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.

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