Defining the SUMO-modified Proteome by Multiple Approaches in Saccharomyces cerevisiae*

J Thomas Hannich,Alaron Lewis,Mary B Kroetz,Shyr-Jiann Li,Heinrich Heide,Andrew Emili,Mark Hochstrasser

doi:10.1074/jbc.m413209200

J Thomas Hannich, Alaron Lewis + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m413209200

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Abstract

SUMO, or Smt3 in Saccharomyces cerevisiae, is a ubiquitin-like protein that is post-translationally attached to multiple proteins in vivo. Many of these substrate modifications are cell cycle-regulated, and SUMO conjugation is essential for viability in most eukaryotes. However, only a limited number of SUMO-modified proteins have been definitively identified to date, and this has hampered study of the mechanisms by which SUMO ligation regulates specific cellular pathways. Here we use a combination of yeast two-hybrid screening, a high copy suppressor selection with a SUMO isopeptidase mutant, and tandem mass spectrometry to define a large set of proteins (>150) that can be modified by SUMO in budding yeast. These three approaches yielded overlapping sets of proteins with the most extensive set by far being those identified by mass spectrometry. The two-hybrid data also yielded a potential SUMO-binding motif. Functional categories of SUMO-modified proteins include SUMO conjugation system enzymes, chromatin- and gene silencing-related factors, DNA repair and genome stability proteins, stress-related proteins, transcription factors, proteins involved in translation and RNA metabolism, and a variety of metabolic enzymes. The results point to a surprisingly broad array of cellular processes regulated by SUMO conjugation and provide a starting point for detailed studies of how SUMO ligation contributes to these different regulatory mechanisms.

Highlights

SUMO, or Smt3 in Saccharomyces cerevisiae, is a ubiquitin-like protein that is post-translationally attached to multiple proteins in vivo
We use a combination of yeast two-hybrid screening, a high copy suppressor selection with a SUMO isopeptidase mutant, and tandem mass spectrometry to define a large set of proteins (>150) that can be modified by SUMO in budding yeast
The results point to a surprisingly broad array of cellular processes regulated by SUMO conjugation and provide a starting point for detailed studies of how SUMO ligation contributes to these different regulatory mechanisms

Summary

TABLE I Yeast strains

␣ his3-⌬200 leu112 lys801 trp ura3–52 ␣ his3-⌬200 leu112 lys801 trp ura ulp2⌬::HIS3 ␣ his3-⌬200 leu112 lys801 trp ura smt3⌬::HIS3 ulp1⌬::HIS3͓pVT102U-SMT3gg YCp50-ULP1͔ ␣ his3-⌬200 leu112 lys801 trp ura smt3⌬::HIS3 ulp1⌬::HIS3͓YRTAG310-H6-SMT3gg ␣ his3-⌬200 leu112 lys801 trp ura smt3⌬::HIS3 ulp⌬::HIS3͓YRTAG310-HFT-SMT3gga trp901 leu112 ura his3-⌬200 gal4⌬ gal80⌬ LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ. Yeast topoisomerase II is sumoylated in vivo, and this modification, while not required for the essential function of the enzyme, may contribute to the cohesive properties of the centromere [12]. SUMO is ligated to the DNA replication/repair protein PCNA (Pol30), and sumoylation inhibits the DNA repair activity of PCNA [13, 14]. All of these substrate proteins are essential for growth, in no case is their sumoylation required for viability. A much more comprehensive list of sumoylated proteins in this organism must first be obtained Toward this end, we have taken several independent genetic and proteomic approaches. The different methods of substrate identification have yielded overlapping but distinct groups of proteins, underscoring the value of using multiple approaches to establish the SUMO proteome

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

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