title: Substrate Selectivity of Lysophospholipid Transporter LplT Involved in Membrane Phospholipid Remodeling in Escherichia coli.
abstract: Lysophospholipid transporter (LplT) was previously found to be primarily involved in 2-acyl lysophosphatidylethanolamine (lyso-PE) recycling in Gram-negative bacteria. This work identifies the potent role of LplT in maintaining membrane stability and integrity in the Escherichia coli envelope. Here we demonstrate the involvement of LplT in the recycling of three major bacterial phospholipids using a combination of an in vitro lysophospholipid binding assay using purified protein and transport assays with E. coli spheroplasts. Our results show that lyso-PE and lysophosphatidylglycerol, but not lysophosphatidylcholine, are taken up by LplT for reacylation by acyltransferase/acyl-acyl carrier protein synthetase on the inner leaflet of the membrane. We also found a novel cardiolipin hydrolysis reaction by phospholipase A2 to form diacylated cardiolipin progressing to the completely deacylated headgroup. These two distinct cardiolipin derivatives were both translocated with comparable efficiency to generate triacylated cardiolipin by acyltransferase/acyl-acyl carrier protein synthetase, demonstrating the first evidence of cardiolipin remodeling in bacteria. These findings support that a fatty acid chain is not required for LplT transport. We found that LplT cannot transport lysophosphatidic acid, and its substrate binding was not inhibited by either orthophosphate or glycerol 3-phosphate, indicating that either a glycerol or ethanolamine headgroup is the chemical determinant for substrate recognition. Diacyl forms of PE, phosphatidylglycerol, or the tetra-acylated form of cardiolipin could not serve as a competitive inhibitor in vitro. Based on an evolutionary structural model, we propose a "sideways sliding" mechanism to explain how a conserved membrane-embedded α-helical interface excludes diacylphospholipids from the LplT binding site to facilitate efficient flipping of lysophospholipid across the cell membrane.
authors.affiliation: From the Center for Membrane Biology, Department of Biochemistry and Molecular Biology, The University of Texas Houston Medical School, Houston, Texas 77030 and.
authors.forename: Yibin
authors.initials: Y
authors.lastname: Lin
authors.affiliation: From the Center for Membrane Biology, Department of Biochemistry and Molecular Biology, The University of Texas Houston Medical School, Houston, Texas 77030 and.
authors.forename: Mikhail
authors.initials: M
authors.lastname: Bogdanov
authors.affiliation: From the Center for Membrane Biology, Department of Biochemistry and Molecular Biology, The University of Texas Houston Medical School, Houston, Texas 77030 and.
authors.forename: Shuilong
authors.initials: S
authors.lastname: Tong
authors.affiliation: Department of Biochemistry, Duke University Medical School, Durham, North Carolina 27703.
authors.forename: Ziqiang
authors.initials: Z
authors.lastname: Guan
authors.affiliation: From the Center for Membrane Biology, Department of Biochemistry and Molecular Biology, The University of Texas Houston Medical School, Houston, Texas 77030 and lei.zheng@uth.tmc.edu.
authors.forename: Lei
authors.initials: L
authors.lastname: Zheng
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D002308:Cardiolipins / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D017353:Gene Deletion; D006868:Hydrolysis; D007711:Klebsiella pneumoniae; D008246:Lysophospholipids / Q000737:chemistry*; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009154:Mutation; D010715:Phosphatidylglycerols / Q000737:chemistry; D054467:Phospholipases A2 / Q000737:chemistry; D049294:Phospholipid Transfer Proteins / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D021381:Protein Transport; D013379:Substrate Specificity
chemical_list: D002308:Cardiolipins; D029968:Escherichia coli Proteins; C000608268:LplT protein, E coli; D008246:Lysophospholipids; D010715:Phosphatidylglycerols; D049294:Phospholipid Transfer Proteins; D054467:Phospholipases A2
keywords: cardiolipin; cardiolipin deacylation; lipid flipping; lipid transport; lysophospholipid; lysophospholipid transporter; phospholipid remodeling; phospholipid turnover; substrate specificity