An Optogenetic Approach to Dynamically Study Membrane Confinement of Prestin

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The membrane protein prestin plays a central role in the mammalian auditory system by enabling outer hair cells in the cochlea to actively respond to electrical signals. Prestin belongs to the SLC26A family of membrane proteins, which all have a large cytosolic terminus containing a conserved region known as the STAS domain (Sulfate Transporters and Anti-Sigma factor antagonist), whose function remains unknown. We hypothesize that molecular interactions of the STAS domain are responsible for confining the lateral mobility of prestin. To test this hypothesis, we designed prestin constructs inserted with a Tobacco Etch Virus (TEV) protease recognition linker (tevS) into different sites, including the interface between the last transmembrane domain and the STAS domain, the disordered region from 563aa to 637aa, and after the STAS domain, and then tested for membrane expression and prestin function. We subsequently employed a PhyB-PIF6 dimerization system and a split-TEV strategy to regulate TEV protease activity via exposure to red and far red light. We found that the interface between the last transmembrane domain and cytoplasmic C-terminus is critical for molecular interactions and for prestin's functionality. We also inserted tevS between amino acids 596/597 and 620/621 in prestin; these modifications retained wild type membrane expression and function, and both constructs were successfully cleaved by the TEV protease. However, the 596/597 cleaved C-terminal tail localized near the membrane, whereas the 620/621 cleaved tail diffused into the cytosol, therefore suggesting the presence of a potential binding site between amino acids 597 and 620 in prestin. This work demonstrates the advantages of employing optogenetic approaches to dynamically study the role of the STAS domain in prestin's function and membrane organization.