Volume 33, Issue 1, Pages 124-135 (January 2009) Global Map of SUMO Function Revealed by Protein-Protein Interaction and Genetic Networks  Taras Makhnevych, Yaroslav Sydorskyy, Xiaofeng Xin, Tharan Srikumar, Franco J. Vizeacoumar, Stanley M. Jeram, Zhijian Li, Sondra Bahr, Brenda J. Andrews, Charles Boone, Brian Raught  Molecular Cell  Volume 33, Issue 1, Pages 124-135 (January 2009) DOI: 10.1016/j.molcel.2008.12.025 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Overview of the SUMO Interactor, Conjugate, and SGA Screens (A) Graphical overview of the SUMO conjugation/deconjugation pathway in yeast. (1) SUMO is translated as a proprotein, and maturation requires the removal of three C-terminal amino acids (effected by the SUMO protease Ulp1p). SUMO conjugation is effected by three SUMO-specific enzymes: (2) the E1 (consisting of an Aos1p/Uba2p heterodimer in budding yeast), (3) an E2 (Ubc9p), and (4) an E3 (the three known SUMO E3 ligases in S. cerevisiae are Siz1p, Siz2p/Nfi1p, and Mms21p). (5) Removal of SUMO from a substrate is effected by SUMO-specific proteases (Ulp1p and Ulp2p). (B) The number of hits (as detected by SGA analysis) contributed by each of the eight SUMO query strains, sorted into 15 broad biological processes of the SUMO system. (C) Venn diagram depicting the degree of overlap between the SUMO SGA, interactor, and conjugate data sets. (D) Percentage of total hits for each of the three different SUMO data sets (SGA, interactors, and conjugates) falling within each of the 15 biological processes. Molecular Cell 2009 33, 124-135DOI: (10.1016/j.molcel.2008.12.025) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 The SUMO Pathway SGA Network Genetic interaction network of eight SUMO query strains (siz1Δ, siz2Δ, siz1Δ siz2Δ, mms21-sp, ubc9-2, ulp1-333SGG, ulp2-DAmP, and smt3-331) representing 1415 synthetic lethal/sick interactions with 526 genes, as determined by SGA analysis. All of the interactions were confirmed by random spore analysis. Hits were sorted into 15 broad biological processes. Gene names highlighted in blue were also identified in our SUMO interactor screen, green were previously identified as putative SUMO conjugates, and red appeared in all three screens. Molecular Cell 2009 33, 124-135DOI: (10.1016/j.molcel.2008.12.025) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 The SUMO Pathway and Protein Catabolism (A) An SGA subnetwork highlighting genetic interactions between the SUMO pathway mutant alleles and genes involved in protein catabolism. (B) Proteins with roles in protein catabolism that interact with SUMO (SUMO interactors) or are modified by SUMO (SUMO conjugates). Proteins highlighted in blue appeared in more than one screen. (C) Cdc48p is a SUMO target in yeast. WT and ubc9-2 strains expressing TAP-tagged Cdc48p were transformed with a pHF-SMT3 plasmid encoding a gal-inducible His-Flag tagged SUMO protein (lanes 1 and 2). HF-Smt3 expression was induced for 4 hr at 25°C. Prior to collection, cultures were shifted to 37°C for 2 hr. The Cdc48-TAP strain (without the SUMO plasmid) was also grown for 2 hr at 37°C, then treated with 5 μg/ml tunicamycin for an additional 1 hr (lanes 3 and 4). Cdc48-TAP was affinity-purified from whole cell lysates and analyzed by western blotting using anti-Smt3p antibodies. Both modified and unmodified Cdc48-TAP are detected by this antibody due to the presence of a Protein A moiety in the TAP tag, which binds to IgG present in the anti-Smt3 rabbit antiserum. (D) Association of Cdc48p with ER membranes is dependent on SUMO function. WT and ubc9-2 cells expressing Cdc48-GFP and cNLS-RFP were grown at 26°C or shifted to 37°C for 3 hr. Cdc48-GFP localization was examined by confocal microscopy. Molecular Cell 2009 33, 124-135DOI: (10.1016/j.molcel.2008.12.025) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 The SUMO Pathway Regulates Nuclear Accumulation of Mga2p (A) GFP-Mga2 and cNLS-RFP localization was characterized in WT cells at 26°C and 37°C. (B) WT, ubc9-2, and cdc48-3 cells harboring GFP-Spt23 or GFP-Mga2 were grown in synthetic media supplemented with raffinose to an OD600 of 0.3. Protein expression was induced for 3 hr by the addition of galactose at either 26°C or 37°C. Localization was determined using confocal microscopy. (C) Percentage of cells in which GFP-Spt23 or GFP-Mga2 were localized to the nucleus in WT, ubc9-2, and cdc48-3 strains at 37°C (∗p < 0.001). Data represent the mean ± standard deviation from four independent experiments. (D) Processing of GFP-Mga2 is not affected by defects in the SUMO pathway. GFP-Mga2 processing was evaluated at 26°C or 37°C in WT and ubc9-2 strains by western blotting, using anti-GFP antiserum. Molecular Cell 2009 33, 124-135DOI: (10.1016/j.molcel.2008.12.025) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 The SUMO Pathway and DNA Replication and Repair (A and B) SGA subnetworks highlighting genetic interactions between SUMO pathway mutants and genes involved in DNA replication and repair. (C) Proteins involved in DNA replication and repair that interact with SUMO (SUMO interactors) or are directly modified by SUMO (SUMO conjugates). Proteins highlighted in blue appear in more than one screen. (D and E) Dna2p subcellular distribution is dependent upon an intact SUMO pathway. Dna2-GFP and Rad52-YFP localization were visualized in WT and ubc9-2 backgrounds at 26°C and 37°C by confocal microscopy. Molecular Cell 2009 33, 124-135DOI: (10.1016/j.molcel.2008.12.025) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 The SUMO Pathway Is Required for DNA-Damage Checkpoint Recovery (A) DNA-damage checkpoint signaling in yeast in response to defects in the SUMO system. (B) Viability of the ubc9-2 strain does not depend upon functional DDC. 10 × serial dilutions of log-phase cells of the indicated genotypes were spotted onto YPD plates and incubated at 30° or 32°C for 2 days. (C) Rad53p is phosphorylated in a Rad9p-dependent manner when the SUMO pathway is compromised. Rad53p phosphorylation in whole-cell lysates prepared from log-phase cells of the indicated genotypes at 26°C or 32°C was analyzed by western blotting, using Rad53p-specific antibodies. (D) Experimental system for the analysis of recovery from DDC. The YMV2 strain was engineered by deletion of HO endonuclease cleavage sites at MAT and HML(R), and insertion of an HO cleavage site at the LEU2 ORF (leu2::cs). A partial duplication of LEU2 was inserted 30 kb away. Gal-induced HO endonuclease creates a DSB in the LEU2 locus. Processive 5′ to 3′ resection produces ssDNA and reaches the region of homology to LEU2, 30 kb from the HO cleavage site. Single-strand annealing (SSA) occurs when this homology is used to anneal the two DNA strands. After successful SSA, the DNA-damage checkpoint is inactivated. P1 and P2 are primers used for PCR analysis. (E) Rad53p dephosphorylation is delayed in the ubc9-2 mutant. Rad53p phosphorylation was evaluated in the WT (YMV2) and YMV2 ubc9-2 mutant at the indicated time points by western blotting using Rad53p-specific antibodies. (F) DSB repair by SSA is effected at similar rates in the WT and ubc9-2 strains. Molecular Cell 2009 33, 124-135DOI: (10.1016/j.molcel.2008.12.025) Copyright © 2009 Elsevier Inc. Terms and Conditions