[page 87↓]

7  Summary

In the plasma membrane of bacteria, phospholipids are synthesized on the cytoplasmic leaflet of the plasma membrane. To ensure balanced growth and thus, stability of biogenic membranes, half of the newly synthesized lipids must move to the opposing leaflet. It is known that this phospholipid transmembrane movement (flip-flop) is rapid, head-group independent and possibly protein mediated. However, the exact mechanism of this process remains elusive.

To investigate these fundamental transbilayer phospholipid transport processes in biogenic membranes, a novel stopped-flow BSA back-exchange assay was utilized to characterize the transmembrane movement and transbilayer distribution of fluorescent labeled, short-chain phospholipid analogues in ex vivo membranes. This approach is based on stopped-flow fluorescence spectroscopy, and the fact that BSA is able to extract fluorescent labeled, short-chain phospholipid analogues from the outer leaflet of (bio)membranes. We chose isolated inverted inner membrane vesicles (IIMV) derived from E.coli wild type MG1655, both for their simple membrane organization and for their suitability as a simple model organism for phospholipid flip-flop.

We observed that fluorescent-labeled, short-chain analogues of the major phospholipids in E.coli, phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), rapidly redistributed across the IIMV bilayer with half-times of less than three minutes. Furthermore, fluorescent, short-chain phospholipid analogues of phosphatidylcholine (PC) and phosphatidylserine (PS), which are not naturally occurring phospholipids in E.coli membranes, behaved similar to the PE and PG analogues. Surprisingly, we found that at equilibrium all fluorescent analogues were asymmetrically distributed between the two leaflet of the IIMV membranes. Approximately 23% of the PE, 18% of the PC, 34% of the PG and 26% of the PS analogues were located in the cytoplasmic leaflet. In conclusion, our analysis showed that the transmembrane movement of the phospholipid analogues across the membrane of IIMV was very rapid, bi-directional and head-group independent. These results were confirmed by an alternative fluorescence quenching assay, which is based on the chemical reduction of the fluorescence by dithionite. Analysis of proteoliposomes, containing fluorescent, long-chain or [page 88↓]fluorescent head-group labeled PE analogues revealed that the chain length did not influence the rapid flip-flop of phospholipid analogues.

To analyze the relevance of proteins for the transmembrane movement of fluorescent analogues, we measured flip-flop of phospholipid analogues in untreated and proteinase K treated vesicles generated from protein detergent extracts of IIMV. We found that the rapid transbilayer movement of phospholipid analogues across the membrane was maintained in untreated reconstituted vesicles. However, the flip-flop of fluorescent PG and PE analogues was eliminated in proteinase K treated vesicles. To further characterize this protein dependency, we reconstituted vesicles with increasing amounts of IIMV proteins. While we detected no flip-flop in protein-free liposomes, we observed that in reconstituted proteoliposomes containing more than 100 µg/ml of IIMV proteins, the flip-flop of short-chain analogues of PE and PG was as rapid as we found in isolated IIMV (half-times less than 2 min). We also observed that all fluorescent analogues were extracted from proteoliposomes containing more than 100 µg/ml of the IIMV proteins, similarly to what we found in IIMV. However, the amount of extractable fluorescent phospholipids analogues correlated with the amount of protein reconstituted into the proteoliposomes, strongly indicating, that protein concentrations below 100 µg/ml were not sufficient to equip every vesicle with proteins that facilitate the transmembrane movement of the fluorescent analogues. These data clearly demonstrated that the transmembrane movement of phospholipids must be facilitated by (a) protein(s).

To identify the molecular basis of the protein-mediated, rapid transmembrane movement of phospholipids across IIMV membranes, we used ion exchange chromatography (IEC) to separate the IIMV proteins. Detergent extracts from IIMV were applied on a strong anion exchanger, and the resulting fractions were reconstituted separately into proteoliposomes. To our surprise, we did not observe an enhanced flip-flop activity in any of the fractions, indicating that at least two proteins with possibly opposite netto charges or several subunits, which were not separable by AEC, are involved. Further analysis using different protein separation techniques will be necessary to identify the putative flippase complex.

[page 89↓]

Nevertheless, the presented data supplies strong evidence, that the bi-directional transmembrane movement of phospholipids is protein mediated, head-group and ATP independent.

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