Back to article: Identification of SUMO conjugation sites in the budding yeast proteome

FIGURE 2: SUMO-acceptor lysines detected in our mass spectrometry analysis were compared with SUMO-acceptor lysines already described in previous studies done in S. cerevisiae. (A) Previous studies were based on either MS or site-directed muta­genesis/immunoblotting, or a combination of both. Most of the SUMO-acceptor lysines previously found were also detected in this study. SUMO substrates (total of 257) identified in the yeast proteome of cells grown asynchronously. Previously published SUMO-substrates were obtained [8,15-21]. (B) To ensure that diglycine-modified lysines detected by mass spectrometry after Smt3 purification are not due to modification by ubiquitin or Nedd8, a centromeric plasmid 8His-SMT3-KallR-REQIGG-pRS415 expressing the Smt3 variant used previously for the Smt3 purification protocol with the difference that the RGG conjugating terminus was replace for the native RIEQGG C-terminus was employed. SUMO-acceptor lysines modified by the 8His-Smt3-KallR-REQIGG keep a side chain of 5 aa after trypsin digestion (EQIGG). Therefore, any diglycine-modified lysines detected by mass spectrometry under these conditions can only be due to either false positive hits, or to ubiquitinated or neddylated contaminants. A large culture of 9 l of the strain expressing the 8His-SMT3-KallR-REQIGG variant was grown in YPD and harvested at O.D. 0.9. Smt3 purification and mass spectrometry analysis was performed as described in Material and Methods. We detected 23 diglycine-modified lysines. None of these corresponded with previously detected diglycine-modified lysines in our Smt3-RGG pulldowns. In addition none of these diglycine-modified lysines are within SUMO consensus sequences. This strongly indicates that sumo-acceptor lysines identified after purification of Smt3-RGG pulldowns represent bona fide SUMOylation sites.

8. Johnson ES, Blobel G (1999). Cell cycle-regulated attachment of the ubiquitin-related protein SUMO to the yeast septins. (J Cell Biol 147(5): 981-994.
15. Albuquerque CP, Yeung E, Ma S, Fu T, Corbett KD, Zhou H (2015). A Chemical and Enzymatic Approach to Study Site-Specific Sumoylation. (PLoS One 10(12): e0143810.
16. Denison C, Rudner AD, Gerber SA, Bakalarski CE, Moazed D, Gygi SP (2005). A proteomic strategy for gaining insights into protein sumoylation in yeast. (Mol Cell Proteomics 4(3): 246-254.
17. Zhou W, Ryan JJ, Zhou H (2004). Global analyses of sumoylated proteins in Saccharomyces cerevisiae. Induction of protein sumoylation by cellular stresses. ( J Biol Chem 279(31): 32262-32268.
18. Silver HR, Nissley JA, Reed SH, Hou YM, Johnson ES (2011). A role for SUMO in nucleotide excision repair. (DNA Repair (Amst) 10(12): 1243-1251.
19. Chen XL, Silver HR, Xiong L, Belichenko I, Adegite C, Johnson ES (2007). Topoisomerase I-dependent viability loss in Saccharomyces cerevisiae mutants defective in both SUMO conjugation and DNA repair. (Genetics 177(1): 17-30.
20. Nathan D, Ingvarsdottir K, Sterner DE, Bylebyl GR, Dokmanovic M, Dorsey JA, Whelan KA, Krsmanovic M, Lane WS, Meluh PB, Johnson ES, Berger SL (2006). Histone sumoylation is a negative regulator in Saccharomyces cerevisiae and shows dynamic interplay with positive-acting histone modifications. (Genes Dev 20(8): 966-976.
21. Sacher M, Pfander B, Hoege C, Jentsch S (2006). Control of Rad52 recombination activity by double-strand break-induced SUMO modification. (Nat Cell Biol 8(11): 1284-1290.

By continuing to use the site, you agree to the use of cookies. more information

The cookie settings on this website are set to "allow cookies" to give you the best browsing experience possible. If you continue to use this website without changing your cookie settings or you click "Accept" below then you are consenting to this. Please refer to our "privacy statement" and our "terms of use" for further information.