Name: functional characterization of ring-type e3 ubiquitin ligases in vitro and in planta
Uploaded: 2019-12-05T13:30:00
Duration: 10 min 27 s
Description: Watch this Scientific Journal Video about Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta at JoVE.com

Our work was made possible by the financial support from the Agriculture and Food Research Initiative competitive grant (2017-67014-26197; 2017-67014-26591) of the USDA National Institute of Food and Agriculture, USDA-NIFA Farm Bill, Northwest Potato Consortium, and ISDA Specialty Crop.

1. Site-directed mutagenesis (Figure 1)
<ol>
	<li>Identify the conserved Cys and His amino acids in the RING domain (Figure 1A) and design primers carrying the substitution codon of interest flanked by 15 base pairs on either side of the mutation site (Figure 1B).</li>
	<li>Introduce the desired mutation by PCR-based amplification of the plasmid harboring the gene of interest using mutagenic primers and high-fidelity DNA polymerase containing Pfu in 50 &#181;L of total PCR reaction volume as shown in Table 1 and Table 2 according to the manufacturer&#39;s protocol.</li>
	<li>Digest the Escherichia coli-derived parental methylated and semi-methylated DNA by adding 3 &#181;L of DpnI restriction enzyme directly to the PCR reaction (step 1.2) and incubating at 37 &#176;C for 2 h. 
	NOTE: Methylation is a posttranscriptional protein modification that is added to the plasmid produced and isolated from bacteria. New copies of PCR-generated plasmid lack methylation, therefore, the new copies will remain intact during DpnI treatment.</li>
	<li>Purify the mutagenized plasmids using a commercial DNA extraction kit based on spin column technology and elute the DNA with 50 &#181;L of water.</li>
	<li>Transform DH5&#945; E. coli chemically competent cells with 0.5 &#181;L of the recovered mutagenized plasmid DNA according to the manufacturer&#39;s protocol. In brief, incubate competent cells with DNA on ice for 30 min, then heat-shock them for 20 s at 42 &#176;C, and place tubes again on ice for 2 min. Incubate cells with 500 &#181;L of LB media at 37 &#176;C for 1 h at 250 rpm and then spread them on selective pates.</li>
	<li>Verify the desired mutation by Sanger sequencing the DNA plasmids isolated from E. coli.</li>
</ol>
2. Recombinant protein purification and in vitro ubiquitination assay
<ol>
	<li>Clone the wild type RING and mutated RING genes of interest into the pMAL-c2 vector (follow the manufacturer&#39;s protocol; Table 3) to fuse these genes with the MBP epitope tag that permits one-step purification using amylose resin. Introduce the resulting constructs into the E. coli BL21 strain as described in step 1.5.</li>
	<li>Grow the E. coli strain BL21 harboring the desired construct in 50 mL LB liquid medium at 37 &#176;C for 2&#8211;3 h until it reaches the logarithmic phase (OD600 of 0.4&#8211;0.6).</li>
	<li>Add IPTG to a final concentration of 0.1&#8211;1 mM to induce the expression of MBP-tagged recombinant protein of interest and incubate the E. coli culture for 2&#8211;3 h at 28 &#176;C. Place the culture on ice after incubation. 
	NOTE: Perform steps 2.4&#8211;2.13 on ice to protect proteins from degradation.</li>
	<li>To check for the induction efficiency, collect 1.5 mL of induced cells, spin them down at 13,000 x g for 2 min, remove the supernatant, and resuspend the cells in 20 &#181;L of 2x SDS-PAGE loading buffer (24 mM Tris-HCl pH 6.8, 0.8% SDS, 10% (v/v) glycerol, 4 mM DTT, 0.04% (w/v) bromophenol blue).</li>
	<li>Boil the samples for 5 min and run them on a 10% SDS-PAGE gel. To visually evaluate accumulation of MBP-fusion protein (molecular weight of protein of interest + 42.5 kDa MBP), stain the gel for 20 min by agitating with Coomassie staining buffer (50% methanol, 10% acetic acid, 0.1% Coomassie blue) and destaining overnight with the destaining buffer (20% methanol, 10% acetic acid).</li>
	<li>Harvest the remaining E. coli cells by centrifugation at 1,350 x g for 6 min, discard the supernatant, and resuspend the cell pellet with 5 mL of column buffer (20 mM Tris HCl, 200 mM NaCl, 1 mM EDTA, bacterial protease inhibitor). 
	NOTE: This is a good place to stop the protocol overnight. The frozen cells can be stored up to 1 week at -20 &#176;C.</li>
	<li>Break down E. coli cells by placing the tube containing the bacteria in an ice-water bath and applying 10 sonication cycles: 10 s sonication at 30% amp followed by 20 s breaks.</li>
	<li>Centrifuge sample at 13,000 x g at 4 &#176;C for 10 min and save the supernatant (crude extract).</li>
	<li>Prepare 500 &#181;L of amylose resin in a 15 mL tube. Wash the resin by adding 10 mL of cold column buffer and centrifuging at 1,800 x g, 4 &#176;C for 5 min. Do this 2x.</li>
	<li>Add 5 mL of crude extract to the tube with the amylose resin and incubate overnight at 4 &#176;C.</li>
	<li>Centrifuge at 1,800 x g at 4 &#176;C for 5 min and discard the supernatant.</li>
	<li>Add 10 mL of column buffer to the resin pellet and incubate for 20 min. Then centrifuge at 1,800 x g at 4 &#176;C for 5 min. Repeat this step 2x.</li>
	<li>Elute the fusion protein with 0.5 mL of column buffer containing 10 mM maltose by incubating a sample for 2 h at 4 &#176;C. Centrifuge at 1,800 x g at 4 &#176;C for 5 min and collect the eluted protein. Repeat this step 2x.</li>
	<li>Dialyze 1 mL of the protein fraction against the cold PBS. Aliquot protein into single-use tubes (10&#8211;20 &#181;L) to avoid freeze-thawing and store at -80 &#176;C until needed.</li>
	<li>Measure the protein concentration using the Bradford assay9.</li>
	<li>Set up the in vitro ubiquitination reaction in a total volume of 30 &#181;L by mixing up 40 ng of E1 (e.g., AtUBA1), 100 ng of E2 (e.g., AtUBC8, SlUBC1/4/6/7/12/13/17/20/22/27/32), 1 &#181;g MBP-RING type protein, and 2 &#181;g FLAG-Ub (or HA-Ub) in the ubiquitination buffer (50 mM Tris-HCl pH 7.5, 2 mM ATP, 5 mM MgCl2, 30 mM creatine phosphate, 50 &#181;g/mL creatine phosphokinase). Incubate the mixture at 30 &#176;C for 2 h. 
	NOTE: Premake 20x ubiquitination buffer and store it up to 6 months at -20 &#176;C in small aliquots for a single use. The creatine phosphokinase easily loses its enzymatic activity when the buffer is thawed and frozen repeatedly.</li>
	<li>Terminate the reaction by mixing the 30 &#181;L samples with 7.5 &#181;L of 5x SDS-PAGE loading buffer (60 mM Tris-HCl pH 6.8, 2% SDS, 25% (v/v) glycerol, 10 mM DTT, 0.1% (w/v) bromophenol blue) and boiling for 5 min.</li>
	<li>Separate the proteins with 7.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), then transfer onto the PDVF membrane, and detect ubiquitination by Western blotting using the anti-FLAG (or anti-HA).</li>
	<li>Stain the PVDF membrane with Coomassie blue to verify the equal loading of tested MBP-RING-type protein.</li>
</ol>
3. Agrobacterium-mediated transient protein expression in Nicotiana benthamiana leaves and in planta ubiquitination assay
<ol>
	<li>Streak appropriate Agrobacterium tumefaciens strains carrying the epitope-tagged gene of interest (e.g., HA-RHA1B, HA-RHA1BC135S, HA-RHA1BK146R, HA-Ub) and empty vector as a control on the LB medium containing the appropriate selection antibiotics.</li>
	<li>After 2 days of growth at 28 &#176;C, pick up single colonies and grow them in LB liquid medium with the appropriate antibiotics at 28 &#176;C/250 rpm for another 24 h.</li>
	<li>Transfer 100 &#181;L of agrobacterial culture to 3 mL fresh LB with the appropriate antibiotics and incubate the culture for an additional 4&#8211;6 h at 28 &#176;C with rotation (250 rpm) to the late exponential growth phase.</li>
	<li>Spin down agrobacterial cells at 1,800 x g for 6 min, discard the supernatant, and resuspend cells with 3 mL of wash buffer (50 mM MES pH 5.6, 28 mM glucose, 2 mM NaH2PO4). Repeat this step 2x.</li>
	<li>After the second wash, resuspend the cells in the induction buffer (50 mM MES pH 5.6, 28 mM glucose, 2 mM NaH2PO4, 200 &#181;M acetosyringone, 37 mM NH4Cl, 5 mM MgSO4.7H2O, 4 mM KCl, 18 &#181;M FeSO4.7H2O, 2 mM CaCl2). Incubate the cells with induction buffer for an additional 10&#8211;12 h at 28 &#176;C. 
	NOTE: Acetosyringone induces T-DNA transfer.</li>
	<li>Centrifuge the cells at 1,800 x g for 6 min, discard the supernatant, and resuspend the cells with 2 mL of infiltration buffer (10 mM MES pH 5.5, 200 &#181;M acetosyringone). 
	NOTE: If Agrobacteria aggregate after incubation with induction buffer, let the aggregated cells sink to the bottom of the tube by leaving it on the bench for a few minutes, and transfer the clear Agrobacterium suspension to a new tube before proceeding with step 3.6.</li>
	<li>Measure the concentration of bacteria using the OD600 value (the optical density at absorbance of 600 nm). Adjust OD600 values to the desired ones. 
	NOTE: Usually an OD600 value between 0.2&#8211;0.4 works best for a single agrobacterial stain expression. If a combination of different agrobacterial strains is applied, the total OD600 values of Agrobacterial strains should not exceed 1.</li>
	<li>Agroinfiltrate 4-week-old N. bethamiana leaves by gently pricking them with a needle, followed by hand-injecting Agrobacterium with a syringe without the needle. Circle the infiltrated leaf area with the marker (usually 1&#8211;2 cm in diameter).</li>
	<li>Collect the infiltrated leaf tissues 36 h post-infiltration. Grind the tissue to a fine powder with liquid nitrogen.</li>
	<li>Resuspended tissue powder with 300 &#956;L of protein extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mM DTT, 10% glycerol, 1% polyvinylpolypyrrolidone, 1 mM PMSF, plant protease inhibitor cocktail) and centrifuge at 15,000 x g for 15 min at 4 &#730;C.</li>
	<li>Transfer the supernatant to a new tube. Add 5x SDS-PAGE loading buffer to a final concentration of 1x and boil for 5 min.</li>
	<li>Separate crude proteins on 10% SDS-PAGE gels, transfer onto PVDF membranes, and probe with anti-HA to detect in planta ubiquitination.</li>
</ol>
4. Establishing the link between enzymatic activity and function in planta
NOTE: For example, RHA1B promotes degradation of resistant protein Gpa2 to suppress HR cell death. This step shows how to verify that those virulent activities of RHA1B are E3-dependent.
<ol>
	<li>Streak appropriate Agrobacterium tumefaciens strains carrying tagged genes of interest (in this example HA-RHA1B, HA-RHA1BC135S, HA-RHA1BK146R, myc-Gpa2, RBP1) and empty vector as a control. Follow steps 3.1&#8211;3.8 for Agrobacterium preparation and injection on N. bethamiana leaves.</li>
	<li>For the E3-dependent substrate protein degradation, follow steps 3.9&#8211;3.12 and perform the Western blotting using appropriate antibodies to detect the protein accumulation in plant cells (e.g., anti-HA and anti-MYC).</li>
	<li>For the E3-dependent hypersensitive response (HR) mediated cell death inhibition, monitor the agroinfiltrated leaves for HR cell death symptoms 2&#8211;4 days post-infiltration.</li>
</ol>

<ol>
	<li>Freemont, P. S., Hanson, I. M., Trowsdale, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+gysteine-rich+sequence+motif.">A novel gysteine-rich sequence motif.</a> Cell. 64, 483-484 (1991).</li><li>Borden, K. L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RING+fingers+and+B-boxes:+Zinc-binding+protein-protein+interaction+domains.">RING fingers and B-boxes: Zinc-binding protein-protein interaction domains.</a> Biochemistry and Cell Biology. 76, 351-358 (1998).</li><li>Barlow, P. N., Luisi, B., Milner, A., Elliott, M., Everett, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+C3HC4+Domain+by+1H-nuclear+Magnetic+Resonance+Spectroscopy:+A+New+Structural+Class+of+Zinc-finger.">Structure of the C3HC4 Domain by 1H-nuclear Magnetic Resonance Spectroscopy: A New Structural Class of Zinc-finger.</a> Journal of Molecular Biology. 237, 201-211 (1994).</li><li>Borden, K. L. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+solution+structure+of+the+RING+finger+domain+from+the+acute+promyelocytic+leukaemia+proto-oncoprotein+PML.">The solution structure of the RING finger domain from the acute promyelocytic leukaemia proto-oncoprotein PML.</a> The EMBO Journal. 14, 1532-1541 (1995).</li><li>Lorick, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RING+fingers+mediate+ubiquitin-conjugating+enzyme+(E2)-dependent+ubiquitination.">RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination.</a> Proceedings of the National Academy of Sciences. 96, 11364-11369 (1999).</li><li>Jim&#233;nez-L&#243;pez, D., Mu&#241;&#243;z-Belman, F., Gonz&#225;lez-Prieto, J. M., Aguilar-Hern&#225;ndez, V., Guzm&#225;n, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repertoire+of+plant+RING+E3+ubiquitin+ligases+revisited:+New+groups+counting+gene+families+and+single+genes.">Repertoire of plant RING E3 ubiquitin ligases revisited: New groups counting gene families and single genes.</a> PLoS ONE. 13, 1-28 (2018).</li><li>Sacco, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cyst+Nematode+SPRYSEC+Protein+RBP-1+Elicits+Gpa2-+and+RanGAP2-Dependent+Plant+Cell+Death.">The Cyst Nematode SPRYSEC Protein RBP-1 Elicits Gpa2- and RanGAP2-Dependent Plant Cell Death.</a> PLoS Pathogens. 5, 1-14 (2009).</li><li>Kud, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potato+cyst+nematode+effector+RHA1B+is+a+ubiquitin+ligase+and+uses+two+distinct+mechanisms+to+suppress+plant+immune+signaling.">The potato cyst nematode effector RHA1B is a ubiquitin ligase and uses two distinct mechanisms to suppress plant immune signaling.</a> PLoS Pathogens. 15, 1007720 (2019).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Analytical Biochemistry. 72, 248-254 (1976).</li><li>Sigrist, C. J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+and+continuing+developments+at+PROSITE.">New and continuing developments at PROSITE.</a> Nucleic Acids Research. 41, 344-347 (2013).</li><li>Dove, K. K., Stieglitz, B., Duncan, E. D., Rittinger, K., Klevit, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+insights+into+RBR+E3+ligase+ubiquitin+transfer+mechanisms.">Molecular insights into RBR E3 ligase ubiquitin transfer mechanisms.</a> EMBO Reports. 17, 1221-1235 (2016).</li><li>Metzger, M. B., Pruneda, J. N., Klevit, R. E., Weissman, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RING-type+E3+ligases:+Master+manipulators+of+E2+ubiquitin-conjugating+enzymes+and+ubiquitination.">RING-type E3 ligases: Master manipulators of E2 ubiquitin-conjugating enzymes and ubiquitination.</a> Biochimica et Biophysica Acta - Molecular Cell Research. 1843, 47-60 (2014).</li><li>Xie, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SINAT5+promotes+ubiquitin-related+degradation+of+NAC1+to+attenuate+auxin+signals.">SINAT5 promotes ubiquitin-related degradation of NAC1 to attenuate auxin signals.</a> Nature. 419, 167-170 (2002).</li></ol>

The authors have nothing to disclose.

Elucidating the biochemical and mechanistic basis of RING type E3 ubiquitin ligases can contribute greatly to our understanding of their biological significance in development, stress signaling, and maintenance of homeostasis. The protocol described here couples a mutagenesis approach with in vitro and in planta functional studies. By introducing a single amino acid substitution in the conserved residues of the RING domain through site-direct mutagenesis, the resulting E3-deficient mutant can be tested in parallel with wild type protein to link enzymatic activity with functionality.
It is critical to properly identify the RING domain, particularly its conserved Cys and His residues. Online tools such as PROSITE can be used to do so10. To destabilize the RING domain responsible for recruiting the E2 enzyme, Cys is normally substituted with Ser, which is its closest structural replacement lacking the ability to create a disulfide bond used for zinc coordination. Lorick et al. showed that the mutation in any of those critical Cys residues would abolish the ubiquitination activity of the single subunit RING-type E3 ligases5. Although some Cys residues are also important for multiunit E3 ligase complexes containing RING-type proteins, due to the multifaceted and dynamic three-dimensional structure of those ubiquitination complexes and different role of RING-type proteins, single substitutions of conserved residues in the RING domain in multiunit E3 ligase has not been successful in generating a ligase deficient phenotype11.
For the site-directed mutagenesis, we found that using smaller plasmid vectors and lower amplification cycles usually yielded higher efficiency for mutagenesis. The Pfu enzyme can be substituted with any other high-fidelity and high processivity DNA polymerase. Furthermore, if the gene of interest contains rare codons, another E. coli stain, Rosetta, can be used to achieve higher yield of the recombinant protein. Additionally, both incubation time and temperature for IPTG induction can be further optimized. Lower temperatures reduce E. coli division rate, which might be favorable for expression of certain proteins. Although higher concentration of IPTG could improve protein expression, it also inhibits E. coli division processes and is not recommended.
Single subunit RING type E3 ligases not only function as a molecular scaffold that positions the E2-Ub intermediate in close proximity to the substrate but also stimulate the ubiquitin transfer activity of their cognate E2s. Furthermore, given that an E2/E3 combination is important for the length and linkages of the polyubiquitin chain that determines the fate of a modified substrate, any consideration of RING-type E3s must include their enzymatic partners, E2s12. As shown in Figure 3B, not all tested E2s are compatible with the RHA1B ligase. Therefore, in vitro ubiquitination assays should be carried in parallel with multiple E2 enzymes representing different E2 classes to avoid false negative results.
Presented here is the in vitro enzymatic assay that detects the self-ubiquitination ability of tested RING-type proteins. However, with small modifications, this protocol can be easily adapted to detect in vitro ubiquitination of substrates. To this end, the in vitro ubiquitination mixture from step 2.15 should be supplemented with the recombinant protein of the potential E3 ligase substrate (500 ng). Following a 2 h incubation at 30 &#176;C, ubiquitinated protein should be captured using 15 &#181;L of anti-HA affinity matrix (if HA-Ub is used, or anti-FLAG affinity matrix if FLAG-Ub is used) by agitation for 2 h at 4 &#176;C. After washing the beads 4x times with the cold Ub wash buffer (20 mM Tris pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 0.05% Tween 20, 1x PMSF), discard all but 40 &#181;L of the buffer and move to step 2.16. The ubiquitination signal, detected by antibodies specific to the epitope-tagged Ub and substrate, respectively, emerging from molecular weight of the substrate protein, confirms the substrate/enzyme specificity.
Furthermore, identification of E3 ligase substrates in vivo is usually associated with multiple challenges due to transient enzyme-substrate interaction and rapid degradation of ubiquitinated target protein. Using an E3 ligase deficient mutant, which still interacts with its target but no longer ubiquitinates it13, is a very useful alternative to addition of proteasomal inhibitor MG132, which does not always sufficiently interfere with 26S proteasome function.
A common characteristic of RING-type E3 ligases is a tendency to form and function as homo- and/or heterodimers. Interestingly, the substitution in the conserved residues of the RING domain is usually associated with a dominant negative phenotype where mutated RING-type E3 ligase blocks enzymatic activity of a native wild type protein13. Thus, overexpression of RING mutants in planta may be an alternative approach to knocking out the E3 ligase gene.

The vast majority of E3 ubiquitin ligases belong to RING (Really Interesting New Gene)-type proteins. The RING-finger domain was originally identified by Freemont et al.1 and functionally described as a domain mediating protein-protein interaction2. The canonical RING finger is a special type of zinc coordinating domain defined as a consensus sequence of eight conserved Cys (C) and His (H) specifically spaced by other amino acid residues (X), C-X2-C-X9&#8211;39-C-X1&#8211;3-H-X2&#8211;3-C/H-X2-C-X4&#8211;48-C-X2-C. Two Zn2+ ions are stabilized by core C and H residues through unique "cross-brace" topology with C1/C2 and C/H5/C6 coordinating the first Zn2+ ion whereas C3/H4 and C7/C8 bind the second (Figure 1A)3,4. Depending on the presence of either C or H in the fifth Zn2+-coordination site, two canonical subclasses of RING-finger proteins were defined: C3HC4 and C3H2C3 (RING-HC and RING-H2, respectively). Because the RING domain of E3 ubiquitin ligase mediates the interaction between E2 conjugating enzymes and substrates, mutation of these essential C and H residues has been shown to disrupt the ligase activity5. An additional five less common subclasses of RING E3 ligases have been described (RING-v, RING-C2, RING-D, RING-S/T, and RING-G)6. The RING-type E3 ubiquitin ligases can be further subdivided into simple and complex E3 enzymes. The simple single subunit RING E3 ligases contain both the substrate recognition site and the E2-binding RING domain. By contrast, the multisubunit RING-type E3 complex either recruit's substrate or mediates binding of the E2-ubiquitin intermediate to the E3 complex. The RING domain Lys residue(s) that serves as a primary ubiquitin attachment site(s) for self-ubiquitination might also be important for the E3 ligase activity.
Not all RING-containing proteins function as E3 ligases. Thus, the bioinformatic prediction of RING-finger domain and the capacity for E2-dependent protein ubiquitination must be biochemically validated and linked to the biological role of the tested protein. Here, we describe a step-by-step protocol outlining how to detect and functionally characterize the enzymatic activity of RING-type E3 ubiquitin ligases, both in vitro and in planta, through a site-directed mutagenesis approach. The representative results from this pipeline are shown for the RING-type E3 ligase RHA1B. RHA1B is an effector protein produced by the plant parasitic cyst nematode Globodera pallida to suppress plant immunity and manipulate the morphology of plant root cells. To protect themselves from pathogen/parasite invasion, plants have evolved nucleotide-binding domain and leucine-rich repeat (NB-LRR) type immune receptors that detect the presence of a pathogen or parasite and, as a consequence, develop the hypersensitive response (HR), which is a form of rapid and localized cell death occurring at the infection site to arrest colonization of pathogens. One such immune receptor is the potato Gpa2 protein that confers resistance to some isolates of G. pallida (field populations D383 and D372)7.
Using the presented protocols, it has been recently found that RHA1B interferes with plant immune signaling in an E3-dependent manner by targeting the plant Gpa2 immunoreceptor for ubiquitination and degradation8.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetosyringone</td>
			<td>Sigma-Aldrich</td>
			<td>D134406</td>
			<td></td>
		</tr>
		<tr>
			<td>Amylose resin</td>
			<td>NEB</td>
			<td>E8021S</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma-Aldrich</td>
			<td>A1852</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial protease inhibitor</td>
			<td>Sigma-Aldrich</td>
			<td>P8465</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromphenol Blue</td>
			<td>VWR</td>
			<td>97061-690</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C1016</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>model: Avanti J-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Commassie Blue</td>
			<td>VWR</td>
			<td>97061-738</td>
			<td></td>
		</tr>
		<tr>
			<td>Creatine phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>P7936</td>
			<td></td>
		</tr>
		<tr>
			<td>Creatine phosphokinase</td>
			<td>Sigma-Aldrich</td>
			<td>C3755</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA clean &#38; concentrator Kit</td>
			<td>ZYMO RESEARCH</td>
			<td>D4029</td>
			<td></td>
		</tr>
		<tr>
			<td>DpnI</td>
			<td>NEB</td>
			<td>R0176S</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma-Aldrich</td>
			<td>D0632</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli BL21</td>
			<td>Thermo Fisher Scientific</td>
			<td>C600003</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli DH5&#945; competent cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>18265017</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>324504</td>
			<td></td>
		</tr>
		<tr>
			<td>FeSO4 7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>F7002</td>
			<td></td>
		</tr>
		<tr>
			<td>FLAG-Ub</td>
			<td>BostonBiochem</td>
			<td>U-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>VWR</td>
			<td>188</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>HA-Ub</td>
			<td>BostonBiochem</td>
			<td>U-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat block</td>
			<td>VWR</td>
			<td>model: 10153-318</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>VWR</td>
			<td>model: 1525 Digital Incubator</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator shaker</td>
			<td>Thermo Fisher Scientific</td>
			<td>model: MaxQ 4000</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Roche</td>
			<td>10724815001</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td>Sigma-Aldrich</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquide nitrogen</td>
			<td>university chemistore</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Maltose</td>
			<td>Sigma-Aldrich</td>
			<td>63418</td>
			<td></td>
		</tr>
		<tr>
			<td>MES</td>
			<td>Sigma-Aldrich</td>
			<td>M3671</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>63138</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4 7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>63138</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>model: 5424</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniprep plasmid purification kit</td>
			<td>ZYMO RESEARCH</td>
			<td>D4015</td>
			<td></td>
		</tr>
		<tr>
			<td>monoclonal anti-FLAG antibody</td>
			<td>Sigma-Aldrich</td>
			<td>F3165</td>
			<td></td>
		</tr>
		<tr>
			<td>monoclonal anti-HA antibody</td>
			<td>Sigma-Aldrich</td>
			<td>H9658</td>
			<td></td>
		</tr>
		<tr>
			<td>monoclonal anti-MYC antibody</td>
			<td>Sigma-Aldrich</td>
			<td>WH0004609M2</td>
			<td></td>
		</tr>
		<tr>
			<td>Mortar</td>
			<td>VWR</td>
			<td>89038-144</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>S8282</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop</td>
			<td>Thermo Fisher Scientific</td>
			<td>model: 2000 Spectrophotometer</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-826-5C</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Sigma-Aldrich</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR machine</td>
			<td>Bio-Rad</td>
			<td>model: C1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Pestle</td>
			<td>VWR</td>
			<td>89038-160</td>
			<td></td>
		</tr>
		<tr>
			<td>Pfu Ultra</td>
			<td>Agilent Technologies</td>
			<td>600380</td>
			<td></td>
		</tr>
		<tr>
			<td>Plant protease inhibitor coctail</td>
			<td>Sigma-Aldrich</td>
			<td>P9599</td>
			<td></td>
		</tr>
		<tr>
			<td>pMAL-c2</td>
			<td>NEB</td>
			<td>N8076S</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma-Aldrich</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinylpolypyrrolidone</td>
			<td>Sigma-Aldrich</td>
			<td>P6755</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma-Aldrich</td>
			<td>1614363</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Qsonica Sonicators</td>
			<td>model: Q125</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Thermo Fisher Scientific</td>
			<td>22-253-260</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td></td>
		</tr>
	</tbody>
</table>

functional characterization of ring-type e3 ubiquitin ligases in vitro and in planta

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent attachment of 76 amino acid ubiquitin modifiers to a target protein, depending on the length and topology of the polyubiquitin chain, can result in different outcomes ranging from protein degradation to changes in the localization and/or activity of modified protein. Three enzymes sequentially catalyze the ubiquitination process: E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase. E3 ubiquitin ligase determines substrate specificity and, therefore, represents a very interesting study subject. Here we present a comprehensive approach to study the relationship between the enzymatic activity and function of the RING-type E3 ubiquitin ligase. This four-step protocol describes 1) how to generate an E3 ligase deficient mutant through site-directed mutagenesis targeted at the conserved RING domain; 2&#8211;3) how to examine the ubiquitination activity both in vitro and in planta; 4) how to link those biochemical analysis to the biological significance of the tested protein. Generation of an E3 ligase-deficient mutant that still interacts with its substrate but no longer ubiquitinates it for degradation facilitates the testing of enzyme-substrate interactions in vivo. Furthermore, the mutation in the conserved RING domain often confers a dominant negative phenotype that can be utilized in functional knockout studies as an alternative approach to an RNA-interference approach. Our methods were optimized to investigate the biological role of the plant parasitic nematode effector RHA1B, which hijacks the host ubiquitination system in plant cells to promote parasitism. With slight modification of the in vivo expression system, this protocol can be applied to the analysis of any RING-type E3 ligase regardless of its origins.

In this section, representative results are provided for the protocol used for examination of a single subunit E3 ubiquitin ligase RHA1B that has a PROSITE-predicted RING-H2 type domain (132&#8211;176 amino acids)10. As shown in Figure 1, in order to obtain an E3-deficient mutant protein, at least one of the eight conserved Cs or Hs in the RING domain (Figure 1A) needs to be mutagenized (Figure 1B). Thus, as a first step, two mutant versions of RHA1B, RHA1BC135S (a substitution of Cys by Ser in the conserved C3 of RING domain) and RHA1BK146R (a substitution of Lys by Arg in the only Lys present in RHA1B) were generated. Although single subunit E3 ligases mediate ubiquitin transfer from ubiquitin harboring E2 to the substrate rather than directly interacting with ubiquitin, self-ubiquitination of E3 at Lys might be required for its maximal enzymatic activity.
The Western blotting results in Figure 2A show a typical positive in vitro ubiquitination assay outcome, with a multibanding smear starting at the molecular weight of the tested protein (e. g., MPB-fused RHA1B ~100 kDa) and progressing upwards. The anti-HA antibody recognized HA-tagged Ub incorporated into the poly-ubiquitination chain of different lengths, creating this typical ubiquitin-associated ladder-like smear. To validate the positive results, Figure 2A also presents all important negative controls missing individual components (E1, E2, Ub, or MBP-RHA1B) or using MBP as control and lacking the smeared ubiquitination signal. Furthermore, the Coomassie blue staining of the PVDF membrane showed equal loading of MBP-RHA1B or MBP in all controls.
Figure 2B shows how in vitro ubiquitination results varied depending on the specific E2/E3 combination. In this example, 11 different E2s representing 10 different E2 families were tested. The detected ubiquitination activity ranged from no signal (no smear) to a multibanding smear starting at different molecular weights, indicating different ubiquitination patterns.
Figure 3 shows ubiquitination assay outcomes for RING- and K-mutant versions of tested protein. Lack of enzymatic activity for RHA1BC135S is supported by its inability to either generate a multibanding smear in vitro (Figure 3A) or promote poly-ubiquitination signal in planta (Figure 3B). It is notable that overexpression of HA-tagged Ub in planta on its own gave basal level ubiquitination in all tested samples, including the vector control, in contrast to the strong ubiquitination signal conferred by the enzymatic activity of wild type RHA1B. Furthermore, the analysis on the RHA1BK146R mutant suggests that the K146 residue is also essential for the E3 activity of RHA1B. Although a marginal self-ubiquitination signal was detected in vitro (Figure 3A), the in planta assay determined the mutant is E3-deficient (Figure 3B, only background ubiquitination signal detected).
After generating and biochemically validating the E3-deficient mutant, functional studies can be designed to determine the E3-associated biological role of the tested RING E3 ubiquitin ligase. In the case of RHA1B, this nematode effector suppresses plant immune signaling, as manifested by suppression of the Gpa2-triggered HR cell death. As presented in Figure 4A, unlike the wild type RHA1B, the RHA1BC135S mutant lacking E3 ligase activity did not interfere with HR cell death. Given that the most common outcome of protein ubiquitination is its proteasome-mediated degradation, mutations residing in the RING domain can also be used to verify an E3-dependent ability to trigger degradation of their direct and/or indirect substrates. Thus, significantly, Western blotting results in Figure 4B confirm that Gpa2 did not accumulate in the presence of wild type RHA1B but RHA1BC135S had no impact on Gpa2 protein stability.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60533/60533fig01.jpg" /> 
Figure 1: Schematic representation of the principle and steps involved in site-directed mutagenesis. (A) RING-CH/H2 domain with conserved Cys and His amino acids highlighted. (B) An example of mutagenic primers design. (C) Steps of site-directed mutagenesis. <a href="https://www.jove.com/files/ftp_upload/60533/60533fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60533/60533fig02.jpg" /> 
Figure 2: Representative in vitro ubiquitination assay. (A) Top gel shows ubiquitination assay including all negative controls, and bottom gel shows equal loading. (B) The range of expected results depending on E2 enzymes. This figure has been modified from Kud et al8. <a href="https://www.jove.com/files/ftp_upload/60533/60533fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60533/60533fig03.jpg" /> 
Figure 3: Ubiquitination assay results for RING- and K- mutants (RHA1BC135S and RHA1BK146R). (A) In vitro ubiquitination results for RHA1BC135S and RHA1BK146R. (B) In planta ubiquitination assay results for RHA1BC135S and RHA1BK146R. This figure has been modified from Kud et al8. <a href="https://www.jove.com/files/ftp_upload/60533/60533fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60533/60533fig04.jpg" /> 
Figure 4: Representative functional study for E3 dependent biological functions. An example of functional studies showing the E3-dependent biological function. (A) E3-dependent HR cell death suppression and (B) degradation of a plant immunoreceptor Gpa2. This figure has been modified from Kud et al8. <a href="https://www.jove.com/files/ftp_upload/60533/60533fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">PCR set up</td>
		</tr>
		<tr>
			<td>1 &#181;L</td>
			<td>plasmid (~100 ng)</td>
		</tr>
		<tr>
			<td>1.5 &#181;L</td>
			<td>F mutagenic primer (10 &#181;M)</td>
		</tr>
		<tr>
			<td>1.5 &#181;L</td>
			<td>R mutagenic primer (10 &#181;M)</td>
		</tr>
		<tr>
			<td>1 &#181;L</td>
			<td>dNTPs (10 mM)</td>
		</tr>
		<tr>
			<td>5 &#181;L</td>
			<td>buffer (10x)</td>
		</tr>
		<tr>
			<td>1 &#181;L</td>
			<td>Ultra Pfu polymerase (2.5 U/&#181;l)</td>
		</tr>
		<tr>
			<td>39 &#181;L</td>
			<td>ddH2O</td>
		</tr>
		<tr>
			<td>50 &#181;L</td>
			<td>TOTAL VOLUME</td>
		</tr>
	</tbody>
</table>
Table 1: PCR reaction set up
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">thermocycler program</td>
		</tr>
		<tr>
			<td>1</td>
			<td>95 &#730;C</td>
			<td>30 s</td>
			<td></td>
		</tr>
		<tr>
			<td>2</td>
			<td>95 &#730;C</td>
			<td>30 s</td>
			<td></td>
		</tr>
		<tr>
			<td>3</td>
			<td>60 &#730;C</td>
			<td>30 s</td>
			<td></td>
		</tr>
		<tr>
			<td>4</td>
			<td>72 &#730;C</td>
			<td>5 min</td>
			<td>repeat 2-4 30 times</td>
		</tr>
		<tr>
			<td>5</td>
			<td>72 &#730;C</td>
			<td>5 min</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: PCR thermocycler program
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">ligation reaction set up for the RHA1B example</td>
		</tr>
		<tr>
			<td>1.5 &#181;L</td>
			<td colspan="2">pMAL-c2::MBP linearized vector by digestion with BamHI and SalI (60 ng)</td>
		</tr>
		<tr>
			<td>7 &#181;L</td>
			<td colspan="2">RHA1B/RHA1BC135S or RHA1BK146R insert digested with BamHI and SalI (25 ng)</td>
		</tr>
		<tr>
			<td>1 &#181;L</td>
			<td colspan="2">T4 ligase buffer (10x)</td>
		</tr>
		<tr>
			<td>0.5 &#181;L</td>
			<td colspan="2">T4 ligase (400 U/&#181;L)</td>
		</tr>
		<tr>
			<td>10 &#181;L</td>
			<td colspan="2">TOTAL VOLUME</td>
		</tr>
	</tbody>
</table>
Table 3: Ligation reaction set up for the RHA1B example.

Watch this Scientific Journal Video about Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta at JoVE.com

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

The goal of this manuscript is to present an outline for the comprehensive biochemical and functional studies of the RING-type E3 ubiquitin ligases. This multistep pipeline, with detailed protocols, validates an enzymatic activity of the tested protein and demonstrates how to link the activity to function.

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent ...

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