Translational Control of Membrane Proteins Figure 5. Membrane localization, cell surface expression and orientation of integral membrane proteins expressed with the LE leader. (A) Over-expressed LE tagged proteins are localized to the membrane fraction in sucrose density gradient centrifugation. The insoluble non- membrane protein EGFL7 has been included as a control. Each lane represents fractions removed from the sucrose gradient and analyzed by SDS- PAGE anti-his immunoblot. (B) Orientation in the E. coli membrane was established by creating two constructs of EG-VEGFR1 with a FLAG-tag epitope at either the N or C terminus. {I} GPCR transmembrane proteins are known to have an exposed N-terminus on the surface of cells. {II} EG-VEGFR1 constructs containing the FLAG-tag epitope at either the N or C terminus express at similar levels in E. coli as confirmed by anti-his immunoblot of whole cell lysates. {III} Only spheroplasts of cells expressing the FLAG tag epitope at the exposed N-terminus were able to pull-down anti-FLAG antibody as detected by anti-murine IgG HRP conjugated antibodies. doi:10.1371/journal.pone.0035844.g005 antibody when placed at either the N or C terminus, we prepared E. coli membrane proteoliposomes where both sides of the membrane are accessible. In each case, approximately equivalent amounts of FLAG antibody were recovered by immuno- precipitation (Figure S4) Addition of the detergent Triton X-100 did not further enhance accessibility of the FLAG tag, as might be expected. Cell membrane expression of CD20 To confirm native folding and membrane expression of human CD20 in a cellular context, spheroplasts of E. coli expressing CD20 were evaluated by fluorescent activated cell sorting (FACS) [32] using the conformation dependent antibody rituximab [33]. The second of the two extracellular loops of CD20 is the binding site for rituximab and this interaction is strongly dependent on the native conformation stabilized by a disulfide bond. FACS analysis showed a large shift in mean fluorescent intensity for CD20 expressing cells as compared to control cells shown in figure 6. The data is consistent with localization of CD20 to the cytoplasmic membrane and correct native like folding of the second exracellular loop of CD20. PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e35844