A Method for SUMO Modification of Proteins in vitro

The Small Ubiquitin-related Modifier (SUMO) is a protein that is post-translationally added to and reversibly removed from other proteins in eukaryotic cells. SUMO and enzymes of the SUMO pathway are well conserved from yeast to humans and SUMO modification regulates a variety of essential cellular processes including transcription, chromatin remodeling, DNA damage repair, and cell cycle progression. One of the challenges in studying SUMO modification in vivo is the relatively low steady-state level of a SUMO-modified protein due in part to the activity of SUMO deconjugating enzymes known as SUMO Isopeptidases or SENPs. Fortunately, the use of recombinant SUMO enzymes makes it possible to study SUMO modification in vitro. Here, we describe a sensitive method for detecting SUMO modification of target human proteins using an in vitro transcription and translation system derived from rabbit reticulocyte and radiolabeled amino acids.

isopeptidases, or SENPs (Mukhopadhyay and Dasso, 2007;Hickey et al., 2012). Although the dynamic cycling between conjugation and deconjugation can result in a relatively low steady-state level of a modified protein, SUMO modification nonetheless produces profound effects on substrate function in a variety of cellular pathways. SUMO modification is most commonly detected by immunoblotting. Like other posttranslational modifications such as ubiquitylation and PARylation, whole cell lysates immunoblotted for SUMO1 or SUMO2/3 reveal high molecular-weight smears due to the large number of cellular conjugates. Notably, SUMO modification of RanGAP1 can be identified as a band appearing on an immunoblot at ~90 kDa, an ~12 kDa shift above the unmodified 70 kDa protein (Matunis et al., 1996). To directly observe SUMO modification of specific substrates, immunoblots may also be performed with substrate-specific antibodies if modification levels are sufficiently high. For example, APC4 is a subunit of the Anaphase Promoting Complex/Cyclosome (APC/C) that is SUMO-modified robustly during the mitotic stage of the cell cycle as observed by immunoblot analysis of whole cell lysates from synchronized cells (Lee et al., 2018). Although SUMO modification by a single protein subunit can be detected as a ~12 kDa shift in molecular weight (Figure 1), this shift cannot be distinguished from modification by other ubiquitin-like proteins without further validation. An alternative method for demonstrating SUMO modification utilizes a cell line with His-SUMO, followed by Ni-NTA purification under denaturing conditions and immunoblotting for co-purifying proteins of interest (Lee et al., 2018). Complimentary approaches for detecting and validating SUMO-modified proteins utilize overexpression of SUMO E1 and E2 enzymes, or the depletion of SENPs, followed by immunoblotting for the protein of interest. Furthermore, recent advances in proteomic methods have made the sensitive identification of SUMO-modified proteins possible, leading to hundreds of SUMO substrates that must be further validated and characterized. (Matic et al., 2010;Schimmel et al., 2014;Cubeñas-Potts, et al., 2015).
In addition to these cellular assays, detection and verification of SUMO modification using biochemically purified components in vitro also represents an important approach to validating and characterizing novel SUMO substrates (Park-Sarge and Sarge, 2008;Werner et al., 2009;Yunus and Lima;Yang et al., 2018). With in vitro analysis of a substrate, mutants can also be particularly valuable in verifying specific modification sites. Here we describe a protocol for in vitro SUMO modification routinely used in our laboratory (Zhu et al., 2008;Reiter et al., 2016), as recently illustrated by our analysis of the APC4 subunit of APC/C (Lee et al., 2018).

Procedure
A. Safe handling of radioactive materials-Please note that this procedure utilizes radioactive [35] S-methionine. All users must be appropriately trained to handle and dispose of radioactive materials. A dedicated laboratory space should also be demarcated for containment. Safe handling and disposal of all tips, tubes, and reagents are important for minimizing the risks of exposure contamination.

B. Preparation of SUMO enzymes from E. coli-Prior to performing the in vitro
SUMO modification assay, recombinant SUMO and pathway enzymes must first be expressed in E. coli and purified. Methods for protein expression and purification have been described elsewhere (Yang et al., 2018).

1.
Remove a tube of rabbit reticulocyte from the kit stored at −80 °C and thaw on ice.

2.
For each substrate, add 20 μl of rabbit reticulocyte to an Eppendorf tube on ice.

3.
Return any remaining rabbit reticulocyte back to −80 °C for future use; avoid freeze/thaw cycles.

5.
Add 500 ng of plasmid DNA for substrate protein of interest to each reaction, keep on ice.

1.
Each reaction will have 28 μl of SUMO Master Mix solution; place in a 1.5 ml Eppendorf tube, at room temperature. It is recommended to prepare fresh SUMO Master Mix solution (see Recipe 2).

2.
Remove 2 μ;l of transcription/translation product and add to the 28 μl SUMO Master Mix, for a total volume of 30 μl, pipetting gently several times up and down to mix, at room temperature.

3.
Incubate each reaction in a 30 °C water bath (see Note C).

5.
Place on a 95 °C heat block for 5 min.

2.
Load 10 μl of completed reaction to a well in a 12.5% SDS-PAGE gel, reserving a lane for 2 μl of protein ladder (lane 1).

3.
Run SDS-PAGE at 70 V, room temperature, for approximately 20 min, or until the bottom dye indicator reaches just below the stacking gel and in the running gel. Run at 120 V, room temperature, for an additional 2 h. Stop electrophoresis before the 10 kDa molecular weight marker reaches the bottom of the gel (or until the bottom dye indicator just reaches the bottom of the gel).

4.
Gently separate gel plates using a spatula. Gently slide surgical blade along sides of the glass plate to release gel and remove the stacking gel away from the running gel with blade and discard.

5.
Carefully remove the gel and place in a small dish or plastic container (a pipet tip box lid is suitable) pre-filled with destaining buffer at room temperature.

6.
Wash with gentle shaking for 10 min, at room temperature.

7.
Carefully discard destaining buffer (free [ 35 S]methionine may be in the solution, so pour in a radiation waste container).

8.
Add MilliQ water and wash with gently shaking for 10 min, at room temperature. Repeat at least 3 times (see Notes).

1.
Lay a piece of Whatman™ paper on a clean benchtop space demarked as a radiation exposure area (room temperature).

2.
Carefully place gel onto the Whatman™ paper so that lane 1 is the protein ladder.

3.
Overlay the gel with a piece of plastic wrap.

4.
Lay the gel and Whatman™ paper onto the gel dryer (the Whatman™ paper is placed directly onto the foam of the gel dryer and the plastic wrap is on top).

5.
Gently cover the gel and dry for approximately 30 min (see Notes).

6.
Carefully remove the plastic wrap from the gel and place on benchtop demarked as a radiation exposure area for 5 min.

7.
In a dark room protected from light, place the dried gel affixed to the Whatman™ paper inside the cassette.

8.
Carefully place a sheet of X-ray film on top of the gel.

9.
Carefully close the cassette. Do not bring into contact with light.

10.
Keep the cassette in a dark area overnight at room temperature.

11.
Develop the exposed film according to manufacturer's instructions.
Note: For a video and detailed protocol for how to perform autoradiography, please refer to Karra et al. (2017).

1.
Overlay developed film over dried gel and use molecular weight markers to demarcate molecular weights using a permanent pen.

2.
Locate the unmodified protein of interest and any shifts in molecular weight (~12 kDa shift represents modification by a single SUMO, 24 kDa shift represents modification by two SUMOs, etc.) (Figure 2). It is possible for several SUMO consensus sites to be present in the protein of interest, and several SUMOmodified bands may be represented.

3.
If amino acid substitutions have been made to SUMO consensus site lysines, high molecular weight band shifts should decrease in number until a complete SUMO mutant is generated (Figure 2). (Zhao et al, 2014) that identifies potential SUMO conjugation sites or SIM Motifs (Yunus and Lima, 2009). Input of the amino acid sequence in FASTA format is used.

B. Preparation of SUMO enzymes from E. coli-Depending
on whether the protein of interest is modified by SUMO1 or SUMO2/3, the appropriate recombinant protein should be used in the assay. If unknown, it is recommended that modification by both SUMO1 and SUMO2/3 be tested. Of note, SUMO2/3 can generate polymeric chains due to a SUMO consensus site at K11 in both SUMO2 and SUMO3.
In human cells, the SUMO E1 enzyme is expressed as a heterodimer Aos1/Uba2. Coexpression and purification of these two proteins can be prepared in batch. The conjugating enzyme, Ubc9, is used in relatively high concentrations and may circumvent the requirement of an E3 ligating enzyme. Caution should be used, however, in possible misinterpretation of results and the possibility that E3's may be critical for site-selective modification. It is also possible for some substrates to require an E3 ligase (such as the PIAS family of proteins [Zhu et al., 2008]) for efficient SUMO modification in vitro.

C.
In vitro SUMO Modification-time-course analysis-In general, the time required for SUMO modification at 30 °C must be determined empirically, as the concentrations of SUMO enzymes in addition to increases in temperature (up to 37 °C) can change the efficiency of conjugation. Generally, with this protocol, SUMO substrates are modified within one hour. However, a time-course is recommended for each protein of interest, as some proteins are more readily SUMO-modified than others. Substrate recognition by SUMO conjugating enzymes or properties inherent in the SUMO protein itself can affect the kinetics of modification. For example, the presence of SUMO Interacting Motifs (SIMs) in the substrate protein can enhance SUMO modification. SIMs are composed of hydrophobic amino acids (V/1)-x-(V/1)-(V/1) flanked by an acidic residue. The hydrophobic amino acids in SIMs interact non-covalently with a hydrophobic pocket situated in the β 2 strand on the surface of SUMO, creating a parallel or antiparallel β-strand conformation. SIM Motifs can also be identified through GPS SUMO (Zhao et al., 2014).

D. In vitro SUMO Modification-enzyme concentrations-
The concentrations of enzymes used can also be empirically adjusted. For example, BLM and APC4 are readily SUMO-modified with the concentrations of SUMO enzymes described above, but RanGAP1 is more efficiently SUMO-modified and therefore requires a lower concentration of conjugation enzymes and shorter incubation periods to achieve complete modification. For example, 15 mM E1, 45 nM E2, and 0.5 μM SUMO proteins are required for complete SUMO modification of RanGAP1 within 5-10 min of incubation at 30 °C.
E. SDS polyacrylamide gel electrophoresis-It is critical to sufficiently wash the gel following destaining. The presence of destaining solution can cause the gel to crack during drying.
A 12.5% gel is recommended because free SUMO is a relatively small protein and this composition has proven to resolve proteins well in our experiments. A 4-15% gradient gel can also be used.
F. Autoradiography-Time for gel drying can vary. Monitor carefully to sufficiently dry the gel but taking care not to over-dry and generate cracks.  Immunoblot to APC4 shows unmodified APC4 at ~100 kDa and SUMO-modified forms indicated by asterisks (*). Glyceraldehyde 3-phosphate dehydrogenase (GAPD) immunoblot is used as a loading control.  APC4 is a 808 amino acid protein with two consensus SUMO sites at K772 and K798 (A). Full-length wild-type APC4 (B) or the indicated lysine to alanine substitution mutants (C, D, E) were expressed in rabbit reticulocyte lysate in the presence of [ 35 S]-methionine and incubated for the indicated times in modification reactions containing SUMO E1 and E2 enzymes and SUMO2. Proteins were detected by SDS-PAGE and autoradiography. Asterisks indicate sumoylated forms of APC4. Please note that we have rearranged the panels of Figure 2. Lee et al. Page 13 Bio Protoc. Author manuscript; available in PMC 2018 November 28.