Studying Protein Complexes by the Yeast Two-Hybrid System

Methods. 2012 Dec;58(4):392-9. doi: 10.1016/j.ymeth.2012.07.015. Epub 2012 Jul 24.


Protein complexes are typically analyzed by affinity purification and subsequent mass spectrometric analysis. However, in most cases the structure and topology of the complexes remains elusive from such studies. Here we investigate how the yeast two-hybrid system can be used to analyze direct interactions among proteins in a complex. First we tested all pairwise interactions among the seven proteins of Escherichia coli DNA polymerase III as well as an uncharacterized complex that includes MntR and PerR. Four and seven interactions were identified in these two complexes, respectively. In addition, we review Y2H data for three other complexes of known structure which serve as "gold-standards", namely Varicella Zoster Virus (VZV) ribonucleotide reductase (RNR), the yeast proteasome, and bacteriophage lambda. Finally, we review an Y2H analysis of the human spliceosome which may serve as an example for a dynamic mega-complex.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Bacteriophage lambda / metabolism
  • Caenorhabditis elegans Proteins / metabolism
  • Crystallization
  • DNA Polymerase III / metabolism
  • Escherichia coli / enzymology
  • Escherichia coli Proteins / metabolism
  • Herpesvirus 3, Human / enzymology
  • Humans
  • Models, Molecular
  • Proteasome Endopeptidase Complex / metabolism
  • Protein Interaction Mapping
  • Protein Interaction Maps
  • Protein Structure, Quaternary
  • Protein Subunits / metabolism
  • Reference Standards
  • Repressor Proteins / metabolism
  • Ribonucleotide Reductases / chemistry
  • Ribonucleotide Reductases / metabolism
  • Spliceosomes / metabolism
  • Two-Hybrid System Techniques / standards*
  • Viral Proteins / chemistry
  • Viral Proteins / metabolism


  • Caenorhabditis elegans Proteins
  • Escherichia coli Proteins
  • MntR protein, E coli
  • Protein Subunits
  • Repressor Proteins
  • Viral Proteins
  • Ribonucleotide Reductases
  • DNA Polymerase III
  • Proteasome Endopeptidase Complex