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>

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	<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. 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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. 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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><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 &amp;amp; concentrator Kit</td><td>ZYMO RESEARCH</td><td>D4029</td><td></td></tr><tr><td>&lt;em&gt;DpnI&lt;/em&gt;</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&amp;alpha; 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>&lt;em&gt;Pfu Ultra&lt;/em&gt;</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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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 ...

functional-characterization-ring-type-e3-ubiquitin-ligases-vitro

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.8K Views.  University of Idaho. Dit stapsgewijze protocol laat zien hoe de enzymatische activiteit van RING-type E3 ubiquitine ligas, zowel in vitro als in planta, kan worden gedetecteerd en functioneel kan worden gekenmerkt door een locatiegerichte mutagenesis-benadering. Door mutatie in een RING-domein te introduceren via site-directed mutagenesis, kan de resulterende E3-deficiënte mutant parallel met het wild-type eiwit worden getest om de enzymatische activiteit te koppelen aan functionaliteit. Het toewerken van de bio...

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

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological functions. Such interactions can be highly dynamic, meaning the interacting subunits are constantly associated and dissociated at certain rates. While measuring the binding affinity using techniques such as quantitative pull-down reveals the strength of the interaction, studying the binding kinetics provides insights on how fast the interaction occurs and how long each complex can exist. Furthermore, measuring the kinetics of an interaction in the presence of an additional factor, such as a protein exchange factor or a drug, helps reveal the mechanism by which the interaction is regulated by the other factor, providing important knowledge for the advancement of biological and medical research. Here, we describe a protocol for measuring the binding kinetics of a protein complex that has a high intrinsic association rate and can be dissociated quickly by another protein. The method uses fluorescence resonance energy transfer to report the formation of the protein complex in vitro, and it enables monitoring the fast association and dissociation of the complex in real time on a stopped-flow fluorimeter. Using this assay, the association and dissociation rate constants of the protein complex are quantified.

Protein-protein interactions are critical for biological systems, and studies of the binding kinetics provide insights into the dynamics and function of protein complexes. We describe a method that quantifies the kinetic parameters of a protein complex using fluorescence resonance energy transfer and the stopped-flow technique. &#160;

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Biology

Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases

Ubiquitin is a small 8.6 kDa protein that is a core component of the ubiquitin-proteasome system. Consequently, it can bind to a diverse array of proteins with high specificity but low affinity. Through phage display, ubiquitin variants (UbVs) can be engineered such that they exhibit improved affinity over wildtype ubiquitin and maintain binding specificity to target proteins. Phage display utilizes a phagemid library, whereby the pIII coat protein of a filamentous M13 bacteriophage (chosen because it is displayed externally on the phage surface) is fused with UbVs. Specific residues of human wildtype ubiquitin are soft and randomized (i.e., there is a bias towards to native wildtype sequence) to generate UbVs so that deleterious changes in protein conformation are avoided while introducing the diversity necessary for promoting novel interactions with the target protein. During the phage display process, these UbVs are expressed and displayed on phage coat proteins and panned against a protein of interest. UbVs that exhibit favorable binding interactions with the target protein are retained, whereas poor binders are washed away and removed from the library pool. The retained UbVs, which are attached to the phage particle containing the UbV's corresponding phagemid, are eluted, amplified, and concentrated so that they can be panned against the same target protein in another round of phage display. Typically, up to five rounds of phage display are performed, during which a strong selection pressure is imposed against UbVs that bind weakly and/or promiscuously so that those with higher affinities are concentrated and enriched. Ultimately, UbVs that demonstrate higher specificity and/or affinity for the target protein than their wildtype counterparts are isolated and can be characterized through further experiments.

Ubiquitination is a critical protein post-translational modification, dysregulation of which has been implicated in numerous human diseases. This protocol details how phage display can be utilized to isolate novel ubiquitin variants that can bind and modulate the activity of E3 ligases that control the specificity, efficiency, and patterns of ubiquitination.

Ubiquitin is a small 8.6 kDa protein that is a core component of the ubiquitin-proteasome system. Consequently, it can bind to a diverse array of proteins ...

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, including cell survival, proliferation, and differentiation. It is a reversible process that consists of a covalent attachment of ubiquitin to the substrate through a cascade reaction of at least three different enzymes, composed of E1 (Ubiquitin-activation enzyme), E2 (Ubiquitin-conjugating enzyme), and E3 (Ubiquitin-ligase enzyme). The E3 complex plays an important role in substrate recognition and ubiquitylation. Here, a protocol is described to evaluate substrate ubiquitylation in mammalian cells using transient co-transfection of a plasmid encoding the selected substrate, an E3 ubiquitin ligase, and a tagged ubiquitin. Before lysis, the transfected cells are treated with the proteasome inhibitor MG132 (carbobenzoxy-leu-leu-leucinal) to avoid substrate proteasomal degradation. Furthermore, the cell extract is submitted to small-scale immunoprecipitation (IP) to purify the polyubiquitylated substrate for subsequent detection by western blotting (WB) using specific antibodies for ubiquitin tag. Hence, a consistent and uncomplicated protocol for ubiquitylation assay in mammalian cells is described to assist scientists in addressing ubiquitylation of specific substrates and E3 ubiquitin ligases.

We provide a detailed protocol for a ubiquitylation assay of a specific substrate and an E3 ubiquitin-ligase in mammalian cells. HEK293T cell lines were used for protein overexpression, the polyubiquitylated substrate was purified from cell lysates by immunoprecipitation, and resolved in SDS-PAGE. Immunoblotting was used to visualize this post-translational modification.

Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, ...

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular &#8220;tags&#8221; for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this principle, the purification of critical proteins required for the functioning of these pathways along with their native interacting partners has not only allowed the mapping of the protein constituents of these pathways, but has also provided a deeper understanding of how these proteins coordinate to regulate these pathways. Within this context, understanding a protein's spatiotemporal localization and its protein-protein interaction network can aid in defining its role within a pathway, as well as how its misregulation may lead to disease pathogenesis. To address this need, several approaches for protein purification such as tandem affinity purification (TAP) and localization and affinity purification (LAP) have been designed and used successfully. Nevertheless, in order to apply these approaches to pathway-scale proteomic analyses, these strategies must be supplemented with modern technological developments in cloning and mammalian stable cell line generation. Here, we describe a method for generating LAP-tagged human inducible stable cell lines for investigating protein subcellular localization and protein-protein interaction networks. This approach has been successfully applied to the dissection of multiple cellular pathways including cell division and is compatible with high-throughput proteomic analyses.

We describe a method for generating localization and affinity purification (LAP)-tagged inducible stable cell lines for investigating protein function, spatiotemporal subcellular localization and protein-protein interaction networks.

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this ...

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the prediction of family functions based on several features, such as the presence of sequence motifs or of particular sites for post-translational modification and the expression profile of family members in different conditions. This work describes a detailed protocol for gene family characterization. Here, the procedure is applied to the characterization of the Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligase family in grapevine. The methods include the genome-wide identification of family members, the characterization of gene localization, structure, and duplication, the analysis of conserved protein motifs, the prediction of protein localization and phosphorylation sites as well as gene expression profiling across the family in different datasets. Such procedure, which could be extended to further analyses depending on experimental purposes, could be applied to any gene family in any plant species for which genomic data are available, and it provides valuable information to identify interesting candidates for functional studies, giving insights into the molecular mechanisms of plant adaptation to their environment.

This article describes the procedure for the identification and characterization of a gene family in grapevine applied to the family of Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligases.

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the ...

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily through modulating protein-protein interactions. Similar to ubiquitin modification, SUMOylation is directed by a sequential enzyme cascade including E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3-ligase (i.e., PIAS family, RanBP2, and Pc2). However, different from ubiquitination, an E3 ligase is non-essential for the reaction but does provide precision and efficacy for SUMO conjugation. Proteins modified by SUMOylation can be identified by in vivo assay via immunoprecipitation with substrate-specific antibodies and immunoblotting with SUMO-specific antibodies. However, the demonstration of protein SUMO E3 ligase activity requires in vitro reconstitution of SUMOylation assays using purified enzymes, substrate, and SUMO proteins. Since in the in vitro reactions, usually SAE1/SAE2 and Ubc9, alone are sufficient for SUMO conjugation, enhancement of SUMOylation by a putative E3 ligase is not always easy to detect. Here, we describe a modified in vitro SUMOylation protocol that consistently identifies SUMO modification using an in vitro reconstituted system. A step-by-step protocol to purify catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase, is also presented. The SUMOylation activities of the purified K-bZIP are shown on p53, a well-known target of SUMO. This protocol can not only be employed for elucidating novel SUMO E3 ligases, but also for revealing their SUMO paralog specificity.

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

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

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