Analysis of SUMOylation in the RENT Complex by Fusion to a SUMO-Specific Protease Domain

Methods Mol Biol. 2017;1505:97-117. doi: 10.1007/978-1-4939-6502-1_9.

Abstract

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.

Keywords: Budding yeast; Cdc14; Net1; RENT; SUMO; Sir2; Ulp1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • Cloning, Molecular / methods
  • Cysteine Endopeptidases / genetics
  • Cysteine Endopeptidases / metabolism*
  • DNA, Fungal / genetics
  • DNA, Fungal / isolation & purification
  • Mutagenesis, Site-Directed / methods
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Polymerase Chain Reaction / methods
  • Protein Tyrosine Phosphatases / genetics
  • Protein Tyrosine Phosphatases / metabolism*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Silent Information Regulator Proteins, Saccharomyces cerevisiae / genetics
  • Silent Information Regulator Proteins, Saccharomyces cerevisiae / metabolism*
  • Sirtuin 2 / genetics
  • Sirtuin 2 / metabolism*
  • Small Ubiquitin-Related Modifier Proteins / genetics
  • Small Ubiquitin-Related Modifier Proteins / metabolism*
  • Sumoylation
  • Transformation, Genetic

Substances

  • CDC14 protein, S cerevisiae
  • Cell Cycle Proteins
  • DNA, Fungal
  • Net1 protein, S cerevisiae
  • Nuclear Proteins
  • Saccharomyces cerevisiae Proteins
  • Silent Information Regulator Proteins, Saccharomyces cerevisiae
  • Small Ubiquitin-Related Modifier Proteins
  • Protein Tyrosine Phosphatases
  • Cysteine Endopeptidases
  • Ulp1 protease
  • SIR2 protein, S cerevisiae
  • Sirtuin 2