A long-standing problem in understanding the mechanism by which the phospholipid bilayer of biological membranes is assembled concerns how phospholipids flip back and forth between the two leaflets of the bilayer. This is important since the phospholipid biosynthetic machinery typically face the cytosol and deposit newly synthesized phospholipids in the cytosolic leaflet of biogenic membranes such as the ER of eukaryotic cells or the plasma membrane of prokaryotes. These lipids must be moved across the bilayer to ensure membrane growth and integrity. Transport does not occur spontaneously and is assumed to be facilitated by specific membrane proteins - the flippases.
During the last two decades, research efforts on protein-mediated phospholipid transmembrane movement in biogenic membranes focused on eukaryotic cells. Due to the inherent difficulties in the isolation of pure (sub)cellular membranes and in the genetic manipulation of eukaryotic cells, progress on the characterization, identification and purification of the flippase proteins of eukaryotic cells is slow.
The progress on the ultimate goal of identifying, purifying and possibly cloning the eukaryotic flippases is greatly enhanced by using much simpler organisms that are easily handled and manipulated on the genetic level. The Gram-negative bacterium Escherichia coli is a powerful model organism for investigations of biogenic membranes. E.coli does not contain subcellular membranes and therefore, a number of technical difficulties in studying transmembrane movement and distribution in eukaryotic systems, such as the combination of endocytosis and transbilayer movement in yeast (Kean, et al., 1993), are non-existent. Moreover, the protein content of a prokaryotic cell is much smaller than that of eukaryotic cells, hence, allowing an easier identification of an possible flippase. The genome of E.coli has recently been fully sequenced and was assumed to be close to a theoretical minimal genome necessary for ensuring survival of a cell. Since one of the genes within this small genome could be a flippase, the smaller number of total genes would make the identification of a flippase gene more likely.
For these reasons, E.coli, in particular the inner membrane of E.coli, was chosen as a model system for the characterization, identification and purification of flippases in biogenic membranes. Much progress has been made in elucidating the biogenesis and function of proteins in the membranes of E.coli. It has become apparent in the last three decades that lipids also play a role in a variety of processes within the E.coli cell and not only by forming a physical barrier. However, the knowledge about the mechanism by which phospholipids are transported across the membrane after synthesis and what the consequences are for the lipid distribution in the membrane, is still very limited. No biogenic membrane flippases have been identified so far, and there is a controversy as to whether proteins are involved at all, whether any membrane protein is sufficient, or whether non-bilayer arrangements of lipids facilitate phospholipid flip-flop.
The main objectives of this thesis were therefore (i) to introduce an assay with a high time resolution allowing the adequate quantitative characterization of the flip-flop in E.coli, (ii) to explore the transmembrane distribution of the phospholipids in the inner membrane of E.coli, (iii) to provide strong evidence for the hypothesis that the phospholipid flip-flop in the inner membrane of E.coli is protein dependent, has no head-group specificity and does not need energy input and (iv) to possibly, identify and purify the putative flippase of the plasma membrane of E.coli.
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