A TWO-HYBRID SYSTEM FOR USE IN NON-DIVIDING YEAST CELL

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DESCRIPTION (Applicant's abstract): The ability to detect protein-protein interactions in living cells has been a critical component in the deve]opment of genomic analyses. The yeast two-hybrid system has been used to detect and study many types of interactions. e.g. between proteins, proteins and DNA, proteins and peptides etc. from many different organisms. This technology has the potential to contribute to human health in numerous ways, including providing a better understanding of cellular function, structure, and signal transduction leading to the identification of novel antibiotics and theraputics. Current two-hybrid technologies for detecting interacting proteins in yeast require that interactions take place in growing cells and that the interactions be stable enough to provide relatively constant transcriptional activation dunng the period of growth. As a result, interactions cannot be detected that occur only in non-dividing cells, require arrest-specific modifications of one or both partners, require other proteins that are produced only in non-growing cells, or are transient. Because most cells spend the majority of their time in non-dividing states and because novel interactions are known to occur dunng these times, the ability to detect interactions when cells are not dividing is critical for understanding the range of protein-protein interactions that occur in living cells. Although more difficult, the ability to detect transient interactions is also critical, especially for understanding processes such as signal transduction and metabolism. To address the limitations of the current technologies, we propose to develop a modified two-hybrid assay that will allow detection of molecular interactions, including those that occur only in non-dividing cells or are relatively transient. The regulatory and catalytic subunits of cAMP-dependent protein kinase, encoded by the BCY1 and TPK1-3 genes, respectively, will he used to test and refine this system. These proteins were selected because Bcylp and Tpklp interact with each other and are found in novel protein complexes in stationary-phase yeast cells. Also, the proteins that interact with Bcy Ip in quiescent cells have not been detected by classical two-hybrid analysis. Because Tpk catalytic subunits are known to interact transiently with a number of substrates, TPK genes will be used to determine whether the modified system can be adapted for detecting transient interactions. Once established for yeast, this method should be useful in a broad range of cell types to facilitate the genomic-scale analysis of protein-protein interactions during all phases of growth.