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.
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Molecular Oncology Group, Max Planck Institute for Biochemistry, 82152 Martinsried, Germany.
Peterson
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Schaffar
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Stearman
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Klausner
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D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D044842:Cullin Proteins; D000075065:Elongin; D005822:Genetic Vectors; D008025:Ligases; D010449:Peptide Mapping; D010453:Peptide Synthases / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D044843:SKP Cullin F-Box Protein Ligases; D012568:Schizosaccharomyces; D014157:Transcription Factors / Q000378:metabolism*; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein
C105153:CUL2 protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D044842:Cullin Proteins; D000075065:Elongin; D011506:Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D008025:Ligases; D010453:Peptide Synthases