Analysis of SUMOylation in the RENT Complex by Fusion to a SUMO-Specific Protease Domain.
Protein sumoylation is a reversible posttranslational modification that controls multiple processes during cell cycle progression. Frequently, SUMO synergistically targets various subunits in a protein complex to modulate its function, leading to what has been defined as protein group sumoylation. Different subunits in the RENT (regulator of nucleolar silencing and telophase) complex, including Net1, Sir2, and Cdc14, can be coupled to SUMO, making it difficult to ascertain the role of this modification. Here we describe a method to downregulate sumoylation in RENT, consisting in the fusion of a catalytic domain of the Ulp1 SUMO protease (Ulp Domain; UD) to the C-terminus of members in the complex using epitope tags as linkers. Targeting of the UD to specific loci can be simplified by transformation of PCR-amplified cassettes. The presence of the UD in the complex allows the concurrent downregulation of sumoylated species in the RENT complex, what can be easily monitored by pull-down of SUMO conjugates. This methodology can be applied to other protein complexes exhibiting group sumoylation.
IRBLLEIDA, Dept. Ciències Mèdiques Bàsiques, Universitat de Lleida, Av. Alcalde Rovira Roure 80w, Lleida, 25198, Spain.
Neus
N
Colomina
IRBLLEIDA, Dept. Ciències Mèdiques Bàsiques, Universitat de Lleida, Av. Alcalde Rovira Roure 80w, Lleida, 25198, Spain.
Clàudia
C
Guasch
IRBLLEIDA, Dept. Ciències Mèdiques Bàsiques, Universitat de Lleida, Av. Alcalde Rovira Roure 80w, Lleida, 25198, Spain. jordi.torres@cmb.udl.cat.
Jordi
J
Torres-Rosell
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D056565:Sirtuin 2 / Q000235:genetics / Q000378:metabolism*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000378:metabolism*; D058207:Sumoylation; D014170:Transformation, Genetic
C074860:CDC14 protein, S cerevisiae; D018797:Cell Cycle Proteins; D004271:DNA, Fungal; C119300:Net1 protein, S cerevisiae; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D025841:Small Ubiquitin-Related Modifier Proteins; D017027:Protein Tyrosine Phosphatases; D003546:Cysteine Endopeptidases; C118083:Ulp1 protease; C087778:SIR2 protein, S cerevisiae; D056565:Sirtuin 2
Budding yeast; Cdc14; Net1; RENT; SUMO; Sir2; Ulp1