Despite the documentation of rapid phospholipid flip-flop in biogenic membrane systems like bacterial membranes or the ER of eukaryotes, and a variety of persuasive evidence for protein involvement in this process, no biogenic flippase has been isolated and the mechanism(s) of catalyzed flip-flop remain(s) to be described. Previous problems of an adequate assay for such very rapid processes have been overcome with our stopped-flow BSA back-exchange assay which provides an adequate time resolution to measure flippase activity in biogenic membranes.
In order to purify the putative flippase protein(s), we utilized anion exchange chromatography. Surprisingly, we were not able to yield a fraction of E.coli inner membrane proteins, which exhibited an enhanced flip-flop activity after reconstitution of these proteins into eggPC proteoliposomes. The combination of ion exchange chromatography with either glycerol gradient fractionation or other chromatographic methods (e.g. size exclusion, two-dimensional gel electrophoresis) could provide stronger evidences for an involvement of (a) specific protein(s) in the phospholipid flip-flop across biogenic membranes.
Another possibility to identify the putative flippase would be a rather theoretical molecular approach. The entire genomes of several bacteria (e.g. E.coli, Haemophilus influenzae, Mycobacterium tuberculosis) and that of S. cerevisiae are known. Using data base screening, it might be possible to reduce the number of possible candidates, which facilitate the energy independent phospholipid flip-flop. For example, access to the complete, annotated E.coli genome in several data bases allows the specific screening for transmembrane domains. Many of them are associated to ABC domains and were not relevant for the ATP independent transport. In a step by step procedure all transporter with e.g. known function etc. can be discarded. Thus, it should be possible to identify proteins with transmembrane domain(s) and unknown function and specifically investigate these candidates. Additionally, it would be possible to investigate the involvement of strong candidates in the lipid flip-flop in more detail using molecular biological and genetically methods, e.g. by generating point mutations. [page 86↓]Possibly, temperature sensitive mutants provide another tool to research the transbilayer movement of (phospho)lipids in biogenic membranes.
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