Phospholipids transbilayer movement

Dekkers, D. W. C., Comfuiius, P., Schroit, A. J., Bevers, E. M. andZwaal, R. F. A., 1998, Transbilayer movement ofNBD-labeled phospholipids in red blood ceU membranes outward-directed transport by the multidrug resistance protein 1 (MRPl). Biochemistry,... 

Gadella, B.M., Miller, N.G., Colenbrander, B., van Golde, L.M. and Harrison, R, A. 1999, Flow cytometric detection of transbilayer movement of florescent phospholipid analogs across the boar sperm plasma membrane elimination of labeling artifacts. Mol. Reprod. Dev. 53 108-125. 

Zhao, J., Zhou, Q., Wiedmer, T. and Sims, P.J., 1998b, Pahnitoylation of phospholipid scramblase is required for normal function in promoting Ca2+-activated transbilayer movement ofmembrane phospholipids. Biochemistry, 37 6361-6366. 

Schroit, A.J., andZwaal, R.F., 1991, Transbilayer movement of phospholipids in red cell and platelet membranes. Biochim. Biophys. Acta 1071 313-329. 

Liu J, Conboy JC. Direct measurement of the transbilayer movement of phospholipids by sum-frequency vibrational spectroscopy. J. Am. Chem. Soc. 2004 126 8376-8377. 

Lipid transfer proteins have proved to be a useful tool for studying artificial and natural membranes (for a recent review see Bloj and Zilver-smit, 1981a). With the ability of phospholipid transfer proteins to replace selectively the phospholipid molecules on the exposed surfaces of membranes, information about the asymmetric distribution of phospholipids across a bilayer and the rate of transbilayer movement of phospholipid... 

The exchangeable lipid pool in a donor particle can be determined by measuring the labeled lipid remaining in the donor membrane after prolonged incubation of radiolabeled donor membranes with an excess of acceptor particles and transfer protein (Zilversmit and Hughes, 1976). This technique has also been used to determine the asymmetric distribution of phospholipid molecules and their rate of transbilayer movement... 

It is concluded that under equihbrium conditions Upids do not easily undergo transbilayer movement in liposomes or membranes [149-151]. On the other hand, phospholipids and cholesterol have been shown to undergo transmembrane movement in erythrocytes under non-equilibrium conditions or following membrane perturbation such as ghost formation or phospholipase treatment [152-154]. More recently Lange and co-workers have made studies of the rate of transmembrane movement of cholesterol in the membranes of human erythrocytes. Normally, cholesterol in intact erythrocytes is not accessible to cholesterol oxidase [155]. 

The movement of lipids within the cell can be divided into two different general classes of transport intramembrane transport, which entails the transbilayer movement of the lipid molecule and intermembrane transport, which is the movement of lipid molecules from one distinct membrane to another. In some cases, the transmembrane movement of a phospholipid is coupled to a process that also removes the lipid from the membrane in which it was resident, and these events have the character of the lipid being vectorially pumped across and out of the membrane. Extensive reviews of these processes have been published [6,7,9-12]. 

Direct experiments to examine the transbilayer movement of phospholipids (R.D. Kornberg, 1971) made use of spin-labeled analogs of PC in which the choline moiety was replaced with the tempocholine probe,, A -dimethyl-A -(l -oxyI-2, 2, 6, 6 -tetramethyl-4 -piperidyl)-ethanolamine (Fig. 2). These workers found that only the electron spin resonance signal generated by molecules in the outer leaflet of unilamellar liposomes could be rapidly quenched by ascorbate. The electron spin resonance signal from lipid molecules initially residing at the inner leaflet of liposomes was accessible to ascorbate with a r,/2 of >6.5 h, indicating slow transbilayer lipid movement (Fig. 3). 

Thus, studies with model membranes provide clear evidence that the transbilayer movement of phospholipids is a very slow process in this system, whereas the process appears to be rapid for non-polar lipids. The results imply that if transbilayer movement of phospholipids does occur in biological membranes, it must be a facilitated process. 

Transbilayer movement of lipid at the endoplasmic reticulum In eukaryotic systems a detailed pattern of synthetic asymmetry has emerged with respect to the topology of the enzymes of phospholipid synthesis in rat liver microsomal membranes. Protease mapping experiments (D.E. Vance, 1977 R. Bell, 1981) have indicated that the active sites of the phospholipid synthetic enzymes are located on the cytosolic face of the ER. Thus, in both prokaryotic and eukaryotic systems, it appears that the site of synthesis of the bulk of cellular phospholipid is the cytosolic side of the membrane. This asymmetric localization of synthetic enzymes strongly implicates transbilayer movement of phospholipids as a necessary and important event in membrane assembly that is required for the equal expansion of both leaflets of the bilayer [13]. 

The transbilayer movement of phospholipids in microsomal membranes has been measured using several different approaches. Phospholipid transfer proteins were used to probe the transbilayer movement of lipids in preparations of liver microsomes that were first radiolabeled with lipid precursors in vivo (D.B. Zilversmit, 1977). The results from these experiments provided evidence that PC, PE, PS, and PI from both membrane leaflets were exchanged between labeled microsomes and excess acceptor membranes with a maximal t 2 of 45 min. 

Thus, the data from both bacteria and animal cells demonstrate that transbilayer movement of phospholipid occurs on a timescale of minutes, in an ATP-independent fashion in membranes that contain the majority of the enzymes involved in their biosynthesis. These intramembrane transport properties observed in the major biosynthetic membranes, however, are not generally true for other membrane systems. This is especially true of the plasma membrane, Golgi, endosomal, and lysosomal membranes. 

As mentioned in the introduction, there is good evidence that phospholipids are organized in several distinct and discrete pools or populations within the two monolayers of the thylakoid membrane [3,5,6). To get further information about the different PG populations of the membrane, we have designed experimental conditions, under which phospholipid depletion (occurring in the presence of pancreatic phospholipase A2, PLA2) and/or delocalization (via transbilayer movement) took place successively. 

Fig. 2 shows that at 2°C, the hydrolysis of both phospholipids occurs in phospholipase A2 treated spinach thylakoids. After 60 min 70% of PG and 60% of PC are digested. This amount correspondsto phospholipid molecules localized in the outer monolayer. This important phospholipid depletion causes only about 20-25% inhibition of the non-cyclic and PSII electron flow activities which can be restored partially in the presence of BSA. The activity begins to be inhibited greatly only when the hydrolysis of phospholipids located in the inner monolayer occurs (due to rapid transbilayer movement at 20°C, see Fig. 2). In the presence of BSA, the restoration of the activity is only partial suggesting that phospholipid depletion in the inner monolayer of the membrane is the cause of the BSA-insensitive inhibition. 

Williamson, P., Schlegel, R. A. Transbilayer phospholipid movement and the clearance of apoptotic cells. Biochim. Biophys. Acta 1585 53-63, 2002. 

Additional evidence for slow transbilayer phospholipid movement in liposomes came from experiments using [ H]PC-labeled liposomes and PC transfer protein. In the presence of excess unlabeled acceptor membranes, only the PC in the outer leaflet of the liposome membrane was rapidly transferred (J.E. Rothman, 1975). The [ H]PC initially present in the inner leaflet of the membrane moved to the outer leaflet with a r,/2 of... 

Additional work with closed vesicles derived from B. megaterium membranes demonstrates that NBD analogs of PE, PG, and PC can translocate across the membrane with a /i/2 of 30 s at 37°C (S. Hraffnsdottir, 1997). Similar types of experiments conducted with closed vesicles isolated from E. coli inner membrane reveal that NBD phospholipids traverse the bilayer with a of 7 min at 37°C (R. Huijbregts, 1996). This latter process is insensitive to protease and A-ethylmaleimide treatments and does not require ATP. Collectively, the data indicate that transbilayer lipid movement is rapid and does not require metabolic energy in bacterial membranes that harbor the biosynthetic enzymes for phospholipids. The basic characteristics of lipid translocation in the intact cell appear to be retained in isolated membranes. 

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