FIGURE 1.

Organization of AraC and ToxR Fusions Used in TM Interaction Assays. In ToxR-based assays (12–14), constructs are configured with ToxR as an N-terminal fusion to a TM domain of interest, with mature MBP lacking its signal peptide (SP) fused at the C terminus. ToxR is a type II integral membrane protein (18), and therefore the TM domain of interest acts both as a signal peptide to direct membrane trafficking as well as membrane integration with the TM domain in a reverse orientation. In the AraTM assay, the C-terminal orientation of the DNA-binding domain within AraC enables constructs to be expressed with a N-terminal MBP fusion, which includes its native signal peptide to direct membrane integration. Thus, AraTM constructs are expressed in their native orientation, and membrane integration is decoupled from the specific sequence fused to MBP and AraC.