Abstract
In a number of cases, the function of membrane proteins appears to require the presence of specific lipid species in the bilayer. We have shown that the secondary multidrug transporter LmrP requires the presence of phosphatidylethanolamine (PE), as its replacement by phosphatidylcholine (PC) inhibits transport activity and directly affects its structure, although the underlying mechanism was unknown. Here, we show that the effect of PE on the structure and the function of LmrP is mediated by interactions between the lipid headgroup and the protein. We used methyl-PE and dimethyl-PE analogs of PE to show that only replacement of the three hydrogens by methyl moieties leads to changes in the biochemical and biophysical properties of the reconstituted protein. This suggests that LmrP does not depend on the bulk properties of the phospholipids tested but solely on the hydrogen bonding ability of the headgroup. We then show that a single point mutation in LmrP, D68C, is sufficient to recapitulate precisely every biochemical and biophysical effect observed when PE is replaced by PC, including energy transfer between the protein tryptophan residues and the lipid headgroups. We conclude that the negatively charged Asp-68 is likely to participate in the interaction with PE and that such interaction is required for proton gradient sensing, substrate binding, and transport. Because Asp-68 belongs to a highly conserved motif in the Major Facilitator Superfamily (which includes LacY and EmrD), this interaction might be a general feature of these transporters that is involved in proton gradient sensing and lipid dependence.
Highlights
Dependent ATPase [14] and the ABC transporter cdr1p [15]
LmrP transport activity was assayed in PE, methyl PE, dimethyl PE and PC proteoliposomes, by measuring the variation in Hoechst 33342 fluorescence as a function of time (Fig. 1)
When a ΔpH is imposed, the fluorescence of membrane-bound Hoechst 33342 decreases at the same rate in PE, methyl PE and dimethyl PE proteoliposomes indicating that Hoechst 33342 was actively being extruded out of the membrane
Summary
Cell culture and protein purification- The growth of L. lactis NZ9000 in M17 medium (from Difco), the overexpression using the NICE system and the purification of the His-tagged LmrP wt and mutants were performed as previously described [2;16,17,18,19,20]. Protein reconstitution and imposition of an artificial proton gradient (ΔpH) were performed as previously described [16;21]. Fluorescence Resonance Energy Transfer (FRET)5% of dansyl-PE with respect to the total lipid mass was incorporated into proteoliposomes during the reconstitution process described above. Hydrogen/deuterium exchange kinetics- A sample of reconstituted LmrP was deposited on a germanium plate as described elsewhere [22;23]. Proteoliposomes were incubated 20 minutes at room temperature with MPB (3-(Nmaleimidylpropionyl) biocytin, Molecular Probes) added to a final concentration of 200μM from freshly-prepared stock solution to biotinylate LmrP. Proteoliposomes were incubated 10 minutes with AMS (4-acetamido-4′maleimidylstilbene-2,2′-disulfonic acid, Molecular Probes)) to block external cysteine, added from a freshly-prepared stock solution and to a final concentration of 200μM. SuperSignal West Pico chemiluminescent substrate (Pierce) was used to visualize biotinylated proteins
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