Abstract
To determine the specific role lipids play in membrane protein topogenesis in vivo, the orientation with respect to the membrane bilayer of Escherichia coli lactose permease (LacY) transmembrane (TM) domains and their flanking extramembrane domains was compared after assembly in native membranes and membranes with genetically modified lipid content using the substituted cysteine accessibility method for determining TM domain mapping. LacY assembled in the absence of the major membrane lipid phosphatidylethanolamine (PE) does not carry out uphill transport of substrate and displays an inverted orientation for the N-terminal six-TM domain helical bundle (Bogdanov, M., Heacock, P. N., and Dowhan, W. (2002) EMBO J. 21, 2107-2116). Strikingly, the replacement of PE in vivo by the foreign lipid monoglucosyldiacylglycerol (MGlcDAG), synthesized by the Acholeplasma laidlawii MGlcDAG synthase, restored uphill transport and supported the wild type TM topology of the N-terminal helical bundle of LacY. An interchangeable role in defining membrane protein TM domain orientation and supporting function is played by the two most abundant lipids, PE and MGlcDAG, in gram-negative and gram-positive bacteria, respectively. Therefore, these structurally diverse lipids endow the membrane with similar properties necessary for the proper organization of protein domains in LacY that are highly sensitive to lipids as topological determinants.
Highlights
Orientation of TM Domains of LacY—We reported that the introduction of MGlcDAG into PE-lacking E. coli cells restored uphill transport activity of LacY, but the steady-state level of substrate accumulation was significantly lower than that observed in PE-containing cells [17]
When compared with LacY assembled in ϩPE cell membranes, the apparent Vmax for uphill transport of LacY synthesized in membranes without PE is 10-fold less (4 nmol/ min/mg of protein), whereas the apparent Km is almost the SCAMTM was originally used to establish that the topology of the N-terminal six-TM helical bundle of LacY and the N-terminal two-TM hairpin of Phenylalanine permease (PheP) and GabP of E. coli are topologically inverted with respect to the membrane bilayer and that the remainder of each protein displays native topology when assembled in membranes lacking PE [2, 9, 10]
Substitution of the foreign glycolipid MGlcDAG for PE partially corrects many of the lipid-related phenotypes of PE-lacking mutants of E. coli [17]
Summary
LacY, lactose permease; CL, cardiolipin; GabP, ␥-aminobutyric acid permease; MGalDAG, monogalactosyldiacylglycerol; MGlcDAG, monoglucosyldiacylglycerol; MPB, 3-(N-maleimidylpropionyl) biocytin; PheP, phenylalanine permease; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; TM, transmembrane; SCAMTM, substituted cysteine accessibility method for determining TM domain mapping; TMG, methyl--D-galactopyranoside. Regardless of the source of LacY (synthesized in membranes with or without PE), its topology and function are solely dependent on the presence of PE in liposomes independent of other cellular components Another zwitterionic lipid, phosphatidylcholine (PC), can support the native topology of LacY in proteoliposomes, it failed to support the uphill transport function of the permease [7]. In the absence of PE, cells grown in medium supplemented with divalent metal ions are viable, but they display a complex mixture of phenotypes Their growth rate is greatly reduced; the filamentous morphology of these cells indicates a defect in cell division; their outer membrane integrity is compromised; and they are defective in assembly of some membrane proteins, as well as in sugar and amino acid transport [1]. Defining the similarities among lipids with similar functions in the membrane will provide the foundation for understanding the specific properties of lipids required to support different aspects of cell function
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