 1× TBST/1% BSA overnight at 4 °C.

4. Wash the membrane with 1× TBST three times for 10 min at room temperature.

5. Rotate the membrane with appropriate secondary antibody in 1× TBST/0.5% dry milk for 1 h at room temperature.

6. Wash the membrane with 1× TBST three times for 10 min at room temperature.

7. Mix 300 μL substrate A and 300 μL substrate B of the LumiGLO Reserve^® Chemiluminescent Substrate Kit in an 1.5 mL eppendorf tube.

8. Slightly dry the membrane on a paper towel and put in a transparent plastic folder.

9. Pipet mixed chemiluminescent substrate equally onto the member; the whole membrane should be covered with substrate.

10. Let the substrate react with membrane for 90 s at room temperature and image the luminescent signal using an X-ray film or an imaging system such as FluorChem Q.

4. Notes

1. During the bortezomib treatment, the liquid should be minimized to just cover the surface of vertically grown seedlings on the filter paper. Too little media will cause inefficient treatment which results in not blocking the protein degradation process. Too much media will cause seedlings floating around and make sample collection difficult. Usually 5 mL liquid MS is used for 10 cm ø plate. If other sizes of plates are used, the exact amount of liquid MS needed should be tested first.

2. MG132 is a common inhibitor for 26S proteasome mediated degradation. Due to its instability, MG132 efficiency relies on the storage time and condition strictly. Bortezomib is a therapeutic broad proteasome inhibitor used in human and works more efficiently than MG132 for PIF1 studies.

3. This protocol is designed to assess the protein–protein interacting complexes PIF1 mainly is involved in. As mentioned in the introduction, PIF1 is rapidly degraded upon light exposure. Thus the light treatment is with a very short period time of saturating light (3000 μmol m⁻² red light) and samples are collected after 5 min dark incubation. However, this Co-IP assay is suitable for all other factors in the light signaling pathway including other PIFs. The amount of light and the time of dark incubation should be modified related to the protein focused on. An example of in vivo coimmunoprecipitation between PIF1 and phyA/B is shown in Fig. 1.

4. For the native extraction buffer/IP buffer, adding 10 mM iodoacetamide is essential to keep intact phytochromes during preparation of the extract and co-IP. Failure to add 10 mM iodoacetamide might result in truncated version of phyA.

5. When studying the protein complexes containing COP1, adding 0.125–0.25 mM EDTA in the native extraction buffer/IP buffer helps to stabilize the COP1 mediated protein–protein interactions. Lack of EDTA dramatically reduced the Co-IP efficiency.

6. It is very important to keep samples and extracts in darkness all the time before adding 1.5× SDS gel loading buffer into the samples. Any excessive light exposure will affect the proteins interacting complex especially in the study of PIF1 interacting partners.

7. The protein concentration can be measured using different kits. This protocol used the Bio-Rad Protein Assay Dye Reagent Concentrate. Other protein concentration assays such as BCA protein assay could also be used.

8. The concentration of acrylamide in the SDS-PAGE gel should be adjusted according to the size of proteins of interest. Generally with the increase of the acrylamide–bis concentration in the lower gel, the separation is improved for proteins with small molecular weight. Commercially available gradient gel can be used for optimal separation.

Fig. 1.

In vivo coimmunoprecipitation of PIF1 with phyA/B. (a) Schematic diagram showing the bait (LUC-PIF1) and the preys (native phyA and phyB proteins). Firefly luciferase is fused with PIF1 at the N-terminus. APB, phyB binding domain; APA, phyA binding domain; bHLH, basic helix–loop–helix domain. (b) LUC-PIF1 interacts with the Pfr forms of phyA and phyB in in vivo coimmunoprecipitation assays. The input and pellet fractions from in vivo co-immunoprecipitation assays are indicated. Total protein was extracted from four-day old dark-grown seedlings either exposed to a short red light pulse (R; 3000 μmol m⁻²) or kept in the dark. Coimmunoprecipitations were carried out using the anti-PIF1 antibody (lanes 1 and 2) or with an unrelated IgG as a control (lane 3). The immunoprecipitated samples were then probed with anti-phyA, anti-phyB, or anti-LUC antibodies. Reproduced in part from “Shen H, Zhu L, Castillon A, Majee M, Downie B, Huq E (2008) Light-induced phosphorylation and degradation of the negative regulator PHYTOCHROME-INTERACTING FACTOR1 from Arabidopsis depend upon its direct physical interactions with photoactivated phytochromes. Plant Cell 20:1586–1602; www.plantcell.org; Copyright American Society of Plant Biologists”