Phosphatidylserine asymmetric vesicles as eukaryotic plasma membrane model

Lipid asymmetries between the outer and inner leaflet of the lipid bilayer exist in nearly all biological membranes. Although living cells spend great effort to adjust and maintain these asymmetries, little is known about the biophysical phenomena within asymmetric membranes and their role in cellular function. One reason for this lack of insight into such a fundamental membrane property is the fact that the majority of model-membrane studies have been performed on symmetric membranes, since it is quite challenging to prepare stable membranes showing a lipid asymmetry. Our aim is to overcome this problem by employing a targeted enzymatic reaction in order to specifically prepare phosphatidylserine (PS) asymmetric liposomes, which can be used for studying the effects of PS asymmetry on transmembrane proteins or utilized as model systems for eukaryotic plasma membranes in general. To achieve this goal, we use a recombinant version of a water soluble phosphatidylserine decarboxylase (PSD) from Plasmodium knowlesi which selectively decarboxylates PS, converting it to phosphatidylethanolamine (PE). This assay presents a straightforward and easy approach to prepare stable asymmetric liposomes mimicking the natural PS asymmetry of eukaryotic plasma membranes. In addition, the use of a highly specific enzyme carries the advantage that the lipid composition of the lipid bilayer is not a limiting factor in the outcome of the experiment. This allows for the preparation of PS asymmetric liposomes in the presence of various lipids, including cholesterol. Furthermore, we tested if the PSD was still active and PS asymmetry was still stable in the presence of two different transmembrane proteins, we used as models. Therefore, the archaeal ammonium transporter (Amt1) and the murine T cell antigen receptor (TCR) were tested. In the presence of both proteins, PSD activity could be shown. However, the PS asymmetry was not as stable as before. In the Amt1 experiments, the detergent used during the reconstitution procedure was not removed completely, leading to increased lipid flip-flop rates. This problem could be addressed in the future by testing other detergent removal techniques. In the TCR experiments, the PS asymmetry in the TCR-free liposomes was more stable than in the TCR-containing liposomes. This suggests that the detergent removal was complete. However, the TCR itself might influence the lipid behavior in the bilayer, e.g. by disturbing the membrane leading to increased flip-flop rates. Consequently, an adjustment of the liposomal lipid composition could lead to a denser lipid packing around the TCR thus decreasing lipid flip-flop rates. Taken together, the preparation of stable PS asymmetric liposomes was possible with the new established PSD assay. The preparation of, for a longer time period, stable PS asymmetric proteolipomes should be improved.