Molecular Determinants of TMC Protein Biogenesis and Trafficking.

Transmembrane channel-like (TMC) proteins are essential for hearing and balance; however, the molecular mechanisms that regulate their proper folding and membrane targeting remain poorly understood. Here, we establish as a genetically tractable model to dissect TMC-1 trafficking by combining CRISPR knock-in strains, super-resolution microscopy, and genome-wide forward genetic screening. We show that TMC-1 robustly localizes to the plasma membrane in both neurons and muscle cells and identify a conserved valine (V803) in transmembrane domain 9 (TM9) as critical for its biogenesis and trafficking. Structural analyses guided by AlphaMissense and AlphaFold uncover two evolutionarily conserved functional hotspots, one in the extracellular loop adjacent to TM9 and the other in the TMC signature motif, which are interconnected by an evolutionarily conserved disulfide bond. Disrupting this bond in worm TMC-1 abolishes its cell-surface localization and destabilizes the mechanotransduction channel complex. Together, these findings provide a structural framework for interpreting deafness-causing mutations in human TMC1 and highlight disulfide-bond-linked hotspots as key molecular determinants of TMC protein biogenesis and trafficking.