Heterologous expression and cell membrane localization of dinoflagellate opsins (rhodopsin proteins) in mammalian cells

Rhodopsins are now found in all domains of life,and are classified into two large groups: type II,found in animals and type I found in microbes including Bacteria,Archaea,and Eukarya. While type II rhodopsin functions in many photodependent signaling processes including vision,type I among others contains rhodopsins that function as a light-driven proton pump to convert light into ATP as in proteobacteria (named proteorhodopsin). Proteorhodopsin homologs have been documented in dinoflagellates,but their subcellular localizations and functions are still poorly understood. Even though sequence analyses suggest that it is a membrane protein,experimental evidence that dinoflagellate rhodopsins are localized on the plasma membrane or endomembranes is still lacking. As no robust dinoflagellate gene transformation tool was available,we used HEK 293T cells to construct a mammalian expression system for two dinoflagellate rhodopsin genes. The success of expressing these genes in the system shows that this mammalian cell type is suitable for expressing dinoflagellate genes. Immunofluorescence of the expressed protein locates these dinoflagellate rhodopsins on the cell membrane. This result indicates that the protein codons and membrane targeting signal of the dinoflagellate genes are compatible with the mammalian cells,and the proteins' subcellular localization is consistent with proton pump rhodopsins.