1Running title:

2A real-time SUMO-binding assay

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A real-time SUMO-binding assay for the analysis of the SUMO-SIM

6protein interaction network.

78Niina Tanaka1 and Hisato Saitoh1, 2, 3

91011121

Department of Biological Sciences, Graduate School of Science and Technology,

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14Engineering, Kumamoto University, Kumamoto, 2-39-1 Kurokami, Kumamoto 860-8555,

15JapanGlobal COE (Centers of Excellence) Program, Global Initiative Center for Pulsed Power16171819

3To whom correspondence should be addressed:

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Email: hisa@kumamoto-u.ac.jp,

21Full Postal Address: Department of Biological Sciences, Graduate School of Science and22Technology,

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Kumamoto University, Kumamoto, 2-39-1 Kurokami, Kumamoto 860-8555, Japan

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Phone: +81-96-342-3450, Fax: +81-96-342-3450

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Abstract27

SUMO-interacting motifs (SIMs) play a central role in the fate of SUMO-modified proteins.

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Here we report a real-time SUMO-binding assay. It can be applied to the identification of29

SIMs and to screening for the identification of novel SUMO-binding proteins. Using this30

assay, we investigate the SIMs in SETDB1 and MCAF1, to gain insight into the assembly of

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SETDB1-MCAF1-mediated gene silencing.

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Keywords: post-translational modification, SUMO, protein interaction, histone

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methyltransferase, chromatin

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Article Outline
SUMO (small ubiquitin-related modifier) is a post-translational protein modifier. It is38

similar to ubiquitin in terms of its structure and its mechanism of conjugation to target

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proteins. SUMO is involved in many diverse cellular functions1, 2). Recent studies document

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diverse SUMO-interacting proteins that recognize conjugated SUMO moieties via41

SUMO-interacting motifs (SIMs), also known as SUMO-binding domains1-5). SIMs usually42

consist of a short stretch of hydrophobic amino acid residues followed by multiple acidic

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amino acid residues. By analogy to the relationship between ubiquitin and44

ubiquitin-interacting proteins, it has been speculated that SUMO-SIM protein interactions

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determine the fate of SUMOylated species.

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Biochemical interactions between SUMO and SIM-containing proteins have, to date,47

been analyzed by the yeast two-hybrid assay and the glutathione S-transferase

48(GST)-pulldown/immunoblot assay combined with NMR spectrometry3-5). Neither

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procedure is complicated, but they are reasonably time consuming. For example, the yeast

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two-hybrid assay requires several days to grow yeast colonies and the51

GST-pull-down/immunoblot assay takes a minimum of 8 hours. In addition, such methods

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are not sufficiently sensitive to detect low-affinity interactions. For example, in the253

two-hybrid assay, SIMs can easily dissociate from SUMO during yeast cell growth and in the54

GST-pulldown assay, dissociation can occur during the washing steps, potentially yielding

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false negative results.

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We describe here, a fast, economical, simple and small-scale method to detect57

direct binding between SUMO and SIM-containing proteins. This method is based on the58

modification of a ‘low-affinity protein interaction procedure’ or the ‘bead halo assay’,59

described by Rexach and colleagues6, 7). It consists of only three steps prior to the assessment

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of binding (Fig. 1A): (i) preparation of beads attached with the protein of interest (~3061min), (ii) incubation with green fluorescent protein (GFP)-SUMO (~5 min), and (iii)62

observation of green fluorescence under the microscope (~10 min). It is simple, rapid, and63

detects low-affinity interactions in real-time under equilibrium binding conditions. Therefore,64

we refer to the assay as ‘the real-time SUMO-binding assay’. This method is also cheap, as it

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requires only small quantities of glutathione-Sepharose beads, in addition to standard

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chemicals and buffers. Typically, only 30 particles of glutathione-Sepharose beads are used in67

a 4 µl reaction.

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We validated this method by examining GFP-SUMO-3 and GFP for their ability to69

bind immobilized GST-MBD1-containing chromatin-associated factor 1 (MCAF1)-SIMWt

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and GST-MCAF1-SIMMut 3-5). Previous data have indicated that MCAF1 is a SIM-containing

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SUMO-3-interactive protein (Kd = ~1.3 M)5). It was, therefore, expected that72

GST-MCAF1-SIMWt would give a positive result and that GST-MCAF1-SIMMut would give73

a negative result in the real-time SUMO-binding assay. A 50 ml culture of Escherichia coli74(E. coli) carrying pET28-GFP-SUMO-3 was used to express recombinant SUMO-3 fused to75

GFP. The bacterial pellet was dissolved in 5 ml of phosphate-buffered saline (PBS, pH 7.0),

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followed by sonication at maximum output for 30 seconds on ice (TOMY UD201, Tokyo).

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The sonicated bacterial lysate was centrifuged at 9,100  g for 5 minutes and the supernatant78

was stored at –20oC until use. It should be noted that purification of expressed GFP-SUMO-3

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is not necessary for this assay, as the supernatant contains a large amount of full-length3Fig. 1

8081

recombinant GFP-SUMO-3 (approximately 0.01 mg GFP-SUMO-3/ml of bacterial lysate).
The SIM-containing protein of interest was expressed in E. coli as a recombinant82

GST-fusion protein and was then incubated with glutathione-Sepharose 4B beads (GE83

Healthcare, Buchinghamshire, UK). We used 10 µl of packed beads for bacterial lysate

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prepared from 10 ml of bacterial culture. This results in GST-fusion proteins immobilized at85

high concentrations on the surface of the Sepharose beads. (Approximately 80–90 µg of86

GST-fusion proteins were bound to 10 µl of packed beads). The beads were resuspended as a87

50% slurry in PBS and a 2 µl portion of the slurry, in which approximately 30 particles of88

beads were included, was mixed, on a 76  26 mm Micro Slide Glass, (thickness 1.2 mm,

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Matsunami, Osaka), with 2.0 µl of bacterial lysate containing recombinant GFP-SUMO-3.

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As soon as the incubation started, the beads were viewed in real-time under equilibrium

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conditions using an Eclips E66 fluorescence microscope (Nicon, Tokyo). When92

GFP-SUMO-3 binds to an immobilized GST-fusion protein of interest, the interaction is93

visible through GFP filters as a halo of fluorescence around the beads. As shown in Fig. 1B

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and C, GFP-SUMO-3 showed strong green fluorescence around the Sepharose beads when95

incubated with GST-MCAF1-SIMWt beads. By contrast, incubation of GST-MCAF1-SIMMut

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beads with GFP-SUMO-3 did not show any green fluorescence localized to beads. As a97

further control, we demonstrated that GFP protein alone, with no SUMO-moiety, showed no98

specific binding signals (data not shown). These results indicate that this method is applicable99

to detect the interaction of SUMO with the SIM in MCAF1. It should be noted that we also

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demonstrated that this method can determine protein binding to SUMO-1 as well. For

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instance, GFP-SUMO-1 localized efficiently to the beads containing Ran binding protein 2

102(RanBP2)/Nucleoporin 358 (Nup358), a well-characterized SIM-containing polypeptide that103

binds to SUMO-1 (data not shown).

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We next applied this method to characterize the interaction between SUMO-3 and a105

histone methyltransferase, SET domain bifurcated 1 (SETDB1). We chose this protein,106

because we had previously isolated partial cDNA fragments of SETDB1 in a yeast

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two-hybrid screen using a GAL4-SUMO3-SUMO3 fusion protein as bait (our unpublished108data). Furthermore, another group has recently reported the interaction between SUMO, and109

SETDB1, suggesting that SETDB1 is a SUMO-binding protein in chromatin-based

110gene expression8). In addition, SETDB1 forms a heterodimer with MCAF1 and a111

complex of MCAF1 with SETDB1 modulates the histone methylase activity of SETDB1,112

converting it from an H3-K9 dimethylase to a trimethylase9). Therefore, characterization of113

the binding between SUMO and SETDB1 is likely to contribute to the understanding of the114

regulation of methyltransferase activity by MCAF1 and to the function of the115

methyltransferase complex in the context of gene silencing and formation of heterochromatin.

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To determine the region responsible for binding to SUMO2/3, we generated a series of117

deletion mutants of recombinant SETDB1 fused to GST and performed the real-time

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SUMO-binding assay (Fig. 2A and B). To this end, twelve amino acid residues

119(SETDB1120-131), which show similarity to the canonical SIM, a short stretch of hydrophobic

120amino acid residues followed by multiple acidic amino acid residues1-5), were found to121

directly interact with GST-SUMO-3 (Fig. 2A). Moreover, we found that the point-mutants in122

which isoleucine (I) at 122, 123, or 125 was substituted for alanine (A) had remarkably

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reduced affinity to SUMO-3, suggesting that these amino acid residues were important for

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SUMO-binding (Fig. 2B). It should be noted that GST-d1-1-5-1, but not a SIM-deleted

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mutant (GST-d1-1-5-delta SIM), bound to SUMO-3 in the in vitro pulldown assay (Fig. 2D),

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confirming the in vitro interaction observed in the real-time SUMO binding assay described127

above. Although the structural basis for the interaction between SUMO-3 and the SIM in

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SETDB1 requires further elucidation, our results indicate a direct molecular linkage of the129

SUMO pathway with, not only MCAF1, but also SETDB1.

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In conclusion, we describe a method which improves upon current in vitro SUMO

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binding assays and we show application of this method, referred to as the real-time SUMO

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binding assay, for the characterization of MCAF1 and SETDB1, thereby giving an insight133

into SUMOylation-induced gene silencing. Since this method is simple, rapid and cheap, it

5Fig. 2

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may be particularly applicable for screening novel SUMO-binding proteins, as well as for

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biochemical analysis of the SUMO-SIM protein interaction network. It is also highly

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applicable to large-scale screening approaches for the identification of drug(s) that can inhibit

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or enhance SUMO-SIM interaction.

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Acknowledgments
We thank Ms. Yuka Fujimitsu for helping the in vitro binding experiments. This work141

was supported by grants to H.S from the Naito Foundation and from The Japanese142

Government Ministry of Education, Culture, Sport, Science and Technology.

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Figure Legends.

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164Figure 1. Detection of SUMO-SIM interaction by the real-time SUMO binding assay.

165(A) Schematic representation of the procedure for the real-time SUMO binding assay.

166(B) The entire amino acid sequences of MCAF1-SIMWt and MCAF1-SIMMut fused to GST

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are shown. The position of the mutation (I to A) is underlined. Indicated on the right are

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aliquots of immobilized GST-MCAF1-SIMWt and GST-MCAF1-SIMMut, as analyzed by

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SDS-PAGE and stained with Coomassie blue. Note that equal amounts of170

GST-MCAF1-SIMWt and GST-MCAF1-SIMMut, to be used for the assay in B, were171

immobilized on the beads.

172(C) Bacterial lysate containing 0.01 mg/ml of either GFP-SUMO-3 (1st and 2nd columns) or

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GFP (3rd and 4th columns) was mixed with the bead-immobilized GST-MCAF1-SIMWt (1st174and 3rd columns) or the bead-immobilized GST-MCAF1-SIMMut (2ndand 4th columns). An

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aliquot of the recombinant GFP-SUMO-3 or GFP resolved by SDS-PAGE and stained with

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Coomassie blue is shown. GFP signal localized to the beads indicates a positive SUMO-SIM

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interaction (upper panel). Lower-panels show the phase-contrast images of the Sepharose

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beads.

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Figure 2. Detection of a SIM in SETDB1 by the real-time SUMO binding assay.

181(A) Schematic representation of human SETDB1 and a summary of the real-time SUMO

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binding assay. SETDB1 deletion constructs are represented as thin black lines. The Table on

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the right summarizes the interactions of each deletion mutant with GFP-SUMO-3 in the184

real-time SUMO binding assay. + indicates significant binding, - indicates negligible binding.

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Numbers correspond to amino acid residues in SETDB1. The amino acid sequence of186

SETDB1120-131 (d1-1-5-1) is represented as SIM. The amino acid residues which are

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important for SUMO-3 binding are indicated by dots.

188(B) The real-time SUMO binding assay. Bacterial lysate containing 0.01 mg/ml of7

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GFP-SUMO-3 was mixed with bead-immobilized GST-SETDB1 deletion mutant proteins.

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Aliquots of the recombinant GST fusion proteins resolved by SDS-PAGE and stained with

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Coomassie blue are shown. The mixtures were imaged under a fluorescent microscope.

192(C) GST-d1-1-5-1-I122A, -I123A and -I125A mutants abolish SUMO-3-binding in the193

real-time SUMO binding assay. Bacterial lysate containing 0.01 mg/ml of GFP-SUMO-3

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was mixed with bead-immobilized GST- d1-1-5-1 mutant proteins as indicated. An aliquot of195

each recombinant GST fusion protein, resolved by SDS-PAGE and stained with Coomassie

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blue, is shown.

197(D) GST-d1-1-5-1, but not GST--d1-1-5-deltaSIM, binds His6-SUMO-3 in the in vitro

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pulldown assay. GST-d1-1-5-1 or GST--d1-1-5-deltaSIM was incubated with beads

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containing 5 g of His6-SUMO-3. Following incubation, pull-down assays were carried out4),

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and proteins associated with the beads were analyzed by immunoblot analysis using

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anti-GST antibody. Coomassie Brilliant Blue (CBB) stained gel is shown at the bottom to202

show amount of the input proteins.

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