Sumo-dependent substrate targeting of the SUMO protease Ulp1.
BACKGROUND
In the yeast Saccharomyces cerevisiae, the essential small ubiquitin-like modifier (SUMO) protease Ulp1 is responsible for both removing SUMO/Smt3 from specific target proteins and for processing precursor SUMO into its conjugation-competent form. Ulp1 localizes predominantly to nuclear pore complexes 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 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, 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
Our study provides novel insights into how the Ulp1 SUMO protease is actively targeted to its substrates in vivo and in vitro. Furthermore, we found 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.
Biology Department, The College of William & Mary, ISC3047, 540 Landrum Drive, Williamsburg, VA 23185, USA.
Zachary C
ZC
Elmore
Megan
M
Donaher
Brooke C
BC
Matson
Helen
H
Murphy
Jason W
JW
Westerbeck
Oliver
O
Kerscher
D055162:Biocatalysis; D020134:Catalytic Domain; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D009685:Nuclear Envelope / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D058112:Septins / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D058207:Sumoylation; D054875:Ubiquitination
D026901:Membrane Transport Proteins; D050505:Mutant Proteins; C483822:PSE1 protein, S cerevisiae; D018160:Receptors, Cytoplasmic and Nuclear; D029701:Saccharomyces cerevisiae Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D003546:Cysteine Endopeptidases; C118083:Ulp1 protease; D058112:Septins