ATP-dependent asymmetric distribution of spin-labeled phospholipids in the erythrocyte membrane: relation to shape changes.

Abstract

Spin-labeled analogs of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine have been used to study phospholipid transverse diffusion and asymmetry in the human erythrocyte membrane. Ascorbate reduction was used to assess the transbilayer distribution of the labels. All three spin-labeled phospholipids initially incorporated into the outer leaflet of the membrane. On fresh erythrocytes at 50C, the phosphatidylcholine label remained mainly in the outer leaflet. In contrast, the phosphatidylserine and phosphatidylethanolamine labels underwent rapid transverse diffusion that led to their asymmetric distribution in favor of the inner leaflet. The latter effect was reversibly inhibited after ATP depletion of the erythrocytes and could be reproduced on resealed erythrocyte ghosts only if hydrolyzable Mg-ATP was included in the internal medium. It is suggested that an ATPdriven transport of amino phospholipids toward the inner leaflet could be the major cause of the phospholipid asymmetry in the erythrocyte membrane. It is also proposed that the same mechanism could explain the ATP requirement of the maintenance of the erythrocyte membrane discoid shape. that this method can be used successfully in erythrocytes, and the transverse diffusion of phosphatidylcholine has been measured. In the present study, the following phospholipid spin labels have been used.These phospholipids possess an unmodified polar head group R that can be either choline, serine, or ethanolamine and areAn asymmetric distribution of phospholipids exists between the two halves of many biological membranes (for review, see ref. 1). The human erythrocyte membrane appears to be the best characterized in this regard. Numerous studies using chemical labeling (2, 3) and phospholipases (4-6) indicate that, in this membrane, phosphatidylcholine and sphingomyelin are distributed in favor of the outer leaflet, while phosphatidylethanolamine and phosphatidylserine are mainly located in the inner leaflet.These observations raise the problem of how this asymmetry is maintained during the 120-day life span of the erythrocyte. The asymmetric distribution could in principle be maintained if transbilayer diffusion of phospholipids was negligible. However, for phosphatidylcholine, transbilayer "flip-flop" has been shown to occur with a half-time of 8-15 hr in the native membrane (7,8), although no such measurements exist for the three other major phospholipid species. A possible role of cytoskeletal proteins in maintaining the asymmetry in spite of transbilayer diffusion has been proposed by Haest et al. (9,10), but no direct evidence for such a process has been observed in the intact native membrane.In the present study, the formation and maintenance of phospholipid asymmetry in the erythrocyte membrane has been addressed by measuring the transverse diffusion of spin-labeled phospholipids bearing different polar head groups. The method, introduced by Kornberg and McConnell (11), is based on the accessibility of membrane-associated spin labe...

P4-ATPase Requirement for AP-1/Clathrin Function in Protein Transport from the trans-Golgi Network and Early Endosomes

“…As little as a 1-2% difference in surface area between leaflets of a bilayer can have a substantial impact on membrane shape (Lim et al, 2002). Consistent with this hypothesis, bilayer surface area asymmetry induced by insertion of additional phospholipid in the extracellular leaflet of the plasma membrane and their subsequent flip to the cytosolic leaflet, catalyzed by the aminophospholipid translocase, leads to dramatic changes in the shape of red blood cells (Seigneuret and Devaux, 1984;Daleke and Huestis, 1985). Translocase-mediated influx of phospholipid to the cytosolic leaflet of the plasma membrane also stimulates endocytosis (Farge et al, 1999).…”

Glycerolipid transfer for the building of membranes in plant cells

“…They maintain bilayer symmetry by contrast with ATP-dependent 8 translocases that can accumulate specific lipid classes against equilibrated gradients. ATPdependent translocases are involved in the preservation of membrane asymmetry [16].…”

Spontaneous lipid transfer between organized lipid assemblies

“…12-16), with spinlabeled lipids (e.g., Refs. [17][18][19][20][21][22]. and with lipids bearing fluorescent dyes (e.g., [23][24][25][26][27][28][29][30].…”

“…As was pointed out during my earlier discussion of cholesterol interbilayer exchange, the membrane lipid composition does influence the distribution of individual lipids between membranes rather dramatically. Furthermore, it is now clear that certain membranes contain energy-dependent lipid flippases that are capable of altering the composition of specific lipids within each half of the membrane [19,[58][59]. Thus, potential mechanisms for minimizing random intracellular spontaneous lipid transfer do exist.…”