Methods to study SUMO dynamics in yeast

CHAPTER NINE

Methods to study SUMO dynamics in yeast € ring, Natasha Petreska, Stefan Pabst, Lennard-Maximilian Do € rgen Dohmen* R. Ju Institute for Genetics, Center of Molecular Biosciences, University of Cologne, Cologne, Germany *Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. General equipment, yeast strains, and plasmids 2.1 General equipment 2.2 Yeast strains 2.3 Plasmids 3. Western blot analysis of cellular SUMO conjugates to study effects of stress conditions and mutations affecting enzymatic functions in the SUMO system 3.1 Theory 3.2 Buffers and reagents 3.3 Procedure 4. Probing the role of desumoylation, SUMO chain formation, and proteolysis in SUMO dynamics using HA-tagged SUMO variants 4.1 Theory 4.2 Reagents 4.3 Procedure 5. Purification of 8xHis-SUMO conjugates using cobalt beads 5.1 Theory 5.2 Buffers and reagents 5.3 Procedure Acknowledgments References

188 190 190 191 191 192 192 192 193 196 196 197 198 203 203 203 204 208 208

Abstract Covalent modification of proteins with the small ubiquitin-related modifier (SUMO) is found in all eukaryotes and is involved in many important processes. SUMO attachment may change interaction properties, subcellular localization, or stability of a modified protein. Usually, only a small fraction of a protein is modified at a given time because sumoylation is a highly dynamic process. The sumoylated state of a protein is controlled by the activity of the sumoylation enzymes that promote either their mono- or poly-sumoylation (SUMO chain formation), by SUMO proteases that reverse these modifications, and by

Methods in Enzymology, Volume 618 ISSN 0076-6879 https://doi.org/10.1016/bs.mie.2018.12.026

#

2019 Elsevier Inc. All rights reserved.

187

188

Stefan Pabst et al.

SUMO-targeted ubiquitin ligases (STUbL, ULS) that mediate their degradation by the proteasome. While some organisms, such as humans, express multiple isoforms, budding yeast SUMO is encoded by a single and essential gene termed SMT3. The analysis of the simpler SUMO system in budding yeast has been instrumental in the identification of enzymes acting on this modification and controlling its dynamics. Sumoylation of proteins changes dramatically during the cell division cycle and under various stress conditions. Here we summarize various approaches that employ Saccharomyces cerevisiae as a model system to study the dynamics of sumoylation and how it is controlled.

1. Introduction SUMO modification is considered to be a molecular switch that regulates the function of proteins mainly by altering their interaction properties, which may affect their subcellular localization, their enzymatic function, or their stability (Flotho & Melchior, 2013; Heun, 2007; Praefcke, Hofmann, & Dohmen, 2012). Sumoylation has been implicated in a variety of cellular processes including the cell division cycle, meiosis, protein targeting and organization of subnuclear structures, transcription, DNA damage repair, and protein quality control (Flotho & Melchior, 2013; Galanty et al., 2009; Heun, 2007; Johnson, 2004; Morris et al., 2009; Psakhye & Jentsch, 2012; Tatham, Matic, Mann, & Hay, 2011; Wilson & Rangasamy, 2001). SUMO modification of proteins is a dynamically regulated and reversible process. For most SUMO target proteins, only a small fraction is modified at any given time, which often makes the functional characterization of these modifications difficult ( Johnson, 2004). Conditions such as heat, oxidative stress, or DNA damage can induce dramatic changes in the sumoylated proteome (Golebiowski et al., 2009; Miller & Vierstra, 2011; Seifert, Schofield, Barton, & Hay, 2015; Tempe, Piechaczyk, & Bossis, 2008; Zhou, Ryan, & Zhou, 2004). The sumoylation state of a protein is controlled by the action of opposing activities in the SUMO system (Schwienhorst, Johnson, & Dohmen, 2000; Sriramachandran & Dohmen, 2014). Similar to other proteins of the ubiquitin family, such as ubiquitin itself, SUMO is posttranslationally conjugated to its substrates by specific enzymes. This process starts with the maturation of the carboxyl terminus of SUMO by the action of a SUMO protease, revealing the C-terminal diglycine motif. The responsible enzyme in Saccharomyces cerevisiae is the Ulp1 protein (Li & Hochstrasser, 1999). Formation of an isopeptide bond between the carboxyl group of SUMO’s

SUMO dynamics

189

C-terminal glycine residue and an ε-amino group of a substrate lysine residue is then mediated by an enzymatic (E1–E2–E3) cascade (Flotho & Melchior, 2013; Johnson, 2004; Ulrich, 2009). The enzymes that mediate protein sumoylation are related to those that catalyze ubiquitylation or conjugation of other ubiquitin family modifiers; they were first discovered in yeast and are conserved among eukaryotes ( Johnson, 2004). The enzymatic cascade leading to protein sumoylation begins with the activation of the modifier by the SUMO-activating enzyme (SUMO E1), a heterodimeric protein (Aos1–Uba2) with a domain organization similar to that of the monomeric ubiquitin-activating enzyme (Uba1) ( Johnson, Schwienhorst, Dohmen, & Blobel, 1997). In this ATP-requiring process, the C-terminal glycine residue of SUMO, after initial formation of an adenylate, is ultimately linked to a cysteine residue of Uba2 via an energy-rich thioester bond. The second step is a transesterification reaction, in which activated SUMO is linked to the active-site cysteine residue in SUMOconjugating enzyme (Ubc9; E2) ( Johnson & Blobel, 1997). Transfer of SUMO from Ubc9 to substrates is promoted by SUMO ligases (E3s), of which there are three known to be expressed in vegetatively growing S. cerevisiae (Siz1, Siz2, and Mms21) with overlapping and apparently partly redundant functions ( Johnson & Gupta, 2001; Pichler, Fatouros, Lee, & Eisenhardt, 2017; Reindle et al., 2006; Zhao & Blobel, 2005). Mms21 is part of the oligomeric “Smc5–Smc6 complex” with roles in DNA repair, chromosome organization and the nucleolus (Kim et al., 2016; Zhao & Blobel, 2005). A substrate can either be modified on a single lysine residue (mono-sumoylation), on multiple lysine residues (multisumoylation), or by formation of lysine-linked SUMO chains (poly-sumoylation) (Bylebyl, Belichenko, & Johnson, 2003; Flotho & Melchior, 2013; Pichler et al., 2017). The sumoylated form of a protein can be dynamically regulated by two principle mechanisms. The first one is desumoylation by SUMO proteases, of which there are two in S. cerevisiae, Ulp1 and Ulp2 (Hickey, Wilson, & Hochstrasser, 2012; Li & Hochstrasser, 1999, 2000). Aside from processing the SUMO precursor, Ulp1 can release single SUMO moieties from substrates, and at least in vitro also cleave SUMO chains at any of its linkages (Eckhoff & Dohmen, 2015; Li & Hochstrasser, 1999). By contrast, Ulp2 acts only on SUMO chains, which it depolymerizes from their distal ends leaving di-SUMO on the substrate as the terminal product (Bylebyl et al., 2003; Eckhoff & Dohmen, 2015). The second mechanism is proteolytic targeting of sumoylated proteins mediated by SUMO-targeted ubiquitin ligases

190

Stefan Pabst et al.

(STUbLs, ULS, or E3-S), which recognize their substrates via SUMO interaction motifs (SIMs) and promote their ubiquitin-dependent degradation by the proteasome (Perry, Tainer, & Boddy, 2008; Sriramachandran & Dohmen, 2014). In S. cerevisiae, two such STUbLs with multiple SIMs have been identified, Uls1 and the heterodimeric Slx5–Slx8 (Uls2) (Uzunova et al., 2007; Xie et al., 2007). Polysumoylation appears to have a critical function in this pathway of SUMO target control, because SUMO chains promote recognition by STUbLs (Tatham et al., 2008; Uzunova et al., 2007). While the enzymatic functions of many of the aforementioned proteins or their relatives in other species have been intensively studied in recent years, many open questions remain regarding their dynamic interplay and how this controls the SUMO proteome as well as individual SUMO targets. Learning more about the complex mechanisms that control SUMO dynamics and their regulation is therefore an important challenge for future studies. In this chapter, we have compiled several approaches and methods that can be used to investigate SUMO dynamics in S. cerevisiae. Similar approaches can be applied to other species. An important aspect in the analysis of cellular SUMO conjugates is that they are rapidly lost upon cell lysis. We therefore describe methods that employ denaturing cell lysis protocols that preclude unwanted enzymatic desumoylation. Using various mutant yeast strains with defects in SUMO system enzymatic functions, one can then investigate how they contribute to the control of SUMO dynamics under various conditions. We also use this protocol to probe the effects of various stress conditions on cellular SUMO conjugates. We employ tagged SUMO that enables purification of its targets under denaturing conditions (Wohlschlegel, Johnson, Reed, & Yates, 2004) and a protease-resistant SUMO variant for enrichment of cellular SUMO conjugates (Bekes et al., 2011) to demonstrate the contribution of STUbLs in their dynamic control.

2. General equipment, yeast strains, and plasmids 2.1 General equipment • • • • • •

Incubator shaker Standard spectrophotometer Standard (micro)centrifuge tubes Protein LoBind Tubes, Eppendorf Vacuum aspirator Glass beads (425–600 μm)

191

SUMO dynamics

• • • • • • • •

Spinning wheel for microcentrifuge tubes Odyssey infrared imaging system, LI-COR SDS-PAGE system for large gels, Hoefer SE 600 series SDS-PAGE system for mini gels, Bio-Rad Mini-Protean 3 Cell Electrophoresis Power Supply EPS 301, Amersham Pharmacia Biotech Trans-Blot SD Semi-Dry Transfer Cell, Bio-Rad Nitrocellulose membrane 0.2 μm Whatman paper

2.2 Yeast strains Yeast strains

Parent strain

Genotype

JD47-13C (wild type)

MATa leu2-3,112 lys2-801 his3-Δ200 trp1-Δ63 ura3-52

JD53

MATα leu2-3,112 lys2-801 his3-Δ200 trp1-Δ63 ura3-52

JD90-1A-1ts9

JD53

MATα uba2-Δ::HIS3, uba2-ts9 on CEN/LEU2 plasmid

YKU1

JD47-13C

MATa ulp2-Δ::HIS3

YKU121

JD53

MATα slx5-Δ::HIS3 uls1 Δ::KanMX4

YKU149

JD47-13C

MATa uls1-Δ::KanMX4

YKU87

JD47-13C

MATa slx5-Δ::TRP1

EJ337

JD53

MATα 8His-SMT3::TRP1

2.3 Plasmids Plasmid

Details

Source

YCplac111

Shuttle vector for cloning and yeast transformation (CEN/LEU2/Amp)

Gietz and Sugino (1988)

pMM43

PGAL1-2xHA-SMT3(CEN/LEU2/Amp)

Dohmen laboratory collection

pNP3

PGAL1-2xHA-smt3-Q95P (CEN/LEU2/Amp)

Dohmen laboratory collection

pNP4

PGAL1-2xHA-smt3-Q95P, K11,15,19R (CEN/LEU2/Amp)

Dohmen laboratory collection

192

Stefan Pabst et al.

3. Western blot analysis of cellular SUMO conjugates to study effects of stress conditions and mutations affecting enzymatic functions in the SUMO system 3.1 Theory Conjugation and deconjugation of SUMO to its target proteins are highly dynamic processes that are adapted to physiological conditions. Impairing components of the SUMO machinery as well as the exposure to various stress conditions can rapidly influence the amount of SUMO conjugates in the cell. Several S. cerevisiae mutants involved in the regulation of sumoylation have been generated and shown to affect the formation or clearance of SUMO conjugates in vivo. These mutants therefore offer the possibility to better understand the interplay of conjugation and deconjugation in a cellular context. Activation of SUMO is impaired in the uba2-ts mutant, which destabilizes Uba2, the catalytic subunit of the SUMO E1 enzyme; this decreases cellular levels of SUMO-protein conjugates. By contrast, interfering with the removal of SUMO conjugates via mutations in SUMO-dependent ubiquitylation (slx5Δ uls1Δ) or SUMO-protein cleavage (ulp2Δ) increases the levels of SUMO conjugates ( Johnson et al., 1997; Schwienhorst et al., 2000; Uzunova et al., 2007). Exposure of cells to stress conditions such as osmotic stress (e.g., high salt concentrations), oxidative stress (e.g., induced by hydrogen peroxide), or thermal stress (heat) increases SUMO conjugate levels in S. cerevisiae (see Section 1) (Zhou et al., 2004).

3.2 Buffers and reagents •

•

•

Growth medium for yeast cells: Yeast extract–peptone–dextrose medium (YPD): 1% yeast extract, 2% bacto peptone, 2% glucose. For solid growth media, 2% agar is added Resolving gel for SDS-polyacrylamide gel electrophoresis (SDSPAGE): 26.6% of a 30% acrylamide/bisacrylamide solution (29:1), 0.375 M tris(hydroxymethyl)-aminomethane-hydrochloric acid buffer (Tris–HCl) pH 8.8, 0.1% sodium dodecyl sulfate (SDS), 0.1% ammonium persulfate (APS), 0.1% tetramethylethylenediamine (TEMED) Stacking gel for SDS-PAGE: 20% of a 30% acrylamide/bisacrylamide solution (29:1), 0.125 M Tris–HCl pH 6.8, 0.1% SDS, 0.1% APS, 0.1% TEMED

SUMO dynamics

•

• • • • •

193

Phosphate-buffered saline (PBS) for washing nitrocellulose membranes: 137 mM sodium chloride (NaCl), 2.7 mM potassium chloride (KCl), 8.1 mM disodium hydrogen phosphate (Na2HPO4), 1.5 mM potassium dihydrogen phosphate (KH2PO4) 1
Laemmli loading buffer: 0.0625 M Tris–HCl pH 6.8, 2% SDS, 10% glycerol, 2% β-mercaptoethanol, 0.005% bromophenol blue SDS-PAGE running buffer: 25 mM Tris–HCl, 192 mM glycine, 0.1% SDS, pH 8.3 Blotting buffer: 25 mM Tris–HCl, 192 mM glycine, pH 8.3, 20% methanol Protein size marker (PageRuler™ Plus Prestained Protein Ladder, Thermo Fisher) Antibodies for immunodetection: rabbit anti-Smt3 serum 1:10000, rabbit anti-Pgk1 1:5000, goat anti-rabbit Alexa Fluor plus 800 nm from Thermo Fisher (A32735), goat anti-mouse Alexa Fluor 680 nm from Thermo Fisher (A21057). All antibodies are incubated in PBS containing 4% milk

3.3 Procedure 1. Grow yeast cells overnight in 5 mL YPD at 30°C with shaking. The uba2-ts strain is cultured at 25°C to avoid prolonged slow growth. 2. Measure optical density at 600 nm (OD600). 3. Subculture cells with a starting OD600 of 0.1 for the wild type, and 0.2 for the mutants, in 20 mL YPD. 4. Grow cells to an optical density of 0.5 OD600 at 30°C with shaking. 5. Provide stress stimulus. For an effective induction of sumoylation by stress, add H2O2 to 1 mM for 10 min, add NaCl to 1 M for 10 min, or heat cells to 50°C for 30 min. Nonstressed cultures are grown for 1 h at 30°C instead. 6. Make sure the cell density does not exceed an OD600 of 1 to keep cells in exponential growth phase. 7. After the treatment, determine OD600 and harvest cells by centrifugation for 5 min at 3220
g. 8. Lyse cells by adding 50 μL 1
Laemmli loading buffer per OD600 unit of cells (corresponding to 1 mL of cells with an OD600 ¼ 1) and heat for 5 min at 99°C. 9. Load 50 μL of the lysate per lane on an SDS-polyacrylamide (PAA) gel. To visualize high molecular weight (HMW) Smt3-protein conjugates

194

10.

11.

12.

13.

14.

15.

16.

Stefan Pabst et al.

as well as the free form of Smt3, large gels that are 14 cm
14 cm or bigger (including stacking gel) are recommended. For a reasonable separation of HMW-conjugates, 1 mm thick gels with 6% acrylamide stacking gels and 8% acrylamide resolving gels as described above are recommended. To minimize lane-to-lane variability in migration of the samples, add Laemmli loading buffer on top of the loaded protein ladder size marker and to the empty wells so that all wells contain approximately the same volume of Laemmli loading buffer (with or without sample). After loading, fill the outer (lower) part of the electrophoresis chamber with running buffer supplemented with 0.1 M sodium acetate. The inner (upper) part of the chamber is filled with running buffer without sodium acetate. Run SDS-PAGE at 300 V maximum (should not be reached) and 50 mA (constant current conditions) for approximately 3.5 h. Since free Smt3 runs slightly above 15 kDa, make sure that the corresponding band of the protein ladder does not run off of the gel. After SDS-PAGE, gels are washed with distilled water and incubated in blotting buffer for 5 min at the same time as the nitrocellulose membrane and Whatman paper. To transfer and detect HMW-Smt3 conjugates, it is recommended to blot the separation gel along with the stacking gel. Be careful transferring the gel, since the stacking gel is relatively fragile due to the low percentage of the gel. Perform Western blot transfer of proteins to the membrane with a current (in mA) corresponding to 1.2
the area of the membrane (in cm2) for 2 h (e.g., for a membrane of 14 cm
14 cm, the Western blot was run at 235 mA (constant) with a voltage setting of 25 V as a maximum (not reached)). To avoid a temperature increase and drying of the gel, it is recommended to blot at 4°C. If desired, after blotting and before blocking, add REVERT Total Protein Stain (LI-COR) to the membrane and follow the manufacturer’s instructions to evaluate protein loading. The Total Protein Stain can be used as a loading control; however, we do not recommend its use if another protein is to be immunodetected at 700 nm because even after removal of the stain, residual signal might interfere with immunological detection of proteins in the same channel. After blotting (and possibly application of Total Protein Stain), incubate the membrane in PBS containing 4% milk with shaking for 30 min at room temperature.

SUMO dynamics

195

17. After blocking, incubate overnight with desired primary antibodies (usually one for the detection of SUMO conjugates and one for a loading control) while gently shaking at 4°C. 18. On the next day, wash the membrane four times for 5 min with PBS on a shaker, and then incubate with secondary antibodies for 1 h at room temperature. 19. After four additional wash steps with PBS for 5 min each, detect proteins using the appropriate method for the secondary antibody used in step 18. Secondary antibodies carrying fluorophores are detected by the Odyssey Infrared imaging system (LI-COR). 20. Processing of images obtained by the Odyssey Infrared imaging system and quantitative analysis can be performed using Image Studio Lite 5.2 (Li-COR). Fig. 1 shows how impairing the SUMO pathway or applying stress conditions influences the patterns of sumoylated proteins in yeast cells. Inhibition of Uba2 drastically reduces the overall amount of sumoylated proteins (lane 1), while the deletion of SLX5 and ULS1, or ULP2 (lanes 2 and 3) leads

Fig. 1 Detection of SUMO conjugates under various stress conditions. Cells were grown at 30°C and treated with 1 mM H2O2 for 10 min, 1 M NaCl for 10 min, or 50°C for 30 min as indicated. Anti-Smt3 antiserum (1:10000) and anti-Pgk1 (1:5000) antibodies were used for detection. SDS-PAGE and Western blotting were performed as described in the protocol.

196

Stefan Pabst et al.

to increased amounts of SUMO conjugates. (Note that the relatively long blotting time used here to compare SUMO conjugates is not ideal to monitor changes in the relative amounts of free SUMO, for which a much shorter blotting time would be more suitable (Schwienhorst et al., 2000).) Similar to these deletions, applying oxidative (lane 5), osmotic (lane 6), or thermal stress (lane 7) increases the amount of sumoylated proteins in the cell compared to the untreated wild type (lane 4). Interestingly, not only an increase of overall sumoylated protein levels is observable, but also an even stronger increase of HMW conjugates in the stacking gel. These results illustrate that the levels of SUMO conjugates rapidly respond to various events challenging the equilibrium of SUMO conjugation and deconjugation. Variations of this protocol, e.g., with respect to the duration of stress, inclusion of recovery periods, or analyzing the effects in combination with individual mutations such as the ones described here or potential novel ones identified in genetic screens, can be employed to learn more about the control of SUMO dynamics under stress.

4. Probing the role of desumoylation, SUMO chain formation, and proteolysis in SUMO dynamics using HA-tagged SUMO variants 4.1 Theory As described above, the cellular SUMO system is highly dynamic and the abundance of sumoylated substrates is the net result of various processes, including SUMO conjugation, SUMO chain formation, cleavage of SUMO from substrates, and proteolytic targeting of sumoylated proteins by STUbLs and the proteasome. By manipulating these processes, their effects on overall sumoylation and potentially on single substrates can be investigated. In the following, several plasmid-encoded SUMO variants are described; these can be introduced into any yeast strain that allows selection for the respective plasmid. All of these SUMO variants are encoded on low-copy centromeric (CEN) plasmids (Gietz & Sugino, 1988). In these plasmids, the SMT3 gene, or modified versions of it, are expressed from the carbon source-regulated PGAL1 promoter (repressed in the presence of glucose, induced in the absence of glucose when galactose is present). The advantage of using this promoter is the possibility of tightly controlling expression of each SUMO variant. This allows cultivation of yeast under

SUMO dynamics

197

conditions where the promoter is not induced prior to the actual experiment, which is particularly important when working with strains for which constitutive expression of a particular SUMO variant is toxic (see Fig. 3). The Smt3 versions described here are expressed with an N-terminal 2xHA tag, which allows specific detection of sumoylated proteins with various commercially available antibodies. The tag, in addition, may be helpful in the Western blot characterization of a specific SUMO-modified form of a given target protein by producing a detectable band-shift when compared to modification by endogenous SUMO. Notably, the plasmid-encoded SMT3 gene encodes mature SUMO lacking the C-terminal residues ATY present in the wild-type SUMO precursor such that conjugation does not require prior maturation. In these constructs, the encoded SUMO variants are either wild-type Smt3 (lacking the terminal propeptide), Smt3-Q95P, or Smt3-Q95P, K11,15,19R. The Q95P mutation prevents cleavage by desumoylating enzymes once this SUMO variant has been conjugated to a substrate. Thus, this mutation in SMT3 has an effect comparable to the mutation Q90P in mammalian SUMO2, for which it was shown that it is protected from cleavage by SUMO-specific proteases (Bekes et al., 2011). Therefore, Smt3Q95P can be applied in order to globally increase the abundance of sumoylated proteins, as demonstrated below. The variant Smt3-Q95P, K11,15,19R, in addition to the features described above, has the lysine residues K11, K15, and K19 mutated to arginines. These lysine residues are located in the flexible N-terminal extension of Smt3 and are the major sites for Smt3 chain elongation (Bylebyl et al., 2003). Therefore, overexpression of Smt3-Q95P, K11,15,19R is a useful tool for increasing the relative abundance of mono- or multisumoylated proteins. To demonstrate the effects of expressing these different SUMO variants, the wild type was compared to cells deficient in the proteolysis of sumoylated proteins because they are lacking ULS1 and SLX5. It should be noted that these strains still express endogenous SMT3. However, the galactose-induced expression of HA-tagged SUMO variants is relatively strong compared to endogenous SUMO.

4.2 Reagents • • •

Polyethylene glycol (PEG) (MW ¼ 3015–3685 g/mol) (Sigma) Lithium acetate (LiAc) Carrier DNA (e.g., calf thymus DNA from Sigma)

198

•

• •

• • •

Stefan Pabst et al.

Media for yeast:  SD -Leu (synthetic minimal medium with dextrose (glucose), lacking leucine) plates: Yeast nitrogen base (6.7 g/L), glucose (20 g/L), tryptophan (40 mg/L), histidine (10 mg/L), arginine (20 mg/L), isoleucine (60 mg/L), lysine (40 mg/L), methionine (10 mg/L), phenylalanine (60 mg/L), threonine (50 mg/L), adenine (in 0.1 M NaOH) (20 mg/L), uracil (in 0.1 M NaOH) (40 mg/L), agar (for plates) (20 g/L), Note: Leucine is omitted for selection of YCplac111-based plasmid transformants, but if another auxotrophic marker is used for selection, omit the respective compound, and add leucine (60 mg/L). It is recommended to autoclave the agar in water and add the other ingredients to the autoclaved agar afterwards. For this purpose, glucose and yeast nitrogen base are autoclaved individually and the amino acids and nucleobases are filter sterilized  SR -Leu (synthetic minimal medium with raffinose, lacking leucine) liquid medium: same formula as SD -Leu but instead of glucose, use raffinose (20 g/L) and omit agar for liquid medium  SG -Leu (synthetic minimal medium with galactose, lacking leucine) plates: same formula as SD -Leu, but instead of glucose, use galactose (20 g/L). Add agar for plates  Sterile galactose (300 g/L) Liquid nitrogen Antibodies for immunodetection: monoclonal (rat) anti-HA antibody (3F10; Roche) at 1:1000, goat anti-rat 800 nm from Rockland at 1:5000, and anti-rat peroxidase (POD)-conjugated antibody from Abcam (ab97057) at 1:5000. All antibodies are diluted in PBS containing 4% milk SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher) For film developing: developer and fixation solution (Agfa G153) Reagents for SDS-PAGE and Western blot analysis, see Section 3

4.3 Procedure Expression of SUMO variants and preparation of cell lysates for SDS-PAGE and Western blot analysis: 1. Transform yeast strains, in this case wild type and uls1Δ slx5Δ, with the plasmids encoding the SUMO variants 2xHA-Smt3 (pMM43), 2xHASmt3Q95P (pNP3), and 2xHA-Smt3Q95P, K11,15,19R (pNP4), or

SUMO dynamics

2.

3.

4. 5.

6.

7. 8.

9. 10.

11. 12.

199

the empty control vector YCplac111 (Gietz & Sugino, 1988). The transformation is carried out following the LiAc/PEG method (Gietz & Schiestl, 2007). Select transformants on SD plates lacking leucine. (As described above, the constructs are available with other auxotrophic markers as well.) Incubate plates at 30°C for approximately 3 days until single colonies are visible and large enough for subsequent streakouts. Streak out cells from isolated colonies on fresh SD plates lacking leucine. To ensure reproducibility of results, at least three individual single colonies of each transformation should be streaked out and analyzed. It is recommended not to work with exceptionally large colonies of what are usually slow-growing strains to avoid unwanted selection of suppressor mutants. Incubate the plates at 30°C for 2–3 days. Inoculate yeast cells in 2 mL SR -Leu liquid medium, and incubate overnight at 30°C in a shaking incubator. Raffinose is used instead of glucose as a carbon source in order to allow rapid galactose induction in the subsequent experiment. Since yeast cells divide more slowly in medium containing raffinose as the sole carbon source compared to glucose, start overnight cultures with a comparably higher amount of yeast cells. On the following day, measure the OD600 of the overnight cultures and inoculate 10 mL SR -Leu in an Erlenmeyer flask with cells from the overnight cultures to obtain a starting OD600 of 0.25. Incubate the cells at 30°C with shaking. When the respective cultures have reached an OD600 of 0.5, add galactose from a sterile 30% galactose stock to yield a final concentration of 2%. For wild-type strains, it takes approximately 4 h to reach an OD600 of 0.5 (one doubling) after starting the culture at 0.25. Upon addition of galactose, expression of the SUMO variant is induced. Incubate galactose-induced main cultures for three additional hours at 30°C with shaking and measure their OD600 values. Take a culture volume equivalent to 7 OD600 units (for example, a culture volume of 7 mL of a culture with an OD600 of 1) and transfer into a 15 mL polypropylene centrifuge tube. Centrifuge sample at room temperature for 5 min at 3220
g. Aspirate supernatant and make sure that no substantial amounts of liquid remain on top of the yeast pellet. Snap freeze the cell pellets in the 15 mL tubes in liquid nitrogen.

200

Stefan Pabst et al.

13. Store the frozen yeast pellets at 80°C. 14. For preparation of denatured extracts, take pellets out of the 80°C freezer, transfer to the bench on ice, and immediately add 350 μL of 1
Laemmli loading buffer. To avoid premature thawing of pellets, make sure to not take too many samples at once from the freezer. Once Laemmli loading buffer is added, the samples are no longer kept on ice. 15. Vortex pellets until the cells are evenly distributed in the loading buffer. 16. Transfer the suspensions to 1.5 mL microcentrifuge tubes and heat at 99°C for 5 min. 17. Let sample cool at room temperature for 5 min. 18. Centrifuge briefly (8 s in microcentrifuge) to pellet cell debris, and transfer supernatants into fresh microcentrifuge tubes. 19. Resolve lysates by SDS-PAGE and perform Western immunoblot analysis as described in Section 3. For chemiluminescent detection of POD-coupled antibodies, a chemiluminescent substrate such as SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher) is applied to the membrane, followed by signal detection on photographic film (e.g., Fujifilm Super RX-N). Fig. 2 shows the results of expressing plasmid-encoded SUMO variants in wild-type and uls1Δ slx5Δ cells. In general, galactose-induced 2xHASmt3 is efficiently conjugated to substrate proteins and sensitively and specifically detected by the anti-HA (3F10) antibody. As expected, an increase of SUMO conjugates is observed in uls1Δ slx5Δ cells, especially in the stacking gel (Fig. 2A and B) due to impaired proteasomal degradation of these species. The 2xHA-Smt3-Q95P variant increases the amount of sumoylated proteins, apparently due to resistance to the action of desumoylating enzymes. This effect is most prominent in the medium molecular weight range in wild-type cells (Fig. 2A, lane 3 vs 5). The 2xHA-Smt3-Q95P, K11,15,19R variant, similarly to Q95P alone, increases overall sumoylation in wild-type cells. The most striking effect of this variant, which is impaired in SUMO chain formation, is the absence of HMW-SUMO conjugates detected in the stacking gel in the samples from uls1Δ slx5Δ cells, indicating that these conjugates depend on the ability to form SUMO chains. These findings demonstrate that the abundance of substrates carrying SUMO chains is controlled by STUbLs, as concluded earlier (Uzunova et al., 2007). Also note that free 2xHA-Smt3-Q95P, K11,15,19R migrates faster in the SDS-PAGE gel than 2xHA-Smt3-Q95P, an effect similar to what was previously reported when these lysines were mutated (Bylebyl et al., 2003). High resolution separation of bands achieved by using long SDS-PAA gels

SUMO dynamics

201

Fig. 2 Sumoylation changes in response to expression of SUMO variants and deletion of STUbLs. For Western blot analyses (8% SDS-PAGE) using immunological detection of highly abundant HA-Smt3 conjugates, an anti-HA 3F10 rat monoclonal (1:1000) was used as a primary antibody and anti-rat Alexa Fluor 800 (1:5000) as secondary antibody (A). For more sensitive detection, anti-rat POD (1:5000) was subsequently applied as a secondary antibody to the upper part of the same membrane followed by chemiluminescence detection (B). Therefore, (A) and (B) show, respectively, signals from the entire membrane, or from the upper part of the same membrane developed with the different antibodies and procedures. (C) As a loading control, the membrane was probed with Total Protein Stain, which was applied before the antibody incubations. A congenic wild-type (wt) control strain was compared to the slx5Δ uls1Δ (ΔΔ) mutant. Treatment of cells, SDS-PAGE, and Western blotting were performed as described in the protocol.

(14 cm) also reveals that certain bands seem to behave opposite to the bulk effects on sumoylation. For example, certain bands are less abundant in uls1Δ slx5Δ than in the wild-type strain although the overall levels of conjugates are higher in the mutant. A possible explanation is that these substrates have undergone chain elongation and thus have shifted to a different molecular weight range. Therefore, not only the bulk abundance but also the pattern of sumoylation changes. An interesting question is why different substrates apparently respond differently to the conditions applied here. Overall, such approaches demonstrate the dynamic nature of the SUMO system and ways to manipulate its balance in order to gain more insight into the mechanisms controlling cellular SUMO homeostasis. For example, it could be investigated in more detail, how cells adjust sumoylated levels of distinct substrates

202

Stefan Pabst et al.

to certain physiological alterations (such as the aforementioned stress conditions) by controlling the activities of sumoylating enzymes, desumolyting enzymes or STUbLs. The presence of a plasmid encoding 2xHA-Smt3-Q95P does not affect growth of yeast strains on SD plates because expression is not induced (data not shown). Growth assays on SG plates, in contrast, reveal the effects of 2xHA-Smt3-Q95P induction on growth of yeast cells. While the wild-type and uls1Δ strains seem to be largely unaffected by expression, a severe growth defect is caused by induction of this SUMO variant in slx5Δ cells and, even more strongly, in the uls1Δ slx5Δ double mutant. These findings suggest that the Ulp-resistant SUMO conjugates formed upon 2xHA-Smt3Q95P expression (shown in Fig. 2) have a toxic effect if they are not controlled by STUbLs (Fig. 3). These findings are consistent with the view that SUMO conjugates, once formed, can either be controlled by desumoylation or by STUbL-mediated proteolytic elimination. If both mechanisms are impaired in the same cells, as is the case in slx5Δ or slx5Δ uls1Δ cells expressing Smt3-Q95P, conjugates can cause severe growth inhibition. The growth inhibition caused by the Q95P mutation is slightly reduced by introduction of the K11,15,19R mutations that are known to inhibit

Fig. 3 Effect of inducible overexpression of SMT3 variants on growth of yeast strains with defects in proteolysis of sumoylated proteins. The indicated strains harboring an empty vector or plasmids expressing 2xHA-SMT3 variants (wild-type, Q95P, Q95P K11,15,19R) from PGAL1 were pregrown on SR (raffinose) media and then grown on SD (glucose) and SG (galactose) media lacking leucine. On SD, where the SUMO variants are not expressed, the plasmids had no effects on the growth of the yeast strains (not shown). On SG, expression of 2xHA-Smt3-Q95P variants is induced causing a severe growth defect in slx5Δ and uls1Δ slx5Δ strains when compared to the vector control of transformants expressing 2xHA-Smt3 without the Q95P mutation. The plate shown on the left with transformants of the wild type and uls1Δ was incubated for 3 days at 30° C; the plate on the right with transformants of the slx5Δ and uls1Δ slx5Δ transformants was incubated for 6 days.

SUMO dynamics

203

Smt3 chain formation (Bylebyl et al., 2003) indicating that formation of polymeric Smt3 conjugates that are neither processed by SUMO proteases nor targeted by STUbLs is particularly harmful.

5. Purification of 8xHis-SUMO conjugates using cobalt beads 5.1 Theory Purification of cellular SUMO conjugates (as well as free SUMO) can have various useful applications. One obvious use is the purification of SUMO conjugates with subsequent detection of a putative substrate. This represents the reverse approach to the more common approach of affinity purifying a putative SUMO substrate with subsequent detection of SUMO. One potential advantage of a pulldown of global cellular SUMO conjugates is that, in principle, the same purified material can be analyzed for the presence of various SUMO substrates, provided that suitable antibodies are available. In particular, if antibodies against endogenous proteins without additional protein tags are available, this is a reasonable strategy for investigation of several substrates with a single purification. Furthermore, such a purification of global SUMO conjugates can be used for mass spectrometric identification of SUMO substrates as demonstrated in previous studies (Denison et al., 2005; Hannich et al., 2005; Panse, Hardeland, Werner, Kuster, & Hurt, 2004; Wohlschlegel et al., 2004; Zhou et al., 2004). In the following, a procedure for the purification of 8xHis-SUMO/Smt3 from yeast is described. In this strain, the Smt3 protein expressed from its authentic genomic locus was N-terminally tagged with an 8His-tag; therefore, all expressed Smt3 molecules in the cell bear the tag (Wohlschlegel et al., 2004). The purification is conducted under denaturing conditions in order to avoid cleavage of Smt3 from substrate proteins during sample handling. Furthermore, denaturing conditions help to ensure that only proteins covalently bound to Smt3 are purified, and not associated proteins.

5.2 Buffers and reagents •

• •

Growth medium for yeast cells: Yeast extract–peptone–dextrose medium (YPD): 1% yeast extract, 2% bacto peptone, 2% glucose. For solid growth media, 2% agar is added Distilled water Liquid nitrogen

204

• • • • • • • • •

Stefan Pabst et al.

Lysis buffer: 8 M urea, 100 mM NaH2PO4, 10 mM Tris–HCl, pH 8.0. Prepare fresh Wash buffer: 8 M urea, 100 mM NaH2PO4, 10 mM Tris–HCl, pH 6.5. Prepare fresh Elution buffer: 8 M urea, 100 mM NaH2PO4, 10 mM Tris–HCl, pH 4.5. Prepare fresh Cobalt beads (TALON® Metal Affinity Resin, Clontech/Takara) 5
Laemmli loading buffer: 0.3125 M Tris–HCl, pH 6.8, 10% SDS, 50% glycerol, 10% β-mercaptoethanol, 0.025% bromophenol blue 2
Laemmli loading buffer: 0.125 M Tris–HCl, pH 6.8, 4% SDS, 20% glycerol, 4% β-mercaptoethanol, 0.01% bromophenol blue For SDS-PAGE and Western blot reagents, see Section 3 SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher) For film developing: developer and fixation solution (Agfa G153)

5.3 Procedure Generation of yeast samples: 1. Streak out the EJ337 (8xHis-SMT3) strain and a congenic wild-type strain (the latter serves as a negative (untagged) control for the purification) on a YPD plate and incubate at 30°C for 2 days. 2. Inoculate 5 mL liquid YPD medium with yeast cells taken from the YPD plate and incubate overnight at 30°C while shaking. 3. On the following day, measure the OD600 of the cultures. Inoculate 60 mL YPD in an Erlenmeyer flask with a volume of the overnight culture to obtain a starting OD600 of 0.25. 4. Incubate the cultures at 30°C with shaking. 5. Harvest the cultures when they reach an OD600 of approximately 1.0. Calculate the exact culture volume corresponding to 15 OD, and transfer this amount into a 50 mL centrifuge tube. The original 50 mL culture, when grown as suggested, should be sufficient for three 15 OD aliquots, which should be collected in individual tubes, so that three independent purifications can be conducted with cells originating from the same culture. Larger culture volumes (1 L) may be required, depending on the experimental objective. 6. Centrifuge samples at room temperature for 5 min at 3220
g. 7. Aspirate off the supernatant, and snap freeze the yeast pellet in liquid nitrogen in the 50 mL plastic conical tube. Store tubes at 80°C.

SUMO dynamics

205

Purification of 8xHis-Smt3 and attached proteins under denaturing conditions using cobalt beads: 8. Transport yeast pellets on ice, and as soon as possible add 500 μL lysis buffer to all tubes. From now on, all described steps are performed at room temperature. 9. Resuspend pellets in lysis buffer and transfer to 1.5 mL LoBind safelock tubes. 10. Add 150 μL glass beads (Ø ¼ 0.425 mm  0.6 mm) to the 500 μL suspension. 11. Vortex at room temperature 4
1 min, with 1 min pauses in between. 12. Centrifuge at 30,000
g for 20 min at room temperature. 13. Meanwhile, equilibrate cobalt beads. For this purpose, add 150 μL cobaltbead slurry into a 1.5 mL LoBind safe-lock tube, with one tube for each experimental condition. For pipetting the slurry, cut 0.5 cm off from a 200 μL pipette tip with scissors before use. Add 500 μL lysis buffer to beads. Centrifuge at 100
g for 1 min and remove supernatant. Two more times, add 500μL lysis buffer, centrifuge and remove the supernatant. 14. Remove 450 μL cell supernatant from step 12 and transfer the extract into a fresh microcentrifuge tube. Transfer of cell debris should be avoided. 15. Dilute sample with 450 μL additional lysis buffer (yielding in total 900 μL lysate). The dilution is done because a larger volume facilitates even mixing of the cobalt beads. For a later analysis of binding efficiency, take 50μL of this sample (named “input”) and add to 12.5 μL 5
Laemmli loading buffer. Heat this “input” sample at 99°C for 5 min, let it sit at room temperature for 5 min, and store at 20°C. 16. The remaining 850 μL lysate are added to equilibrated TALON beads, and the binding reaction is conducted by rotation of the 1.5 mL LoBind safe-lock tubes on a spinning wheel at room temperature overnight. 17. On the next day, centrifuge samples at 100
g for 1 min. For the later analysis of binding efficiency, take 50 μL of the supernatant (named “unbound”) and add to 12.5 μL 5
Laemmli loading buffer. Heat this “unbound” sample at 99°C for 5 min, let it sit at room temperature for 5 min, and store at 20°C. The remaining supernatant is removed from the beads and stored at room temperature in case TALON bead binding is going to be repeated. 18. Add 1 mL wash buffer to the beads. 19. Rotate on a spinning wheel for 5 min. 20. Centrifuge samples at 100
g for 1 min. For a later analysis of wash efficiency, you may take 100μL of the supernatant (named “wash 1”)

206

21. 22. 23. 24. 25. 26.

27.

28.

29.

30.

Stefan Pabst et al.

and add to 25 μL 5
Laemmli loading buffer. Heat this “wash 1” sample at 99°C for 5 min, let it sit at room temperature for 5 min, and store at 20°C. The remaining supernatant is removed from the beads. Again, add 1 mL wash buffer, rotate on wheel, centrifuge and remove supernatant. Transfer cobalt beads with bound proteins to a fresh 1.5 mL LoBind safe-lock tube by using a cut pipette tip with a wider opening. Once more, add 1 mL wash buffer, rotate on wheel, centrifuge and remove supernatant. After removal of wash buffer, add 200 μL elution buffer to the beads and rotate on spinning wheel for 20 min at room temperature. Centrifuge samples at 100
g for 1 min. For analysis by SDS-PAGE, take 190 μL eluate and add to 47.5 μL 5
Laemmli loading buffer. Heat this “pH eluate” sample at 99°C for 5 min, let it sit at room temperature for 5 min, and store at 20°C. If analysis by SDS-PAGE is not desired, the eluate can be directly used for further applications. The remaining 10 μL eluate on the beads should be discarded since pipetting in this region increases the risk of accidentally taking up beads. For assessing the efficiency of elution, 237.5 μL 2
Laemmli loading buffer may be added to the cobalt beads for a harsh elution of all proteins that might still be bound to the beads. Heat the sample at 99°C for 5 min (named “boiled beads”), let it sit at room temperature for 5 min, and store at 20°C. For analysis by SDS-PAGE, thaw samples at room temperature, subsequently heat them at 99°C, and let them sit at room temperature for 5 min. Before loading for SDS-PAGE, briefly centrifuge samples (8 s at top speed) to pellet insoluble material. For the experiment shown in Fig. 4A and B, 17.5 μL (1.5% of input material) of both input and unbound protein were loaded on the same gel. On the gel in Fig. 4C, 20 μL of the “pH eluate” (8%) were loaded. Perform SDS-PAGE and immunoblotting as described in Section 3. Alternatively, if only the success of the purification is of interest, instead of the described large SDS-PAA gels, standard mini-SDS-PAGE systems can be used, as was done in the example shown in Fig. 4. Detect SUMO conjugates by using anti-Smt3 or another suitable antibody.

SUMO dynamics

207

Fig. 4 Purification of 8His-Smt3 (8H-SUMO) and its conjugates by Co2+ beads. Specific binding of 8H-Smt3 from a yeast strain expressing the genomically tagged 8H-SMT3 allele. Shown are the results of Western blot analyses of cellular proteins separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was probed with anti-Smt3 (1:10000; rabbit) and anti-rabbit POD (1:5000), with chemiluminescence detection. (A) Cell extracts after glass bead lysis under denaturing conditions. (B) Loss of 8H-Smt3 signal in the nonbound material indicates efficient binding of 8H-Smt3 to beads. (C) Specific elution of 8H-Smt3 and its conjugates using low pH. (A) and (B) both show the upper and the lower parts of the same membrane but with different exposures times.

Fig. 4 shows a successful purification of 8xHis-Smt3-protein conjugates using the described procedure. In the input, similar levels of sumoylated proteins are detected in both strains, EJ337 and the untagged control (Fig. 4A). Notably, 8xHis-Smt3 runs slightly slower than untagged Smt3. As expected, in the untagged control, the amounts of SUMO conjugates in the input and unbound fractions are very similar. In contrast, 8xHisSmt3 gives a very weak signal in the unbound fraction, suggesting that most of the 8xHis-tagged Smt3 conjugates and free Smt3 have bound to the cobalt beads (Fig. 4B). The third panel shows the elution of 8xHis-Smt3 conjugates from the cobalt beads; in the untagged control, as expected, no SUMO conjugates are detected after purification (Fig. 4C).

208

Stefan Pabst et al.

Acknowledgments We thank Erica S. Johnson for providing a yeast strain with genomically expressed 8xHistagged Smt3, Ingrid Schwienhorst for the uba2-ts mutant, Kristina Uzunova for uls mutants, Kerstin N€ urrenberg for help with generating plasmids expressing HA-tagged Smt3, and Maria Miteva for raising anti-Smt3 antibodies. This work is supported by the Deutsche Forschungsgemeinschaft (DFG) as part of the priority programme CRC 1218.

References Bekes, M., Prudden, J., Srikumar, T., Raught, B., Boddy, M. N., & Salvesen, G. S. (2011). The dynamics and mechanism of SUMO chain deconjugation by SUMO-specific proteases. The Journal of Biological Chemistry, 286(12), 10238–10247. Bylebyl, G. R., Belichenko, I., & Johnson, E. S. (2003). The SUMO isopeptidase Ulp2 prevents accumulation of SUMO chains in yeast. The Journal of Biological Chemistry, 278(45), 44113–44120. Denison, C., Rudner, A. D., Gerber, S. A., Bakalarski, C. E., Moazed, D., & Gygi, S. P. (2005). A proteomic strategy for gaining insights into protein sumoylation in yeast. Molecular & Cellular Proteomics, 4(3), 246–254. Eckhoff, J., & Dohmen, R. J. (2015). In vitro studies reveal a sequential mode of chain processing by the yeast SUMO (small ubiquitin-related modifier)-specific protease Ulp2. The Journal of Biological Chemistry, 290(19), 12268–12281. Flotho, A., & Melchior, F. (2013). Sumoylation: A regulatory protein modification in health and disease. Annual Review of Biochemistry, 82, 357–385. Galanty, Y., Belotserkovskaya, R., Coates, J., Polo, S., Miller, K. M., & Jackson, S. P. (2009). Mammalian SUMO E3-ligases PIAS1 and PIAS4 promote responses to DNA doublestrand breaks. Nature, 462(7275), 935–939. Gietz, R. D., & Schiestl, R. H. (2007). High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nature Protocols, 2(1), 31–34. Gietz, R. D., & Sugino, A. (1988). New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene, 74(2), 527–534. Golebiowski, F., Matic, I., Tatham, M. H., Cole, C., Yin, Y., Nakamura, A., et al. (2009). System-wide changes to SUMO modifications in response to heat shock. Science Signaling, 2(72), ra24. Hannich, J. T., Lewis, A., Kroetz, M. B., Li, S. J., Heide, H., Emili, A., et al. (2005). Defining the SUMO-modified proteome by multiple approaches in Saccharomyces cerevisiae. The Journal of Biological Chemistry, 280(6), 4102–4110. Heun, P. (2007). SUMOrganization of the nucleus. Current Opinion in Cell Biology, 19(3), 350–355. Hickey, C. M., Wilson, N. R., & Hochstrasser, M. (2012). Function and regulation of SUMO proteases. Nature Reviews. Molecular Cell Biology, 13(12), 755–766. Johnson, E. S. (2004). Protein modification by SUMO. Annual Review of Biochemistry, 73, 355–382. Johnson, E. S., & Blobel, G. (1997). Ubc9p is the conjugating enzyme for the ubiquitin-like protein Smt3p. The Journal of Biological Chemistry, 272(43), 26799–26802. Johnson, E. S., & Gupta, A. A. (2001). An E3-like factor that promotes SUMO conjugation to the yeast septins. Cell, 106(6), 735–744. Johnson, E. S., Schwienhorst, I., Dohmen, R. J., & Blobel, G. (1997). The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer. The EMBO Journal, 16(18), 5509–5519.

SUMO dynamics

209

Kim, D. H., Harris, B., Wang, F., Seidel, C., McCroskey, S., & Gerton, J. L. (2016). Mms21 SUMO ligase activity promotes nucleolar function in Saccharomyces cerevisiae. Genetics, 204(2), 645–658. Li, S. J., & Hochstrasser, M. (1999). A new protease required for cell-cycle progression in yeast. Nature, 398(6724), 246–251. Li, S. J., & Hochstrasser, M. (2000). The yeast ULP2 (SMT4) gene encodes a novel protease specific for the ubiquitin-like Smt3 protein. Molecular and Cellular Biology, 20(7), 2367–2377. Miller, M. J., & Vierstra, R. D. (2011). Mass spectrometric identification of SUMO substrates provides insights into heat stress-induced SUMOylation in plants. Plant Signaling & Behavior, 6(1), 130–133. Morris, J. R., Boutell, C., Keppler, M., Densham, R., Weekes, D., Alamshah, A., et al. (2009). The SUMO modification pathway is involved in the BRCA1 response to genotoxic stress. Nature, 462(7275), 886–890. Panse, V. G., Hardeland, U., Werner, T., Kuster, B., & Hurt, E. (2004). A proteome-wide approach identifies sumoylated substrate proteins in yeast. The Journal of Biological Chemistry, 279(40), 41346–41351. Perry, J. J., Tainer, J. A., & Boddy, M. N. (2008). A SIM-ultaneous role for SUMO and ubiquitin. Trends in Biochemical Sciences, 33(5), 201–208. Pichler, A., Fatouros, C., Lee, H., & Eisenhardt, N. (2017). SUMO conjugation—A mechanistic view. Biomolecular Concepts, 8(1), 13–36. Praefcke, G. J., Hofmann, K., & Dohmen, R. J. (2012). SUMO playing tag with ubiquitin. Trends in Biochemical Sciences, 37(1), 23–31. Psakhye, I., & Jentsch, S. (2012). Protein group modification and synergy in the SUMO pathway as exemplified in DNA repair. Cell, 151(4), 807–820. Reindle, A., Belichenko, I., Bylebyl, G. R., Chen, X. L., Gandhi, N., & Johnson, E. S. (2006). Multiple domains in Siz SUMO ligases contribute to substrate selectivity. Journal of Cell Science, 119(Pt. 22), 4749–4757. Schwienhorst, I., Johnson, E. S., & Dohmen, R. J. (2000). SUMO conjugation and deconjugation. Molecular & General Genetics, 263(5), 771–786. Seifert, A., Schofield, P., Barton, G. J., & Hay, R. T. (2015). Proteotoxic stress reprograms the chromatin landscape of SUMO modification. Science Signaling, 8(384), rs7. Sriramachandran, A. M., & Dohmen, R. J. (2014). SUMO-targeted ubiquitin ligases. Biochimica et Biophysica Acta, 1843(1), 75–85. Tatham, M. H., Geoffroy, M. C., Shen, L., Plechanovova, A., Hattersley, N., Jaffray, E. G., et al. (2008). RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenicinduced PML degradation. Nature Cell Biology, 10(5), 538–546. Tatham, M. H., Matic, I., Mann, M., & Hay, R. T. (2011). Comparative proteomic analysis identifies a role for SUMO in protein quality control. Science Signaling, 4(178), rs4. Tempe, D., Piechaczyk, M., & Bossis, G. (2008). SUMO under stress. Biochemical Society Transactions, 36(Pt. 5), 874–878. Ulrich, H. D. (2009). The SUMO system: An overview. Methods in Molecular Biology, 497, 3–16. Uzunova, K., G€ ottsche, K., Miteva, M., Weisshaar, S. R., Glanemann, C., Schnellhardt, M., et al. (2007). Ubiquitin-dependent proteolytic control of SUMO conjugates. The Journal of Biological Chemistry, 282(47), 34167–34175. Wilson, V. G., & Rangasamy, D. (2001). Intracellular targeting of proteins by sumoylation. Experimental Cell Research, 271(1), 57–65. Wohlschlegel, J. A., Johnson, E. S., Reed, S. I., & Yates, J. R., 3rd. (2004). Global analysis of protein sumoylation in Saccharomyces cerevisiae. The Journal of Biological Chemistry, 279(44), 45662–45668.

210

Stefan Pabst et al.

Xie, Y., Kerscher, O., Kroetz, M. B., McConchie, H. F., Sung, P., & Hochstrasser, M. (2007). The yeast Hex3.Slx8 heterodimer is a ubiquitin ligase stimulated by substrate sumoylation. The Journal of Biological Chemistry, 282(47), 34176–34184. Zhao, X., & Blobel, G. (2005). A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization. Proceedings of the National Academy of Sciences of the United States of America, 102(13), 4777–4782. Zhou, W., Ryan, J. J., & Zhou, H. (2004). Global analyses of sumoylated proteins in Saccharomyces cerevisiae. Induction of protein sumoylation by cellular stresses. The Journal of Biological Chemistry, 279(31), 32262–32268.