Modular assembly of synthetic proteins that span the plasma membrane in mammalian cells

BACKGROUND: To achieve synthetic control over how a cell responds to other cells or the extracellular environment, it is important to reliably engineer proteins that can traffic and span the plasma membrane. Using a modular approach to assemble proteins, we identified the minimum necessary components required to engineer such membrane-spanning proteins with predictable orientation in mammalian cells. RESULTS: While a transmembrane domain (TM) fused to the N-terminus of a protein is sufficient to traffic it to the endoplasmic reticulum (ER), an additional signal peptidase cleavage site downstream of this TM enhanced sorting out of the ER. Next, a second TM in the synthetic protein helped anchor and accumulate the membrane-spanning protein on the plasma membrane. The orientation of the components of the synthetic protein were determined through measuring intracellular Ca(2+) signaling using the R-GECO biosensor and through measuring extracellular quenching of yellow fluorescent protein variants by saturating acidic and salt conditions. CONCLUSIONS: This work forms the basis of engineering novel proteins that span the plasma membrane to potentially control intracellular responses to extracellular conditions. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12896-016-0320-7) contains supplementary material, which is available to authorized users.