Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli

Abstract

Phosphatidylcholine (PC) has been widely used in place of naturally occurring phosphatidylethanolamine (PE) in reconstitution of bacterial membrane proteins. However, PC does not support native structure or function for several reconstituted transport proteins. Lactose permease (LacY) of Escherichia coli, when reconstituted in E. coli phospholipids, exhibits energy-dependent uphill and energy-independent downhill transport function and proper conformation of periplasmic domain P7, which is tightly linked to uphill transport function. LacY expressed in cells lacking PE and containing only anionic phospholipids exhibits only downhill transport and lacks native P7 conformation. Reconstitution of LacY in the presence of E. coli-derived PE, but not dioleoyl-PC, results in uphill transport. We now show that LacY exhibits uphill transport and native conformation of P7 when expressed in a mutant of E. coli in which PC completely replaces PE even though the structure is not completely native. E. coli-derived PC and synthetic PC species containing at least one saturated fatty acid also support the native conformation of P7 dependent on the presence of anionic phospholipids. Our results demonstrate that the different effects of PE and PC species on LacY structure and function cannot be explained by differences in the direct interaction of the lipid head groups with specific amino acid residues alone but are due to more complex effects of the physical and chemical properties of the lipid environment on protein structure. This conclusion is supported by the effect of different lipids on the proper folding of domain P7, which indirectly influences uphill transport function.

Functional dynamics in the voltage-dependent anion channel

“…At the same time the charge and polarity of E73 is strongly correlated with the dynamics and conformation of the membrane environment, suggesting that the dynamics of the lipids might be connected with global protein motions. This finding is interesting as lipid-protein interactions are known to be important for the structure and function of integral membrane proteins (47)(48)(49)(50)(51)(52). In particular, VDAC1 gating and ionic selectivity have been shown to be dependent on lipid composition (53).…”

Substrate Selectivity of Lysophospholipid Transporter LplT Involved in Membrane Phospholipid Remodeling in Escherichia coli

“…32 P-Labeled PG and CL were made in the E. coli AL95 strain (pss 93::kan lacY::Tn9), which lacks the ability to synthesize PE (22). Strain AL95 carrying plasmid pAC-PCS lp -Sp-Gm was used to prepare 32 P-labeled PC (23). This strain is capable of making PC instead of PE due to expression of PC synthase encoded by the Legionella pneumophila pcsA gene under control of an arabinose-inducible promoter (P araB ).…”

Dynamic membrane protein topological switching upon changes in phospholipid environment

“…Strain AL95 (null for PE and LacY synthesis) with (PE-containing) or without (PE-lacking) plasmid pDD72GM (encodes for PE synthesis) and strain AL95 with plasmid pAC-PCSlp-Sp-Gm (encodes for PC synthesis in place of PE) were grown (6,32) and used to prepare lipid extracts for preparation of proteoliposomes, protein-free liposomes, and MLVs (7) as previously described. Strain AL95 with or without plasmid pDD72GM (6) was used as the host for expression of plasmid pT7-5/C-less LacY (ampR, ColE1 replicon) encoding LacY in which Cys residues are replaced by Ser, plasmid pT7-5/C-less LacY/ V331C (ampR, ColE1 replicon) encoding a single Cys replacement at Val331 in otherwise Cys-less LacY, or plasmid pSP72/CscB-WT (ampR, ColE1 replicon) encoding WT CscB; plasmids were provided by H. R. Kaback (University of California, Los Angeles).…”