Studying interactions of four proteins in the yeast two-hybrid system: structural resemblance of the pVHL/elongin BC/hCUL-2 complex with the ubiquitin ligase complex SKP1/cullin/F-box protein.
The yeast two-hybrid system is a powerful technique that detects interactions between two proteins and has been useful in identifying new binding partners. However, the system fails to detect protein-protein interactions that require the presence of additional components of a multisubunit complex. Here we demonstrate that the vector YIpDCE1 can ... be used to express elongins B and C in yeast, and that these proteins form a stable complex that interacts with the von Hippel-Lindau tumor-suppressor gene product (pVHL). Only when pVHL and elongins B and C (VBC) are present does an interaction with the cullin family member, hCUL-2, occur, forming the heterotetrameric pVHL/elongin BC/hCUL-2 complex. This system was then used to map the binding region of hCUL-2 for the VBC complex. The first amino-terminal 108 aa of hCUL-2 are necessary for interaction with the VBC complex. The elongin BC dimer acts as a bridge between pVHL and hCUL-2 because pVHL and hCUL-2 can form distinct complexes with elongins B and C. These results reveal a striking structural resemblance of pVHL/elongin BC/hCUL-2 complex with the E3-like ubiquitin ligase complex SKP1/Cullin/F-box protein with respect to protein composition and sites of interactions. Thus, it seems possible that pVHL/elongin BC/hCUL-2 complex will possess ubiquitin ligase activity targeting specific proteins for degradation by the proteasome.
Mesh Terms:
Binding Sites, Carrier Proteins, Cell Cycle Proteins, Cullin Proteins, Genetic Vectors, Ligases, Peptide Mapping, Peptide Synthases, Polymerase Chain Reaction, Protein Binding, Proteins, SKP Cullin F-Box Protein Ligases, Schizosaccharomyces, Transcription Factors, Tumor Suppressor Proteins, Ubiquitin-Protein Ligases, Von Hippel-Lindau Tumor Suppressor Protein
Binding Sites, Carrier Proteins, Cell Cycle Proteins, Cullin Proteins, Genetic Vectors, Ligases, Peptide Mapping, Peptide Synthases, Polymerase Chain Reaction, Protein Binding, Proteins, SKP Cullin F-Box Protein Ligases, Schizosaccharomyces, Transcription Factors, Tumor Suppressor Proteins, Ubiquitin-Protein Ligases, Von Hippel-Lindau Tumor Suppressor Protein
Proc. Natl. Acad. Sci. U.S.A.
Date: Aug. 17, 1999
PubMed ID: 10449727
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