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.

Fig. 1.

Topological organization of LacY as a function of membrane lipid composition. TMs (Roman numerals), extramembrane domains (P for periplasmic and C for cytoplasmic as in +PE cells), N-terminus (NT) and C-terminus (CT) domains are indicated. The approximate positions and names of amino acids substituted by cysteine and used for topological analysis are indicated near the closed and open symbols in a cysteineless derivative of LacY used in this work. The conformation-sensitive (2, 3), PE-dependent (–8) epitope in domain P7 recognized by mAb 4B1 is noted with the amino acids that are part of this epitope marked with an asterisk. (Top) Denotes orientation in +PE cells; (Bottom) denotes orientation in -PE cells with the upper side of each figure being the side that faces the cytoplasm. TM VII resides in the periplasm in -PE cells. Topological orientation of LacY is taken from ref. .

Fig. 2.

Transport function of wild-type LacY in whole cells. Uphill transport of TMG (A) or downhill transport of lactose (B) normalized to total cell protein was determined as a function of time by AL95/pDD72 (closed squares, +PE), AL95/pAC-PCS_lp-Sp-Gm (open squares -PE + PC), or AL95 (open circles -PE), all induced for expression of a plasmid-borne copy of the wild-type lacY gene. Uphill transport in -PE + PC cells was verified by addition of a protonophore (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) to the assay of TMG uptake (open diamonds), which reduced the uptake to the level of the -PE results (open circles). No uptake of TMG or lactose by AL95/pAC-PCS_lp-Sp-Gm (closed diamonds, -PE + PC ΔLacY) demonstrated that uphill and downhill transport in -PE + PC cells (open squares) were mediated by LacY. Standard deviations for the average of two determinations are shown except for lines where uptake was essentially zero (open diamonds, open circles, and closed diamonds). The reduced level (one-fifth) of LacY in -PE + PC cells relative to the level of LacY in +PE cells (25 μg of protein per lane) is shown by Western blot analysis (B Inset) using anti-LacY polyclonal antibody.

Fig. 3.

Mapping the topology of LacY in -PE + PC cells. All samples were treated with MPB without sonication (-) or during sonication (+). AL95/pAC-PCS_lp-Sp-Gm (-PE + PC) cells expressing single cysteine replacements in the indicated extramembrane loop/amino acid were grown and processed as described in Methods subsequent to Western blotting using avidin-HRP to detect the biotin moiety linked to cysteines (see Fig. 1 for approximate positions of substitutions) that were accessible to MPB during labeling. MBP labeling was performed at pH 7 except for P3/I103C and P5/T163C, which were labeled at pH 9.1.

Fig. 4.

Western and Eastern-Western blotting of LacY used to probe the conformation of domain P7. (A) Inner membrane preparations (duplicate samples of 10 μg of protein) from cells with the indicated lipid composition and expressing a plasmid-borne copy of wild-type LacY were analyzed by Western blot analysis using conformation-specific mAb 4B1. (B–E) Inner membrane preparations from cells with the indicated phospholipid composition and expressing a plasmid-borne copy of wild-type LacY were subjected to SDS PAGE. The region of the gel containing LacY (molecular weight 31,000–33,000) was transferred to the area of a nitrocellulose sheet onto which the indicated phospholipids were transferred from a TLC plate (Eastern blot) followed by Western blotting analysis using mAb 4B1. Single samples contained 10 μg of membrane protein and duplicate samples contained 5 (Left) or 10 (Right) μg of membrane protein. Preblotted phospholipids were as follows: (B) no lipid (none), total lipid extract from -PE + PC cells, diC_18∶1PC (Avanti); (C) PE (Avanti) purified from E. coli (+PE), 70% diC_18∶1PC (Avanti) supplemented with 30% of total lipid extract from -PE cells containing approximately equal amounts of PG and CL, total lipid extract from -PE + PC cells, PC isolated from -PC + PE cells by TLC (EcPC); (D) total lipid extract from -PE + PC cells, PC isolated from -PC + PE cells by DE52 chromatography either alone (EcPC) or 70% PC supplemented with E. coli PG (3%) and CL (27%) from Avanti (EcPC + PG + CL); (E) preblotted PCs (Avanti) with the indicated fatty acid composition are shown. One fatty acid denotes both acyl chains are identical. Two fatty acids indicate fatty acids at positions sn-1 and sn-2, respectively. PCs were supplemented with E. coli PG plus CL (Avanti) in the ratio as indicated for D. None indicates no lipid preblotted. Fatty acids for lanes 1 and 2 were Δ9 trans and Δ9 cis, respectively.