Processing and localization of the mechanotransduction channel subunit TMC1 - PROJECT SUMMARY/ABSTRACT Hearing loss is the most common sensory deficit, often arising from dysfunction of cochlear hair cells. Hair cells express unique protein complexes vital for auditory function, including the mechanotransduction (MET) channel, a heteromeric protein complex which converts mechanical sound waves into electrical signals. Proper expression, assembly, trafficking, and localization of MET proteins to stereocilia are critical for auditory function, yet there is a significant knowledge gap in the molecules and mechanisms that participate in these processes. Numerous forms of hereditary hearing loss are associated with deficits in MET protein localization, making the study of the underlying mechanisms highly significant. The overall goal of the project is to investigate the molecules and mechanisms that regulate processing and localization of MET channel proteins, and the effects of genetic mutations on these processes. TMC1 and TMC2 are pore-forming subunits of the MET channel, with TMC1 predominating throughout most of life. Recently, we discovered that Transmembrane-O-methyltransferase (TOMT) is critical for regulating TMC1 localization, MET channel activity, and auditory function. TOMT is the first molecule known to participate in MET protein localization that is not part of the MET complex. The precise mechanism by which TOMT regulates TMC1 remains unknown, but our preliminary results provide important insights. Specifically, although TOMT localizes to the endoplasmic reticulum (ER) of hair cells and is excluded from stereocilia, it interacts with and stabilizes TMC1. Critically, outside hair cells, TOMT is not sufficient for TMC1 ER exit, suggesting the involvement of unknown partners expressed in hair cells. Importantly, our preliminary studies have uncovered novel molecular chaperones (PEX3, 16, 19, referred to as PEX proteins) that are expressed in hair cells, and that interact with TOMT to enhance TMC1 expression and stability. Notably, mutations in PEX proteins cause hearing loss in peroxisomal biogenesis disorders. Based on our preliminary data, our central hypothesis is that TMC1 processing and localization is regulated by complex hair-cell-specific machinery, involving TOMT and PEX proteins, that is affected by deafness-linked mutations. Using multifaceted approaches that incorporate cutting-edge technologies, we will: 1) define mechanisms by which TOMT facilitates TMC1 ER exit by investigating how TOMT-TMC1 binding domains and deafness-linked mutations contribute to TMC1 stability, localization, and auditory function; and 2) characterize expression patterns and manipulate inner ear gene function of PEX proteins to interrogate the individual and synergistic roles of PEX proteins with TOMT in TMC1 processing and auditory function. Our findings will contribute to a new paradigm of auditory protein complex expression, assembly, trafficking, and localization. We will gain critical insights into the processing and localization of TMC1, a key auditory protein and the pore-forming subunit of the MET channel. By doing so, we anticipate uncovering novel molecules and mechanistic checkpoints to serve as therapeutic targets addressing hearing loss associated with defects in protein processing and/or localization.
