leaders was performed with the non-membrane His-tagged EGFL7, a protein which aggregates in the cytoplasm. Again, the Uni leader reveals a stronger translation rate than the LE leader (Figure 3B). However, if the CD20 culture is pulse-labeled for 5 minutes with 35S cysteine later in the induction (14 hours post IPTG addition), then the translation rate for the LE leader is much higher than that observed for the Uni leader (Figure 3B). Immediately following induction, translation from the Uni leader is higher than from the LE leader; however, the relative rates of the two leaders reverse over time. To examine this observation in more detail, the induction of CD20 or EG- VEGFR1 fused to each of the two leaders was repeated and samples were removed at numerous time points. These samples were analyzed by SDS-PAGE followed by anti-His western blotting to visualize CD20 and EG-VEGFR1 accumulation. As shown in figure 4A and B, the accumulation of both CD20 and EG-VEGFR1 reaches a maximum after approximately 30 min- utes with the Uni leader. By contrast, accumulation of both membrane proteins from the LE leader increased over several hours to outpace accumulation from the Uni leader. Leader amino acid sequence and size are not important To confirm that the translation initiation rate is the crucial variable in expression of these membrane proteins, the length of the LE leader was evaluated for effects on protein accumulation. A series of deletions at the C-terminus of the LE leader were created while preserving the TIR [29] in the first several codons. These constructs were fused to the N-terminus of CD20 and analyzed for their ability to accumulate protein. The results shown in figure 4C reveal that CD20 accumulation does modestly and gradually decrease with size. However, even at the smallest size of 10 amino acids, the accumulation of CD20 with the LE leader is significantly greater than that of the Uni leader (7 amino acids). This suggests that the core TIR of the leader is important for membrane protein accumulation presumably as a function of translational strength. The results imply that the weaker TIR of the LE leader allows continuous membrane protein accumulation over several hours of induction while the stronger TIR of the Uni leader produces an early overload and collapse of the membrane targeting system. Therefore, if transcription levels were equivalently reduced for both leaders, then overload of the membrane targeting system would be avoided and the Uni leader should surpass the LE leader in membrane protein accumulation. To test this hypothesis, cultures with either the Uni or the LE leader fused to CD20 under the control of the tphac promoter were induced by phosphate starvation for 16 hours. Without the addition of IPTG to remove the lac repressor control, a partial induction is achieved, leading to an equivalent drop in transcription/translation in each cell. The results shown in figure 4D bear out this prediction with the Uni leader, resulting in greater membrane protein accumulation than that observed with the LE leader and rule out anything special about their actual amino acid sequences. Membrane association of over-expressed proteins To ascertain the native like expression of proteins fused to the LE leader, the sub-cellular localization of the proteins was evaluated by equilibrium ultracentrifugation as previously de- scribed [15]. Correctly localized membrane proteins should migrate with the bacterial membranes to a density of less than 1.29 g/cm3 (1.75 M sucrose layer), while typical soluble or retractile body proteins, if present, lack membrane association and have a density between 1.33–1.42 g/cm3 and will migrate to the bottom of the sucrose gradient. The results shown in figure 5A indicate that all four LE tagged proteins migrate to above the Translational Control of Membrane Proteins Figure 4. Leader dependent accumulation of CD20 and EG- VEGFR1 following induction of the tphac promoter. (A) Protein accumulation maximizes within thirty minutes of induction with the Uni leader. (B) Protein accumulation continues over 20 hours after induction with the LE leader. (C) The effect of C-terminal truncations on expression from the LE leader. The full 79 amino acid LE leader was truncated from the C-terminus to observe the importance of the translation initiation rate as compared to the length of the LE leader. Truncated leaders were fused to CD20 and the whole cell lysates were immunoblotted with HRP conjugated anti-His antibody. Two film exposures are shown. (D) Reduced promoter activity results in reversal of the relative expression levels from the Uni and the LE leaders fused to CD20. Cultures were grown under partial promoter induction and the whole cell lysates were probed with HRP conjugated anti-His antibody for detection. doi:10.1371/journal.pone.0035844.g004 1.29 g/cm3 density layer, consistent with the 1.15–1.25 g/cm3 density of the E. coli membrane [30]. Correct membrane orientation To determine if the GPCRs with the LE leader are correctly oriented in the cytoplasmic membrane, a FLAG tag was added to either the N- or C- termini of EG-VEGFR1 and the extracellular localization of the FLAG tag was evaluated by immuno- precipitation of E. coli spheroplasts expressing either constructs [31]. These results shown in figure 5B-II demonstrate equivalent expression of both constructs. The FLAG antibody binds to LE- EG-VEGFR1 only when the FLAG tag is expressed at the N- terminus as predicted for a GPCR; however, no binding is observed for LE-EG-VEGFR1 with the FLAG tag at the C- terminus (Figure 5B-III). This indicates the FLAG tag is unavailable for binding, which is consistent with the cytoplasmic localization of EG-VEGFR1 C-terminus (Figure 5B-I). To confirm that the FLAG tag on LE-EG-VEGFR1 is equally accessible to PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e35844