Flippase Activity Detected with Unlabeled Lipids by Shape Changes of Giant Unilamellar Vesicles

Abstract

Transbilayer movement of phospholipids in biological membranes is mediated by energy-dependent and energy-independent flippases. Available methods for detection of flippase mediated transversal flip-flop are essentially based on spin-labeled or fluorescent lipid analogues. Here we demonstrate that shape change of giant unilamellar vesicles (GUVs) can be used as a new tool to study the occurrence and time scale of flippase-mediated transbilayer movement of unlabeled phospholipids. Insertion of lipids into the external leaflet created an area difference between the two leaflets that caused the formation of a bud-like structure. Under conditions of negligible flip-flop, the bud was stable. Upon reconstitution of the energy-independent flippase activity of the yeast endoplasmic reticulum into GUVs, the initial bud formation was reversible, and the shapes were recovered. This can be ascribed to a rapid flip-flop leading to relaxation of the monolayer area difference. Theoretical analysis of kinetics of shape changes provides self-consistent determination of the flip-flop rate and further kinetic parameters. Based on that analysis, the half-time of phospholipid flip-flop in the presence of endoplasmic reticulum proteins was found to be on the order of few minutes. In contrast, GUVs reconstituted with influenza virus protein formed stable buds. The results argue for the presence of specific membrane proteins mediating rapid flip-flop.In lipid bilayers the spontaneous movement of major phospholipids, e.g. of phosphatidylcholine (PC), 6 between the two monolayers is slow, with half-times on the order of hours or even days. However, lipid topology of cellular membranes results from a continuous bidirectional movement (flip-flop) of lipids between the two leaflets in which specific membrane proteins, so called flippases, play an essential role (1, 2). Energyindependent flippases allow phospholipids to equilibrate rapidly between the two monolayers, whereas energy-dependent flippases mediate a net transfer of specific phospholipids to one leaflet of the membrane. Candidates for the latter flippase are members of a conserved subfamily of P-type ATPases (2) as well as ATP binding cassette transporters (3).In eukaryotes the cytoplasmic leaflet of the endoplasmic reticulum (ER) membrane is the major site of phospholipid biosynthesis. To ensure stable membrane growth, energy-independent flippases mediate rapid, bidirectional, and rather unspecific phospholipid flip-flop with half-times of minutes or less (4 -7). A similar flippase activity was also found in the bacterial inner membrane, where lipid synthesis occurs likewise at the cytoplasmic leaflet (8).Techniques to determine transbilayer phospholipid movement as well as the activity of flippases have been critically evaluated (9). Spin-labeled and fluorescent lipid analogues have provided much insight into protein-mediated transbilayer dynamics of phospholipids (9). However, the bulky reporter moieties may affect the absolute values of transbilayer lipid movement.An alternati...

Flip-Flop of Fluorescently Labeled Phospholipids in Proteoliposomes Reconstituted with Saccharomyces cerevisiae Microsomal Proteins

“…Likewise, protein pools with higher specific flippase activities were obtained in this study by fractionation; conversely, other fractions replete with membrane proteins were inactive. In addition, a recent study clearly demonstrated that the presence of membrane proteins in giant unilamellar vesicles per se is not sufficient to facilitate the flip-flop of phospholipids (31). These results reinforce the conclusion that specific proteins are required to facilitate phospholipid flip-flop; the data presented in this paper indicate that the identification of these proteins is feasible.…”

“…Our experiments also allowed us to estimate that the flippase protein represents ϳ2% (wt/wt) of membrane proteins in the TE. Using ER membrane proteincontaining proteoliposomes such as those described here, we recently succeeded in reconstituting flippase activity in giant unilamellar vesicles (31), allowing us to study the time scale of flippase-mediated transbilayer movement of unlabeled phos- FIG. 6.…”

Membrane lipids: where they are and how they behave

“…In model membranes of bilayer-forming lipids, the rate of spontaneous polar lipid movement between bilayer leaflets is slow (hours to days for PtdCho lipids 37,38 ) and is governed by the size, charge and polarity of the head-group. The half life (t 1/2 ) for translocation is days for complex GSLs but seconds for Cer 39 , DAG 40,41 and sterols 42 .…”

Lipid Polymorphisms and Membrane Shape

“…In lipid vesicles, this effect is seen as membrane budding. The budding can be induced by temperature or addition of lipids to the outer monolayer (i.e., by factors changing the monolayers area balance), in this case because of relative expansion of the outer monolayer (Dobereiner et al 1997;Papadopulos et al 2007). To compensate, the vesicle is transformed into a chain of smaller vesicles to match the new area disparity.…”

“…The enzyme adsorption causes changes in the area of the outer monolayer (Papadopulos et al 2007). The reaction products, such as ceramides, tend to aggregate forming rigid membrane domains (Holopainen et al 2000).…”