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. 2007 Nov;6(11):1679-91.
doi: 10.1016/j.dnarep.2007.06.004. Epub 2007 Jul 31.

Stimulation of in vitro sumoylation by Slx5-Slx8: evidence for a functional interaction with the SUMO pathway

Tatsuya Ii  1 , Janet R Mullen, Christopher E Slagle, Steven J Brill
Affiliations

Stimulation of in vitro sumoylation by Slx5-Slx8: evidence for a functional interaction with the SUMO pathway

Tatsuya Ii et al. DNA Repair (Amst). 2007 Nov.

Abstract

The yeast genes SLX5 and SLX8 were identified based on their requirement for viability in the absence of the Sgs1 DNA helicase. Loss of these genes results in genome instability, nibbled colonies, and other phenotypes associated with defects in sumoylation. The Slx5 and Slx8 proteins form a stable complex and each subunit contains a single RING-finger domain at its C-terminus. To determine the physiological function of the Slx5-8 complex, we explored its interaction with the SUMO pathway. Curing 2micro circle from the mutants, suppressed their nibbled colony phenotype and partially improved their growth rate, but did not affect their sensitivity to hydroxyurea. The increase in sumoylation observed in slx5Delta and slx8Delta mutants was found to be dependent on the Siz1 SUMO ligase. Physical interactions between the Slx5-8 complex and both Ubc9 and Smt3 were identified and characterized. Using in vitro reactions, we show that Slx5, Slx8, or the Slx5-8 complex stimulates the formation of SUMO chains and the sumoylation of a test substrate. Interestingly, a functional RING-finger domain is not required for this stimulation in vitro. These biochemical data demonstrate for the first time that the Slx5 and Slx8 complex is capable of interacting directly with the SUMO pathway.

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Figures

Figure 1
Figure 1
Two-micron circle is responsible for the nibbled colony phenotype of slx5Δ and slx8Δ strains. (A) WT, slx5Δ, and slx8Δ strains were cured of 2μ circle and the indicated cultures were spread on YPD plates. Colonies were photographed following 3 days growth at 30°C. Filled arrowheads indicate nibbled colonies. (B) The indicated strains were resuspended at an OD = 3, serially diluted in 10-fold steps, and approximately 5 μl spotted on solid yeast extract-peptone-dextrose (YPD) media in the absence or presence of 0.1 M hydroxyurea (HU). The plates were photographed following 2 (YPD) or 4 (HU) days growth at 30°C. The growth rates of these strains were determined in liquid YPD at 30°C. These doubling times (DT) are presented at right.
Figure 2
Figure 2
SLX5 and SLX8 show genetic interactions with known sumoylation components. (A) Four tetrads from the cross YO174 (ubc9-1) X JMY1604 (slx8Δ) were dissected vertically onto a YPD plate, allowed to germinate for 4 days at 25°C, and photographed. Genotypes of the spore clones were determined and are indicated as follows: WT, wild type; u, ubc9-1; s, slx8Δ; us, ubc9-1 slx8Δ. Note the nibbled colony morphology of slx8Δ and ubc9-1 single mutants. (B) Extracts from the indicated yeast mutants were prepared by the NaOH method [56] and analyzed for Smt3-protein conjugates by 10% SDS-PAGE and immunoblotting using antibodies against Smt3. Identical samples were run on 17% SDS-PAGE, blotted, and probed for free Smt3 or RPA1 as internal loading controls.
Figure 3
Figure 3
Slx5 and Slx8 show physical interactions with known sumoylation components. (A) Yeast Two-Hybrid interactions with SLX5. SLX5 was subcloned into a binding-domain vector (pGAD) and transformed into strain AH109 along with an activating domain plasmid containing the indicated gene. Transformants were streaked onto selective media lacking histidine and adenine as selection for the two reporter genes in this strain. (B) Yeast Two-Hybrid interactions with SLX8 in the binding-domain vector were assayed as in (A). (C) Increasing amounts (12, 48, or 120 pmol) of Slx5 (top panel), Slx8 (second panel), or the Slx5-Slx8 complex (lower panel) were incubated on ice with either no protein, or 24 pmol of GST, GST-Ubc9, or GST-Smt3 as indicated. Bound proteins were detected following glutathione bead pull-down and immunoblotting with Slx5 or Slx8 antisera.
Figure 4
Figure 4
Purified Slx5 and Slx8 stimulate SUMO conjugation in vitro. (A) Standard sumoylation reactions were carried out as described in the Materials and Methods, but included the indicated amounts of Slx5 or Slx8, in addition to the following levels of yeast Aos1/Uba2 (E1, 3.3 ng in lanes 1-14 and 16), Ubc9 (E2, 8.3 ng in lanes 1-15), and HF-Smt3 (SUMO, 150 ng). Following incubation, the reaction products were analyzed by SDS-PAGE and immunoblotting using antibodies against the FLAG epitope to detect SUMO. The positions of monomeric (SUMO) and polymerized SUMO chains (di = S2; tri = S3; multiple = Sn) are indicated. (B) Sumoylation reactions were carried out under standard conditions but contained RPA (50 ng) as substrate and methylated HF-Smt3 (150 ng) in place of HF-Smt3 to limit SUMO chain formation. Reactions contained either no addition (NA), Slx8 (225 ng), no Aos1-Uba2 (-E1), or no RPA (−RPA). Following incubation, the reaction products were analyzed by SDS-PAGE and immunoblotting using antibody against yeast RPA1. Arrowheads indicate the positions of RPA1 and its sumoylated products. (C) Sumoylation reactions were carried out under standard conditions, but contained methylated HF-Smt3 (150 ng) in addition to the following yeast transcription factors as substrates: TFIIA (100 ng, lanes 1 and 2), Ydr1 (100 ng, lanes 3 and 4), Bur6 (100 ng, lanes 5 and 6), or TBP (100 ng, lanes 7 and 8). Reactions were performed in the presence (+) or absence (−) of Slx5 (300 ng). Following incubation the reactions were analyzed as in (A) to detect SUMO. The positions of SUMO and sumoylated Ydr1 products are indicated.
Figure 5
Figure 5
Slx5-dependent sumoylation of Ydr1 in vitro. (A) Standard sumoylation reactions were carried out as described in the Materials and Methods, but where indicated contained Slx5 (300 ng), either Ydr1 (WT, 100 ng) or Ydr1-K141R (M, 100 ng) as substrate, and either HF-Smt3 (WT, 150 ng) or Me-HF-Smt3 (ME, 150 ng). Following incubation, the reactions were analyzed by SDS-PAGE and immunoblotting using antibody against FLAG to detect SUMO (A) or against Ydr1 (B). The identity of the reaction products are indicated. Asterisks in (B) indicate cross-reacting proteins present in the Ydr1-K141R preparation. (C) The specificity of SUMO ligase activity was tested using standard reaction conditions and included Ydr1 (100 ng), methylated HF-Smt3 (150 ng), and the indicated amount of BSA, Slx5, or Slx8. Following incubation, the reactions were analyzed by SDS-PAGE and immunoblotting with antibodies against Ydr1. Arrowheads indicate the positions of sumoylated products and asterisks indicate cross-reacting bands. The cross-reacting band denoted by *8 is His6-Slx8.
Figure 6
Figure 6
Stimulation of SUMO conjugation to Ydr1 by Slx5, Slx8, and the Slx5-Slx8 complex. (A) The SUMO ligase activities of the monomeric and heterodimeric forms of Slx5-Slx8 were compared using standard reactions conditions and either Ydr1 (+, 50 ng) or Ydr1-K141R (M, 50 ng), as indicated. Reactions contained methylated HF-Smt3 (150 ng) and various amounts of either Slx5 (225, 0, 10, 60, 225, 225 ng), Slx8 (225, 0, 10, 60, 225, 225 ng), or Slx5-Slx8 complex (450, 0, 20, 120, 450, 450 ng), as indicated. (B) and (C) Additive effects of Slx5 and Slx8 were tested in reactions containing Ydr1 (100 ng), HF-Smt3 (150 ng), and either Slx5 (0, 180, 30, 60, 120, 180 ng in lanes 1–6), Slx8 (30, 60, 120, 180 ng in lanes 7–10); Slx5 plus Slx8 (constant 30 ng Slx5 plus 30, 90, 150 ng Slx8 in lanes 11–13), or the Slx5-Slx8 complex (60, 120, and 180 ng in lanes 14–16). Incubation and analysis was performed as in Figure 5 using antibodies against Ydr1 (A) and (B) or FLAG (C).
Figure 7
Figure 7
RING-finger function of Slx5 and Slx8 is dispensible for in vitro sumoylation. (A) Slx5 (WT) or Slx5 protein lacking the N-terminal 200 aa (ΔN200), N-terminal 400 aa (ΔN400), or C-terminal 126 aa (ΔC126) was purified from E. coli, and 100 ng was assayed for the ability to stimulate SUMO chain formation using 10% SDS-PAGE. The RING domain is comprised of the C-terminal 126 residues. (B) Slx8 (WT) or Slx8 protein lacking the N-terminal 163 aa (ΔN163), the N-terminal 200 aa (ΔN200), or the C-terminal 74 aa (ΔC74) was titrated into in vitro sumoylation assays and analyzed as in (A) except for the use of 12.5% SDS-PAGE. The RING domain is comprised of the C-terminal 74 residues. (C) and (D) Time course of in vitro Ydr1 sumoylation reactions. Standard sumoylation reactions were carried out as described in the Materials and Methods, but contained 10 ng of the indicated Slx5 or Slx8 protein in addition to Ydr1 (50 ng) and methylated HF-Smt3p (150 ng). Following incubation at 30°C for 0, 10, 30, 60, or 90 min, the reactions were terminated, analyzed by SDS-PAGE, and immunoblotted using anti-Ydr1 antibody. The level of singly-modified Ydr1 was determined by densitometry and is presented as the percent of maximal sumoylation as a function of time. Point mutations are as follows: Slx5-7 (C556S), Slx5-8 (C556S, H558A, C561S), Slx8-2 (C221S), Slx8-3 (C221S, H223A, C226S).
Figure 8
Figure 8
Sumoylation of PCNA and Cdc11 is altered in slx5Δ mutants. Guanidine extracts from wt or slx5Δ strains that express His7-tagged PCNA were partially purified on a Ni resin and treated with or without Ulp1 protease prior to immunoblotting with antibodies against PCNA or Smt3. (B) TCA extracts from wt or slx5Δ strains that express HF-Smt3 as their only source of SUMO were immunoblotted with antibody against Cdc11. (C) Guanidine extracts from the strains in (B) were partially purified on a Ni resin and immunoblotted with antibodies against Cdc11. Ponceau S-stained membranes are shown as gel-loading controls. S1, S2: the relevant protein conjugated to 1 or 2 Smt3 moieties, respectively. Asterisks indicate non-specific cross-reacting bands. Strains: NJY2504, NJY2505, NJY2510, NJY2543.
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