Nucleic Acids Research Advance Access originally published online on November 5, 2007
Nucleic Acids Research 2007 35(21):e142; doi:10.1093/nar/gkm932
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Nucleic Acids Research, 2007, Vol. 35, No. 21 e142
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
A yeast two-hybrid system reconstituting substrate recognition of the von Hippel-Lindau tumor suppressor protein
Max Planck Institute of Biochemistry, Department of Molecular Cell Biology, Am Klopferspitz 18, 82152 Martinsried, Germany
*To whom correspondence should be addressed. Tel: +49 89 8578 3050; Fax: +49 89 8578 3022; Email: buchberg{at}biochem.mpg.de
Received June 22, 2007. Revised October 10, 2007. Accepted October 11, 2007.
The von Hippel-Lindau tumor suppressor protein (pVHL) is inactivated in the hereditary cancer syndrome von Hippel-Lindau disease and in the majority of sporadic renal carcinomas. pVHL is the substrate-binding subunit of the CBCVHL ubiquitin ligase complex that negatively regulates cell growth by promoting the degradation of hypoxia-inducible transcription factor subunits (HIF1/2
). Proteomics-based identification of novel pVHL substrates is hampered by their short half-life and low abundancy in mammalian cells. The usefulness of yeast two-hybrid (Y2H) approaches, on the other hand, has been limited by the failure of pVHL to adopt its native structure and by the absence of prolylhydroxylase activity critical for pVHL substrate recognition. Therefore, we modified the Y2H system to faithfully reconstitute the physical interaction between pVHL and its substrates. Our approach relies on the coexpression of pVHL with the cofactors Elongin B and Elongin C and with HIF1/2 prolylhydroxylases. In a proof-of-principle Y2H screen, we identified the known substrates HIF1/2 and new candidate substrates including diacylglycerol kinase iota, demonstrating that our strategy allows detection of stable interactions between pVHL and otherwise elusive cellular targets. Additional future applications may include structure/function analyses of pVHL-HIF1/2 binding and screens for therapeutically relevant compounds that either stabilize or disrupt this interaction.
Present addresses: Claudia Bex, Department of Biological Sciences, Imperial College London, Imperial College Road, London SW7 2AZ, UK.
Katja Knauth, Institute of Biochemistry, Swiss Federal Institute of Technology Zurich, Schafmattstrasse 18, CH-8093 Zurich, Switzerland.