We describe a method that detects proteins capable of interacting with a known protein and that results in the immediate availability of the cloned genes for these interacting proteins. Plasmids are constructed to encode two hybrid proteins. One hybrid consists of the DNA-binding domain of the yeast transcriptional activator protein GAL4 fused to the known protein; the other hybrid consists of the GAL4 activation domain fused to protein sequences encoded by a library of yeast genomic DNA fragments. Interaction between the known protein and a protein encoded by one of the library plasmids leads to transcriptional activation of a reporter gene containing a binding site for GAL4. We used this method with the yeast SIR4 protein, which is involved in the trinscriptional repression of yeast mating type information. (a) We used the twohybrid system to demonstrate that SIR4 can form homodimers.(ii) A small domain consisting of the C terminus of SIR4 was shown to be sufficient to mediate this interaction. (iii) We screened a library to detect hybrid proteins that could interact with the SIR4 C-terminal domain and identified SIR4 from this library. This approach could be readily extended to mammalian proteins by the construction of appropriate cDNA libraries in the activation domain plasmid.Specific interactions between proteins form the basis of many essential biological processes. Additionally, transforming proteins of tumor viruses in many cases exert their effect through their interactions with cellular proteins; for example, the simian virus 40 (SV40) large tumor (T) antigen binds to the cellular proteins p53 and Rb (1, 2). Consequently, considerable effort has been made to identify those proteins that bind to proteins of interest. Typically, these interactions have been detected by using coimmunoprecipitation experiments in which antibody to a known protein is used to precipitate associated proteins as well. Such biochemical methods, however, result only in the identification ofthe apparent molecular mass of the associated proteins; obtaining cloned genes for these proteins is often a difficult process. In one approach, this problem has been circumvented by the use ofpurified proteins as probes against bacterial expression libraries, where a positive signal for an interacting protein is accompanied by the availability of the corresponding gene (3).We have described a method by which a protein-protein interaction is identified in vivo through reconstitution of the activity ofa transcriptional activator (4). The method is based on the properties of the yeast GAL4 protein, which consists of separable domains responsible for DNA-binding and transcriptional activation (5). Plasmids encoding two hybrid proteins, one consisting of the GAL4 DNA-binding domain fused to protein X and the other consisting of the GAL4 activation domain fused to protein Y, are constructed and introduced into yeast. Interaction between proteins X and Y leads to the transcriptional activation of a reporter gene containing a binding site fo...

The Ci/Gli family of transcription factors mediates Hedgehog (Hh) signaling in many key developmental processes. Here we identify a Hh-induced MATH and BTB domain containing protein (HIB) as a negative regulator of the Hh pathway. Overexpressing HIB down regulates Ci and blocks Hh signaling, whereas inactivating HIB results in Ci accumulation and enhanced pathway activity. HIB binds the N- and C-terminal regions of Ci, both of which mediate Ci degradation. HIB forms a complex with Cul3, a scaffold for modular ubiquitin ligases, and promotes Ci ubiquitination and degradation through Cul3. Furthermore, HIB-mediated Ci degradation is stimulated by Hh and inhibited by Suppressor of Fused (Sufu). The mammalian homolog of HIB, SPOP, can functionally substitute for HIB, and Gli proteins are degraded by HIB/SPOP in Drosophila. We provide evidence that HIB prevents aberrant Hh signaling posterior to the morphogenic furrow, which is essential for normal eye development.

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