ABSTRACT: BACKGROUND: In the yeast Saccharomyces cerevisiae, the essential SUMO protease Ulp1 is responsible for both removing SUMO/Smt3 (small ubiquitin-like modifier) from specific target proteins and for processing precursor SUMO into its conjugation-competent form. Ulp1 localizes predominantly to nuclear pore complexes (NPCs) but has also been shown to deconjugate sumoylated septins at the bud neck of dividing cells. How Ulp1 is directed to bud-neck localized septins and other cytoplasmic deconjugation targets is not well understood. RESULTS: Using a structure/function approach, we set out to elucidate features of Ulp1 that are required for substrate targeting. To aid our studies, we took advantage of a novel, catalytically inactive mutant of Ulp1 that is greatly enriched at the septin ring of dividing yeast cells. We found that the localization of Ulp1 to the septins requires both SUMO and specific structural features of Ulp1's catalytic domain. Our analysis identified a 218 amino acid-long, substrate-trapping mutant of the catalytic domain of Ulp1, Ulp1(3)(C580S), that is necessary and sufficient for septin localization. We also used the targeting and SUMO-binding properties of Ulp1(3)(C580S) to purify Smt3-modified proteins from cell extracts. CONCLUSIONS: In summary, our study provides novel insights into how the Ulp1 SUMO protease is actively targeted to its substrates in vivo and in vitro. Furthermore, we find that a substrate-trapping Ulp1(3)(C580S) interacts robustly with human SUMO1, SUMO2, and SUMO2 chains, making it a potentially useful tool for the analysis and purification of SUMO-modified proteins.
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|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
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