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

TLDR

SUMO (Smt3) is a ubiquitin‑like modifier that attaches to many proteins in yeast, with cell‑cycle‑regulated conjugation essential for viability, yet only a limited number of substrates have been definitively identified, hindering mechanistic studies. The study seeks to comprehensively identify SUMO‑modified proteins in Saccharomyces cerevisiae. To achieve this, the authors combined yeast two‑hybrid screening, a high‑copy suppressor selection using a SUMO isopeptidase mutant, and tandem mass spectrometry. These complementary approaches identified over 150 SUMO substrates, with mass spectrometry yielding the largest set, revealing a potential SUMO‑binding motif from two‑hybrid data, and highlighting diverse functional categories—including conjugation enzymes, chromatin regulators, DNA repair factors, stress proteins, transcription factors, RNA metabolism proteins, and metabolic enzymes—demonstrating that SUMO conjugation broadly regulates cellular processes.

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.