Figure 1
From: Cell-free expression tools to study co-translational folding of alpha helical membrane transporters
The MFS transporters LacY and XylE spontaneously insert into liposomes when expressed cell-free. (a) Crystal structures of LacY and XylE (PDB codes 2V8N64 and 4GBY65). Both have the same two domains of 6 helices, XylE has an additional cytoplasmic domain important for function (coloured red). (b) Schematic of sucrose flotation. The cell-free reaction is mixed with 60% (w/v) sucrose and spun at 200,000 g. The liposomes containing inserted protein float to the buffer:30% sucrose interface, aggregated/misfolded/non-inserted protein and the cell-free kit components remain in the bottom fraction. (c) LacY and XylE were expressed cell-free in the presence of 25:50:25 DOPC:DOPE:DOPG liposomes and a sucrose gradient performed as described in (b). The top (inserted) and bottom (aggregated) fractions were quantified via liquid scintillation counting of incorporated [35S] methionine, and the efficiency of insertion calculated by expressing the % of protein in the top fraction compared to the total protein synthesised in the cell-free reaction. LacY inserted with a yield of 29% and XylE with a yield of 38%. (d) SDS-PAGE of the top (inserted) and bottom (aggregated) fractions of a sucrose gradient following cell-free expression. The gels are from two different experiments; LacY was detected via an anti-HA tag antibody, XylE by phosphorimaging of incorporated [35S] methionine. (e) LacY and XylE were made via cell-free expression in the presence of 25:50:25 DOPC:DOPE:DOPG liposomes. Following flotation on a sucrose gradient, LacY was incubated at 25 °C with 20 ng of thermolysin in 40 mM HEPES-KOH pH 7.6 for 5 or 30 min, either with or without 2% DDM to solubilise the liposomes. XylE was incubated overnight at 4 °C with 10 ng thermolysin in 40 mM HEPES-KOH pH 7.6, either with or without 2% DDM. Both transporters were more digested when solubilised in DDM, but protected from proteolysis when in liposomes, indicating stable insertion into the bilayer. A small amount of oligomer was observed for each protein, which was also susceptible to proteolysis. Protein was detected via an anti-HA tag (LacY) or anti-His tag (XylE) antibody. Representative gels are shown (see Supplementary Information for an example repeat gel with the same trend). The gels in this figure are cropped from the original images but otherwise unadjusted. The original gels for all of those in this figure are in the Supplementary Information and the related gel intensities are in Fig. S2.
