Phospholipid translocator proteins

If recently synthesized phospholipid molecules remained only on the cytoplasmic surface of the ER, a monolayer would form. Unassisted bilayer transfer of phospholipid, however, is extremely slow. (For example, half-lives of 8 days have been measured across artificial membrane.) A process known as phospholipid translocation is now believed to be responsible for maintaining the bilayer in membranes (Figure 12F). Transmembrane movement of phospholipid molecules (or flip-flop), which may occur in as little as 15 seconds, appears to be mediated by phospholipid translocator proteins. One protein (sometimes referred to as flippase) that transfers choline-containing phospholipids across the ER membrane has been identified. Because the hydrophilic polar head group of a phospholipid molecule is probably responsible for the low rate of spontaneous translocation, an interaction between flippase and polar head groups is believed to be involved in phosphatidylcholine transfer. Translocation results in a higher concentration of phosphatidylcholine on the lumenal side of the ER membrane than that... 

Phospholipid translocator proteins, phospholipid exchange proteins, and transition vesicles are involved in the complicated process of membrane synthesis and delivery of membrane components to their cellular destinations. 

Membrane phospholipids are synthesized on the cytoplasmic side of SER membrane. Because the polar head groups of phospholipid molecules make transport across the hydrophobic core of a membrane an unlikely event, a translocation mechanism is used to transfer phospholipids across the membrane to ensure balanced growth. Choline-containing phospholipids are found in high concentration on the lumenal side of ER membrane because a prominent phospholipid translocator protein called flippase preferentially transfers this class of molecule. 

While examples such as these provide evidence that strong interactions of negatively-charged membrane lipids with membrane proteins the role in maintaining asymmetric distributions of lipids aaoss biological membranes is unclear. In any event such effects are likely to be of minor importance relative to actively mediated phospholipid translocation processes. 

The phosphatidylcholine in bile is synthesised in the endoplasmic reticulum of the hepatocyte and must be transported to the canalicular membrane. One possibility involves the nonspecific phosphatidylcholine transfer protein but a mouse null for this protein did not show reduced phosphatidylcholine secretion into bile and there was no compensatory increase in other phospholipids transfer proteins. However, the plasma membrane would receive a ready supply of phospholipid by insertion of vesicles, and the MDR3 protein translocates this molecule from the inner leafiet to the outer surface where there is contact with bile acids, as suggested by Smit and colleagues. The role of this transporter is shown in Figure 2.2. 

Reconstitutions are technically simple, require little training, and teach you how to handle detergents, phospholipids, membranes, and membrane proteins. Once the reconstitution has succeeded, you can observe your protein in a defined environment, free of influences from the cell metabolism. Afterward, you can construct in vitro entire metabolic chains or processes and thus build your own organelle. Sometimes reconstitution of the translocator function and flux assay is a prerequisite to the purification of the translocator protein. Figure 4.2 shows the possibilities of this field. 

To demonstrate an application of TIRF-FLIM, a FRET study of annexin A4 translocation and self-aggregation near the plasma membrane is shown in Fig. 9.4. This is a particularly useful application of TIRF-FLIM, since TIRF provides the spatial contrast of detecting only molecules immediately adjacent to the plasma membrane and the lifetime contrast reports on the aggregation state of annexin A4. Annexins are calcium-dependent lipid-binding domains with a different type of lipid binding domain compared to the common C2 domains (e.g., found in protein kinase C). Annex-ins consist of an N-terminal domain and a core domain binding calcium and phospholipids. The core domain is conserved in the... 

In many eukaryotic plasma membranes, PS resides in the inner leaflet (Schroit and Zwaal, 1991 Zachowski, 1993). This transbilayer distribution of membrane hpids is not a static situation but a result of balance between the inward and outward translocation of phospholipids across the membranes. Recent studies showed that the transbilayer lipid asymmetry is regulated by several lipid transporter proteins, such as aminophospholipid translocase (Daleke and Lyles, 2000), ATP-binding cassette transporter family (van Helvoort et al, 1996 Klein et al, 1999), and phospholipid scramblase (Zhou et al, 1997 Zhao et al, 1998). An increment of intracellular due to cell activation, cell injury, and apoptosis affects the activities of these transporters, resulting in exposure of PS (Koopman et al, 1994 Verhoven et al, 1995) and PE (Emoto et al, 1997) on the cell surface. 

Kamp, D. and Haest, C.W.M., 1998, Evidence for a role of the multidrug resistance protein MRP in the outward translocation of NBD-phospholipids in the erythrocyte membrane, Biochim. Biophys. Acta 1372 91-101. 

Lipids and proteins can shift easily within one layer of a membrane, but switching between the two layers ( flip/flop") is not possible for proteins and is only possible with difficulty for lipids (with the exception of cholesterol). To move to the other side, phospholipids require special auxiliary proteins (translocators, flipases ). 

Figure 15-2. A simplified schematic of cholesterol transport. Cholesterol travels to non-hepatic cells, such as the macrophage, via VLDL and LDL particles, while excess cholesterol is shuttled to the liver via HDL particles. Note that AHCAl mediates nascent HDL formation by translocating cellular cholesterol and phospholipids to apolipoprotein A-I (apoA-I) in an active, energy-dependent reaction. CETP, cholesteryl ester transfer protein LCAT, lecithinxholesterol acyltransferase LDLR, low-density lipoprotein receptor SR-B1, scavenger receptor Bl.

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