Inverted Inner Membrane Vesicles Research Articles

Transbilayer movement of fluorescent phospholipid analogues in the cytoplasmic membrane of Escherichia coli.

We investigated the transmembrane movement of fluorescent labeled phospholipids in inverted inner membrane vesicles (IIMV) of Escherichia coli (E. coli) wild-type strain (MG1655), as well as in proteoliposomes reconstituted from detergent extracts of the IIMV. The transbilayer movement of 1-myristoyl-2-[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphoethanolamine (M-C6-NBD-PE) and -phosphocholine (M-C6-NBD-PC) was measured by a fluorescence stopped-flow back-exchange assay. Both analogues were rapidly translocated across the IIMV membrane, with half-times of <1 min (outward movement) and approximately 3 min (inward movement). No flip-flop was detected in protein-free liposomes, but in IIMV-derived proteoliposomes flip-flop of M-C6-NBD-PE occurred similarly to IIMV and could be largely eliminated by proteinase K treatment.

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°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 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 ΔpH 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.

SecD and SecF are required for the proton electrochemical gradient stimulation of preprotein translocation.

Mutations in secD and secF show impaired protein translocation across the inner membrane of Escherichia coli. We investigated the effect of SecD and SecF (SecD/F) depletion on preprotein translocation into inverted inner membrane vesicles (IMVs). Both IMVs and cells which were depleted of SecD/F were defective in their ability to maintain a proton electrochemical gradient. The translocation of pre-maltose binding protein (preMBP), which is strongly delta microH+ dependent, showed a 5-fold decreased rate with IMVs lacking SecD/F. In contrast, proteolytic processing of preMBP to MBP by leader peptidase was similar in IMVs containing and lacking SecD/F, consistent with earlier findings that only ATP-dependent translocation is required for the initiation of translocation. In the absence of a delta microH+, with ATP as the sole energy source, preMBP translocation into IMVs which contained or were depleted of SecD/F was identical. Translocation of the precursor of outer membrane protein A (proOmpA) in the presence of subsaturating ATP also required a generated delta microH+ and, under these conditions, proOmpA translocation required SecD/F. With saturating concentrations of ATP, where delta microH+ has little effect on in vitro proOmpA translocation, SecD/F also had little effect on translocation. These results explain why SecD/F effects are precursor protein dependent in vitro.