title: Rapid transmembrane movement of C6-NBD-labeled phospholipids across the inner membrane of Escherichia coli.
abstract: In this study we have investigated the transmembrane movement of short chain fluorescently labeled phospholipids across the inner membrane of Escherichia coli. Exogenously added C6-NBD-labeled phospholipids rapidly flip across the inner membrane of E. coli, as was shown by a dithionite reduction assay applied to inverted inner membrane vesicles (IIMV) isolated from wild type E. coli cells. The rate of transmembrane movement of the phospholipid probes incorporated into IIMV is temperature dependent, and shows no phospholipid head group specificity. C6-NBD-labeled phospholipids translocate across the membrane of IIMV incubated at 37 degrees C with a t1/2 of 7 min. After the incorporation into IIMV C6-NBD-PG is partially converted to CL by CL-synthase. If IIMV are pretreated with proteinase K the conversion of this fluorescent probe to C6-NBD-CL is not observed anymore, suggesting that the catalytic domain of CL-synthase is at the cytoplasmic site of the plasma membrane of E. coli. Newly synthesized C6-NBD-CL also flips across the inner membrane although at a slower rate than the other phospholipid probes. The transmembrane movement occurs in both directions and is not influenced by treatment of the IIMV with a sulfhydryl reagent or a proteinase, nor by the presence of ATP, or a deltapH across the membrane of the IIMV. However, the transmembrane movement of the C6-NBD-labeled phospholipid probes is not observed in LUVETs (large unilamellar vesicles made by extrusion technique) prepared of wild type E. coli lipids, indicating that the rapid transmembrane movement of phospholipids across the inner membrane of E. coli is a protein-mediated process.
authors.affiliation: Department Biochemistry of Membranes, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, The Netherlands.
authors.forename: R P
authors.initials: RP
authors.lastname: Huijbregts
authors.forename: A I
authors.initials: AI
authors.lastname: de Kroon
authors.forename: B
authors.initials: B
authors.lastname: de Kruijff
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002463:Cell Membrane Permeability; D004227:Dithionite / Q000378:metabolism / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes; D008565:Membrane Proteins; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012697:Serine Endopeptidases; D013696:Temperature; D017855:Transferases (Other Substituted Phosphate Groups) / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D008081:Liposomes; D008565:Membrane Proteins; D010714:Phosphatidylethanolamines; D011993:Recombinant Fusion Proteins; C052267:1,2-(palmitoyl-NBD-aminocaproyl)phosphatidylethanolamine; D004227:Dithionite; D017855:Transferases (Other Substituted Phosphate Groups); C100091:cardiolipin synthetase; D000469:Alkaline Phosphatase; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase; D009327:4-Chloro-7-nitrobenzofurazan