DETERMINING THE MEMBRANE TOPOLOGY OF TRANSMEMBRANE O-METHYLTRANSFERASE IN ZEBRAFISH HAIR CELLS USING A SPLIT GFP SYSTEM

Abstract

Mechanosensory hair cells are used by the auditory and vestibular systems to convert mechanical vibrations into electrical signals. Given their specialized function and morphology, hair cells express a unique set of genes for which, in some cases, little is known about their protein function. One such gene is called transmembrane o-methyltransferase (tomt). Mutations in tomt have shown it causes non-syndromic deafness in humans, mice, and zebrafish. Tomt is a transmembrane protein localized in the hair cell secretory pathway where it facilitates the trafficking of Tmc1 and Tmc2, which are pore-forming subunits of the hair cell mechano-electrical transduction channel. However, the membrane topology and functional domains of Tomt have not been clearly defined. We will experimentally determine the membrane topology of Tomt in zebrafish hair cells using the split GFP system.

Green fluorescent protein (GFP) is an eleven-stranded beta-barrel that emits green light when excited by blue light photons. The GFP protein can be split into self-assembling fragments consisting of beta strands 1-10 and beta strand 11. The two parts can reconstitute the GFP chromophore. Transgenic lines of fish expressing GFP1-10 in either the cytoplasm or the lumen of the endoplasmic reticulum were created. We tested the functionality of the cytoplasmic and luminal GFP1-10 lines by adding C-terminal GFP11 tags to proteins of known topology or subcellular localization. If the GFP11 tag is localized to the same subcellular compartment as the GFP1-10, then we expect to observe GFP fluorescence. After confirming that the cytoplasmic and luminal GFP1-10 transgenes function as expected, Tomt can be assayed. The GFP11 tag will be fused to the Tomt C-terminus and injected into transgenic fish expressing cytoplasmic or luminal GFP1-10. If Tomt is a type I (C-terminus in the cytoplasm) protein, then we should observe reconstituted GFP only in the cytoplasmic GFP1-10 fish. Conversely, if TOMT is a type II (C-terminus in the lumen) protein, then we expect to see reconstituted GFP in the luminal GFP1-10 line only. Determining the topology of Tomt will help to define its functional domains and provide information on how Tomt facilitates TMC protein folding or trafficking.