Parts-based assembly of synthetic transmembrane proteins in mammalian cells

Transmembrane proteins span cellular membranes such as the plasma membrane and endoplasmic reticulum (ER) membrane to mediate inter- and intracellular interactions. An N-terminal signal peptide and transmembrane helices facilitate recruitment to the ER and integration into the membrane, respectively. Using a parts-based assembly approach in this study, we confirm that the minimum requirement to create a transmembrane protein is indeed only a transmembrane helix (TM). When transfected in mammalian cells, our fusion proteins in the schematic form X-TM-Y were localized to vesicles, the golgi apparatus, the nuclear envelope, or the endoplasmic reticulum, consistent with ER targeting. Further studies to determine orientation showed that X was facing the cytoplasm, and Y the lumen. Lastly, in our fusion proteins with an N-terminal TM, the TM effectively reversed the orientation of X and Y. This knowledge can be applied to the parts-based engineering of synthetic transmembrane proteins with varied functions and biological applications.