1Running title:

2A simple in situ cell-based SUMOylation assay

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A simple in situ cell-based SUMOylation assay with potential application to6drug-screening.

78Miyuki Muramatsu1 Junsuke Uwada1, 2, Naoyuki Matsumoto3 and Hisato Saitoh1, 2, 3, 4.

9101112

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

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Global COE (Centers of Excellence) Program, Global Initiative Center for Pulsed Power Engineering,
Faculty of Science, Kumamoto University, Kumamoto, 2-39-1 Kurokami, Kumamoto 860-8555, Japan151617To whom correspondence should be addressed:

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

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Full Postal Address: Department of Biological Sciences, Graduate School of Science and Technology,

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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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Abbreviations: SUMO, small ubiquitin-related modifier; GFP, green fluorescent protein

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Abstract

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Here, we show that the SUMO conjugation reaction can be visualized by simply incubating

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GFP-SUMO-1 with permeabilized cells in the presence of ATP for 15 min. Neither special

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equipment for protein purification nor trained skills for recombinant technologies are required,30

making the assay potentially applicable to large-scale drug-screening strategies for the identification

31of drug(s) that can inhibit or enhance SUMOylation.

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Keyword: post-translational modification, SUMO, cell-based assay, drug-screening

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Text
SUMO-1 is a small ubiquitin-related protein modifier that is conserved from yeast to human.

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The attachment of SUMO-1 to a target protein, referred to as SUMOylation, is mediated by the40

enzymatic cascade reaction1). SUMOylation begins with a SUMO-activating enzyme (also called an41E1), which consists of an Aos1 and Uba2 heterodimer that carries out an ATP-dependent activation

42of the SUMO-1 C terminus. The activated SUMO-1 is then transferred to a SUMO-conjugating

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enzyme Ubc9 (E2). Finally SUMO-1 is transferred from Ubc9 to the substrate with or without the44assistance of one of several SUMO-1 ligases (E3s).

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Pichler et al.2) and Saitoh et al.3) demonstrated that when a yellow fluorescent (YFP) or green

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fluorescent (GFP) protein fused to SUMO-1 was incubated with permeabilized cells in the presence

47of ATP and recombinant SUMO-E1 and -E2 enzymes, YFP/GFP-SUMO-1 was efficiently

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conjugated to nuclear pore associated factors, including RanBP2/Nup358 and RanGAP1, thereby

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enabling the visualization of SUMOylation inside permeabilized cells2, 3). This assay can detect the50

SUMOylation reaction in situ, and is, therefore, referred to as the in situ SUMOylation assay [3]. As

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shown in Fig. 1, this assay appears relatively simple. However, the preparation of the recombinant

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proteins required, including a heterodimer of Uba2 and Aos1 for SUMO-E1, Ubc9 for SUMO-E2

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and YFP/GFP-SUMO-1, is time-consuming and the purification of these recombinant proteins
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requires specialized equipment and trained skills.
To provide a SUMOylation assay that can be performed by researchers withoutFig. 156

experience of recombinant technologies and protein purification, we simplified and improved the in57

situ SUMOylation assay. Our new procedure is schematically represented in Fig. 1. This method

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requires only cultured cells and crude bacterial lysate containing GFP-SUMO-1. We briefly

59summarize the process as follows. The expression in E. coli of (His)6-GFP-SUMO-1gg and

60(His)6-GFP-SUMO-1g, a SUMOylation deficient mutant, were described previously3, 4). The

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bacterial pellet was harvested by centrifugation and dissolved in 5 ml of TRB [20 mM Hepes (pH627.3), 110 mM KOAc, 2 mM MgCl2, 1 mM EGTA]. The bacteria were then sonicated twice for 3063

sec on ice. The supernatant was collected and stored at -20˚C until use. Approximately 1.0 g of64(His)6-GFP-SUMO-1 was present in 10 l of bacterial lysate. The procedure used to detect in situ

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cell-based SUMOylation was as follows. HeLa cells were grown on a coverslip in Dulbecco’s

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modified Eagle’s medium containing 10% fetal bovine serum and antibiotics at 37oC in a 5% CO267

incubator. The coverslip was briefly rinsed with cold TRB plus 2 mM dithiothreitol (DTT), and the68cells were then permeabilized for 5 min on ice with TRB plus 2 mM DTT and 500 g/ml digitonin,

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a detergent that preferentially permeabilizes the plasma membrane and leaves the nuclear envelope

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intact (Merck-Calbiochem, Darmstadt, Germany). Cells were then rinsed twice with cold TRB. 1071

l of bacterial lysate containing ATP (final concentration: 1 mM) was added to 1 ml of TRB. This72

mixture was then applied to the digitonin-permeabilized cells followed by incubation for 15 min at

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25˚C. The cells were then washed twice for 5 min with pre-warmed TRB and then fixed with 4%74

paraformaldehyde in PBS for 15 min at room temperature. The cells were then rinsed with PBS for

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5 min three times. A coverslip was then applied using mounting medium (2.5% DABCO in 80%76glycerol). During the secondary wash, DNA was stained with 1 g/ml

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5,6-diamidino-2-phenylindole (DAPI). Samples were analyzed with a BIOREVO BZ-9000

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fluorescence microscope (Keyence, Osaka).

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When approximately 50-80% confluent (exponentially growing) or confluent

80(growth-arrested) Hela cells were permeabilized and incubated with the bacterial lysate

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containing (His)6-GFP-SUMO-1gg, GFP signals at the nuclear rim were detected in cells cultured
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under both growth conditions (Figs 2A and C). However GFP signals in this assay were somehow

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weaker than the signals detected in previous methods (data not shown). In contrast, when the84bacterial lysate containing (His)6-GFP-SUMO-1g was used, no prominent signals were detected

85(Fig. 2B). Since SUMO-1g is a SUMOylation deficient mutant3), these results imply that the GFP86signals detected by (His)6-GFP-SUMO-1gg might represent covalent conjugation, rather than87

non-covalent interaction with components at the edge of the nucleus. Unlike previous methods, we88

could detect the GFP-SUMO conjugation at the nuclear rim in the absence of excessive amounts of89

recombinant E1 and E2, suggesting the existence of endogenous E1 and E2 that might remain after

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digitonin-treatment. It should be noted that, when GFP-fused SUMO-3gg was used in this simple in91

situ SUMOylation assay, we found that the distribution of GFP-SUMO-3gg was similar to that of92

GFP-SUMO-1gg; i.e. the nuclear rim was clearly labeled with GFP-SUMO-3gg (data not shown).

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Thus these results indicate that the improved in situ SUMOylation assay is highly applicable for94

studying SUMOylation using cells cultured under the different growth conditions.

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Using a previously developed in situ SUMOylation assay to screen small molecules, Fukuda

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et al. reported the identification of ginkolic acid and kerriamycin B, which inhibit

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SUMOylation4, 5). To demonstrate that our simple cell-based in situ SUMOylation assay

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provides a basis for the development of drugs targeted against diseases involving aberrant

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SUMOylation, we tested whether ginkgolic acid impairs the signals of GFP-SUMO-1 at the nuclear

100rim. As shown Fig. 3A, the GFP signals were significantly reduced in the presence of 100 M of101

ginkgolic acid, supporting the previous observation by Fukuda et al.4) In addition to the effect of102

this drug, we demonstrated that the signals at the nuclear rim were not observed in the absence of103

ATP (Fig. 3B), supporting not only the idea that our assay is useful for investigation of small

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compounds, but also the notion that accumulation of GFP-SUMO-1gg at the nuclear rim represents

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active SUMOylation, rather than non-covalent SUMO-protein interaction.

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Fig. 3In sum, we develop an in situ cell-based SUMOylation method which is simpler, cheaper and107

more rapid than previously described assays. The assay described here will be more easily

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performed by researchers and may be particularly useful in large-scale screening approaches for the109

identification of drug(s) that can inhibit or enhance SUMOylation, thereby contributing to the4110development of therapeutic drugs.

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Acknowledgments

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We appreciate all members of our laboratory for helpful discussions. This work was supported by

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

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References

119[1] Geiss-Friedlander R and Melchior F, Nat. Rev. Mol. Cell Biol., 8, 947-956 (2007).

120[2] Pichler A, Gast A, Seeler JS, Dejean A, and Melchior F, Cell, 108, 109-120 (2002).

121[3] Saitoh N, Uchimura Y, Tachibana T, Sugahara S, Saitoh H, and Nakao M, Exp. Cell Res., 312,122123
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1418-1430 (2006).
[4] Fukuda I, Ito A, Hirai G, Nishimura S, Kawasaki H, Saitoh H, Kimura K, Sodeoka M, andYoshida M, Chem. Biol., 16, 133-140 (2009).
[5] Fukuda I, Ito A, Uramoto M, Saitoh H, Kawasaki H, Osada H, and Yoshida M. J Antibiot(Tokyo), 62, 221-224 (2009).

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Figure Legends.

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Fig. 1

Schematic representation of our improved in situ SUMOylation assay, relative to132

previous assays.

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Typically, cells are grown on coverslips and permeabilized with detergents, such as digitonin. In the134

improved assay (right panel), the permeabilized cells are incubated with GFP-SUMO-1gg in the135

presence of ATP and the reaction is stopped by fixing the cells with paraformaldehyde. Compared136

with previous methods described by Pichler et al. [2] and Saitoh et al. [3] (left panel), the137

requirements of the improved assay are straight forward; i.e. the improved method requires only the5138

bacterial lysate containing GFP-SUMO-1gg, whereas the method of Pichler et al. requires four

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different recombinant proteins, including purified fluorescent-labeled SUMO-1gg, Aos1/Uba2

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heterodimer for SUMO-E1 and Ubc9 for SUMO-E2.

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Fig. 2143

different growth conditions.

144(A) Asynchronously grown HeLa cells (approximately 50% confluent) were permeabilized with

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digitonin and mixed with bacterial lysate containing (His)6-GFP-SUMO-1gg. The GFP and the146

DAPI (inset) signals were observed using fluorescence microscopy.

147(B) Asynchronously grown HeLa cells (approximately 50% confluent) were permeabilized with

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digitonin and mixed with bacterial lysate containing (His)6-GFP-SUMO-1g. The GFP and the DAPI

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(inset) signals were observed using fluorescence microscopy. Identical exposure conditions were150

used to compare SUMO-1g versus SUMO-1gg proteins.

151(C) Hela cells cultured to near confluence (growth arrested state) were tested with the improved

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assay. (His)6-GFP-SUMO-1gg was incubated with the cultured cells as indicated. The GFP and the153

DAPI (inset) signals were observed using fluorescence microscopy. Bars indicate 20 m.

Application of the improved in situ SUMOylation assay to Hela cells cultured under154
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Fig. 3156(A) Asynchronously grown HeLa cells (approximately 50% confluent) were permeabilized with157

digitonin and mixed with bacterial lysate containing (His)6-GFP-SUMO-1gg in the presence (left158panel) or absence (right panel) of 100 M ginkgolic acid. The GFP (larger panel) and the DAPI

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(inset) signals were observed using fluorescence microscopy. Bars indicate 20 m.

160(B) Asynchronously grown HeLa cells (approximately 50% confluent) were permeabilized with161

digitonin and mixed with bacterial lysate containing (His)6-GFP-SUMO-1gg in the presence (left162panel) or absence (right panel) of 1 mM ATP. The GFP (larger panel) and the DAPI (inset) signals

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were observed using fluorescence microscopy.

Application of the improved in situ SUMOylation assay to drug-screening strategies.

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