Materials and Reagents

15 ml conical tube
2 ml Eppendorf tube
1.5 ml Eppendorf tube
Pipette tips
96-well Plate (Thermo Scientific, catalog number: 249935 )
10-day-old P_PAG1-gPAG1-FM transgenic seedlings
Liquid nitrogen
Tris Base (Fisher Scientific, catalog number: BP152-10 )
Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
NaCl (Fisher Scientific, catalog number: BP358-10 )
MgCl₂(Sigma-Aldrich, catalog number: M9272 )
EDTA (Fisher Scientific, catalog number: BP120-1 )
Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43817 )
Glycerol (Sigma-Aldrich, catalog number: V900122 )
PMSF (Sigma-Aldrich, catalog number: 78830 )
Miracloth (Calbiochem, catalog number: 475855 )
Anti-FLAG^®M2 magnetic beads (Sigma-Aldrich, catalog number: M8823 )
3× FLAG peptide (DYKDDDDK) (Sigma-Aldrich, catalog number: F4799 )
Anti-Flag (Sigma-Aldrich, catalog number: F1804 )
Anti-Myc (Sigma-Aldrich, catalog number: C3956 )
Anti-SE (Agrisera, catalog number: AS09 532A)
DMSO (Sigma-Aldrich, catalog number: D4540 )
MG132 (Calbiochem, catalog number: 474787 )
Succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (Suc-LLVY-AMC) (Sigma-Aldrich, catalog number: S6510 )
SilverQuest^TM Staining Kit (Invitrogen, catalog number: LC6070 )
Bradford Reagent (Sigma-Aldrich, catalog number: B6916 )
2× SDS-PAGE loading buffer (see Recipes)
Extraction buffer (see Recipes)
Washing buffer (see Recipes)
Reaction Buffer (see Recipes)

Equipment

-80 °C freezer
Pipettes
Centrifuge
DynaMag^TM-2 (Invitrogen, model: 12321D )
PolyATtract^® System 1000 Magnetic Separation Stand (Promega Corporation, model: Z5410 )
Rugged Rotator (Glas-Col, model: 099A RD4512 )
Thermomixer R (Eppendorf, model: 022679810)
Microplate spectrophotometer (PerkinElmer, model: VICTOR^TM X3)
Gel imaging system (Bio-Rad, model: Universal Hood II )
Vortex mixer (VWR, model: 945300 )

Procedure

Note: Please see Figure 1 for a schematic diagram of the steps described below.

Figure 1. A schematic diagram of procedure

Preparation of 10-day-old P_PAG1-gPAG1-FM stable transgenic plants (Li et al., 2020)
1. Transform a binary vector pBA002a-P_PAG1-gPAG1-FM into Col-0 ecotype of Arabidposis thaliana by the floral-dip transformation method (Zhang et al., 2006) to generate P_PAG1-gPAG1-FM stable transgenic plants.
2. Sterilize and place the seeds from the stable transgenic line expressing P_PAG1-gPAG1-FM on MS medium (Zhang et al., 2006) and stratify seeds by keeping them in the dark at 4 °C for 3 days.
3. Germinate seeds and grow the seedlings under a 12 h light-12 h dark cycle at 22 °C for 10 days.
4. Collect 5 g of 10-day-old seedlings, ground to fine powder in liquid nitrogen, and stored at -80 °C.
Affinity purification of 20S proteasomes (Book et al., 2010)
1. Re-suspend the 5 g powder sample in 8 ml of extraction buffer.
2. Keep the sample on ice for min. Keeping the sample cold, homogenize it well with a vortex mixer 2-3 times.
3. Centrifuge the fully dissolved protein extract at 4 °C for 15 min at 21,000 × g and then filter the protein extract through one layer of pre-wet Miracloth.
4. After filtration, centrifuge the cleared protein extract again at 4 °C for 15 min at 21,000 × g.
5. Collect the supernatant from Step B4 in a pre-cooled 15 ml conical tube and keep it on ice.
6. Prepare Anti-FLAG beads for immunoprecipitation during the centrifugation steps. Add 200 μl bed volume Anti-FLAG^® M2 magnetic beads (for 5 g plant tissue) into a new 2 ml Eppendorf tube on ice.
7. Wash the magnetic FLAG-beads with 600 μl of 0.1 M glycine HCl (pH 3.0) to remove un-conjugated antibody.
8. Invert the tube gently and leave the mixture in the tube for 1.5 min.
9. Immediately re-equilibrate the Anti-FLAG^® M2 magnetic beads with 1 ml buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0).
10. Remove the equilibration buffer, followed by washing the beads with 1 ml extraction buffer for three times using DynaMag10. Remove the equilibration buffer, followed by washing the beads with 1 ml extraction buffer three times using DynaMag^TM-2.
11. Completely remove the extraction buffer and add 200 μl new extraction buffer into the tube to re-suspend the beads.
12. Add 200 μl equilibrated magnetic beads into sample.
13. Rotate the 15 ml tube at 4 °C for 30 min using a rugged rotator.
14. At the end of Step B13, prepare 3× FLAG peptide solution for elution of Flag-4Myc-tagged PAG1 from the Anti-FLAG^® M2 magnetic beads.
15. Add 35 μl 3× FLAG elution buffer stock (4 mg/ml) into 245 μl extraction buffer to make a final concentration 500 ng/μl of 3× FLAG peptide. Mix well and put it on ice.
16. After Step B13 is done, load the 15 ml tube on the PolyATtract^® System 1000 Magnetic Separation Stand for a few seconds, then slowly pour out the supernatant. Supernatant can be discarded.
17. Re-suspend the beads with 6 ml washing buffer and transfer all the beads to a new, clean precooled 15 ml tube.
18. Wash the beads with 6 ml washing buffer three times at 4 °C, each time for 5 min using the rugged rotator.
19. After washing three times, add 2 ml of extraction buffer to a 15 ml tube to re-suspend the beads.
20. Transfer all the beads carefully to a clean 2 ml Eppendorf tube.
21. Load the 2 ml tube into DynaMag^TM-2, and remove the extraction buffer.
22. Add 250 μl 3× FLAG elution buffer into the 2 ml tube, incubate the tube for 30 min at 4°C with 1,200 rpm shaking using Thermomixer R.
23. Load the 2 ml tube into DynaMag^TM-2. Transfer the eluates to the 1.5 ml Eppendorf tube and store at -80 °C.
Silver staining of purified proteasome
1. Add 20 μl 2× SDS-PAGE loading buffer into 20 μl of the eluted sample and boil at 95 °C for 8 min.
2. Load 20 μl eluted sample onto two 10% SDS-PAGE gels and subject to electrophoresis for 2.5 h at 80 V.
3. Stain one of the gels with SilverQuest^TM Staining Kit (Figure 2).
  
  Figure 2. A representative silver-staining image of immunoprecipitated PAG1-FM-containing 20S resolved by SDS-PAGE. The immunoprecipitation was performed using the anti-FLAG^®M2 magnetic beads with the protein extracts prepared from P_PAG1-gPAG1-FM transgenic seedlings (gPAG1-FM) or from Col-0 (control), respectively. The bracket indicates subunits of the 20S core proteasome (CP).
4. Do Western blot analysis for the other gel using anti-Myc or anti-Flag antibodies.
5. Take images of silver staining gel using a gel imaging documentation system.
Proteasome activity assay (Yang et al., 2004; Han et al., 2019)
1. Prepare reaction buffer containing 50 μM Suc-LLVY-AMC substrate, which is widely used as a fluorogenic substrate for measuring the chymotrypsin – like activity of the 20S proteasome (Reidlinger et al., 1997).
2. Add 10 μl eluted sample into 90 μl reaction Buffer, mix well and add into 96-well Plate.
3. Add 10 μl extraction buffer into 90 μl reaction Buffer as a blank control and repeat each reaction three times.
4. Incubate the reaction mixtures at 37 °C for 20, 40, 60, 80, 100, 120 min.
5. Monitor the fluorescence reading of the released AMC at the indicated times using a Microplate spectrophotometer by fluorescence using 380 nm excitation and 440 nm emission wavelengths.
6. Plot proteasome activity in relative fluorescence units per 1 μl of reaction mixtures, using free AMC as a standard.
  Note: You can also use other representations to display your data, such as relative fluorescence units per 10 μl of eluted sample.

In vitro 20S proteasome degradation assay (Hsieh et al., 2015)

Prepare purified proteins for testing.
Estimate the concentration of the purified proteasome and test proteins by the Bradford method (Bradford, 1976). (Method: https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/b6916bul.pdf).

Prepare in vitro 20S proteasome-decay reaction mixtures on ice as follows (Table 1):

Table 1. In vitro 20S proteasome degradation reaction system

One reaction	Reaction mixtures for five indicated times
150 nM Purified test protein	According to the concentration
10 nM Purified 20S proteasome	According to the concentration
50 mM Tris-HCl (pH 7.5)	10 μl [1 M Tris-HCl (pH 7.5) ]
2% DMSO or 50 μM MG132	4 μl (2.5 mM MG132, dissolved in DMSO)
Add H₂O to final volume 40 μl	Add H₂O to final volume 200 μl

Note: The amount of the purified 20S proteasome and test protein in the reaction mixture is only a reference, the reaction condition needs to be optimized for different proteins.

Then distribute the mixtures evenly into five PCR or 1.5 ml tubes and incubate the tubes at 22 °C.
Stop the reaction by adding 40 μl 2× SDS-PAGE loading buffer at the indicated times (0, 5, 10, 20, 30 min) followed by Western blot analysis (Figure 3).

Figure 3. A representative image of in vitro 20S proteasome-mediated protein degradation assay. Recombinant 6xHis-SUMO-SE protein was incubated with the PAG1-FM immunoprecipitate from P_PAG1-gPAG1-FM transgenic plants or control IP from Col-0, respectively. The reaction mixture was stopped at the indicated time intervals. Western blot assay of 6xHis-SE was probed with an anti-SE antibody.

Data analysis

For additional reference images of silver staining and in vitro 20S proteasome degradation assay, see Figures 4c and 4d from Li et al. (2020), respectively. For additional reference images of the proteasome activity assay, see the Extended Data Figure 7b from Li et al. (2020).

Notes

In step B, all the buffers and tubes required for purification should be pre-cooled to 4 °C before use.
In step B, Miracloth is pre-wet with extraction buffer.
In step B, divide the final elution samples into several tubes to avoid freeze-thaw samples in future use, which will effect proteasome activity.
In step E, optimize the in vitro degradation conditions according to test proteins, including pH, degradation time, degradation temperature and the concentration of the 20S proteasome and test proteins.
In step E, prepare and evenly distribute the reaction mixtures quickly and keep on ice. Make sure all time points start the reaction at the same time.

Recipes

2× SDS-PAGE loading buffer
0.125 mM Tris-HCl, pH 6.8
20% glycerol
4% SDS
0.2 M DTT
0.02% bromophenol blue
Extraction buffer
50 mM Tris-HCl, pH 7.5
25 mM NaCl
2 mM MgCl₂
1 mM EDTA
5% glycerol
2 mM PMSF (add just before use)
Washing buffer
50 mM Tris-HCl, pH 7.5
800 mM NaCl
2 mM MgCl₂
1 mM EDTA
5% glycerol
2 mM PMSF (add just before use)
Reaction Buffer
50 mM Tris-HCl, pH 7.5
25 mM NaCl
2 mM MgCl₂
1 mM EDTA
2 mM dithiothreitol (DTT)
5% glycerol
50 μM Suc-LLVY-AMC substrate

Acknowledgments

The work was supported by grants from the NIH (grant GM127742) to X.Z. The protocol presented here was developed from several previous publications (Yang et al., 2004; Zhang et al., 2006; Book et al., 2010; Hsieh et al., 2015; Han et al., 2019) and optimized in our recent work (Li et al., 2020).

Competing interests

The authors declare no competing interests.

References

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