Electromotility is a unique characteristic of the outer hair cells (OHC) of the mammalian cochlea. This electromotility is thought to underlie the exquisite frequency selectivity and sensitivity of mammalian hearing. Recently, we cloned the gene for the protein prestin, and have determined that prestin is the key molecule responsible for OHC electromotility. However, other proteins have been shown to modify prestin's activity, thereby affecting hearing function. Currently, the precise identities of these prestin-associated proteins (PAPs) are unknown, and the interactions between prestin and PAPs have not been described at the molecular level. Thus, the overall aim of our proposed research is to identify PAPs and to characterize their function. First, we propose to identify PAPs using a newly developed membrane-based yeast two-hybrid selective method (Aim I). An OHC-cDNA library suitable for that method will be built to increase the sensitivity and to decrease the false positive clones of library screening. Then, using prestin as "bait," we will use the yeast two-hybrid method to identify PAPs. Because of its highly sensitive nature, the yeast two-hybrid method is able to identify PAPs that can not be recognized by other methods. Second, we propose to identify PAPs using a second method: combined mass spectrometry analysis with prestin coimmunoprecipitation (Aim II). The major advantage of this method is its ability to identify physiologically relevant Prestin-PAP interactions that exist in vivo. Finally, after PAPs have been identified using either the two complementary methods or from other sources, we will determine whether those PAPs reside in OHCs in vivo, and will characterize the mechanisms by which PAPs modify prestin's function (Aim III). Taken together, these experiments will further our understanding of the molecular basis of OHC-based cochlear amplification, and potentially lead to identification of PAP-related deafness genes. This knowledge may make it possible to manipulate OHC function for therapeutic purposes.