Rapid transmembrane movement of C-6-NBD-labeled phospholipids across the inner membrane of Escherichia coli

In this study we have investigated the transmembrane movement of short chain fluorescently labeled phospholipids across the inner membrane of Escherichia coli. Exogenously added C-6-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. C-6-NBD-labeled phospholipids translocate across the membrane of IIMV incubated at 37 degrees C with a t(1/2) of 7 min. After the incorporation into IIMV C-6-NBD-PG is partially converted to CL by CL-synthase. If IIMV are pretreated with t proteinase K the conversion of this fluorescent probe to C-6-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 C-6-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 Delta pH across the membrane of the IIMV. However, the transmembrane movement of the C-6-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.