Cytoplasmic leaflet

Shapiro, A. B., Ling, V., Extraction of Hoechst 33342 from the cytoplasmic leaflet of the plasma membrane by P-glycoprotein, Eur. J. Biochem. 1997, 250, 122-129. 

Because membranes components participate in nearly every cell activity their structures are also dynamic and far from the equilibrium states that are most readily understood in biophysical terms. Newly synthesized bilayer lipids are initially associated with endoplasmic reticulum (Ch.3) whereas phospholipids initially insert into the cytoplasmic leaflet while cholesterol and sphingolipids insert into the luminal endoplasmic reticulum (ER) leaflet. Glycosylation of ceramides occurs as they transit the Golgi compartments, forming cerebrosides and gangliosides in the luminal leaflet. Thus, unlike model systems, the leaflets of ER membranes are asymmetric by virtue of their mode of biosynthesis. 

The membrane-associated small G proteins H-Ras and K-Ras have been studied with respect to their association with cytoplasmic leaflets. These two proteins have nearly identical structures and functions but different membrane anchors, membrane distributions and effector responses. Application of the FRAP method to fluorescent constructs of H-Ras and K-Ras revealed that only H-Ras in its guanosine 5 diphosphate (GDP)-bound form associates with cholesterol-dependent rafts [26]. 

Figure 1. Percent distribution of major phospholipids between the outer and cytoplasmic leaflets of the human erythrocyte membrane. SM, sphingomyelin PC, phosphatidylcholine PE, phosphatidylethanolamine PS, phosphatidylserine PI, phosphatidylethanolamine...

It can be seen from Figure 1 that the choline-containing phospholipids, phosphatidylcholine and sphingomyelin are localized predominantly in the outer monolayer of the plasma membrane. The aminophospholipids, conprising phosphatidylethanolamine and phosphatidylserine, by contrast, are enriched in the cytoplasmic leaflet of the membrane (Bretcher, 1972b Rothman and Lenard, 1977 Op den Kamp, 1979). The transmembrane distribution of the minor membrane lipid components has been determined by reaction with lipid-specific antibodies (Gascard et al, 1991) and lipid hydrolases (Biitikofer et al, 1990). Such studies have shown that phosphatidic acid, phosphatidylinositol and phosphatidylinositol-4,5-fc -phosphate all resemble phosphatidylethanolamine in that about 80% of the phospholipids are localized in the cytoplasmic leaflet of the membrane. 

The maintenance of an asymmetric distribution of phospholipids across the plasma membrane with choline phospholipids predominating on the external surface and amino phospholipids confined to the cytoplasmic leaflet of the membrane has now been well estabhshed. The participation of... 

We have encountered examples of simple lipid bilayers earlier. These bilayers are composed largely of amphipathic molecules. In water, they have their hydrophobic parts occupying the center of the bilayer and their hydrophilic parts occupying the bilayer surface. Such bilayers form a continuous and essential structural feature of virtually all biological membranes. We need to distinguish between that layer which faces out from the cell and is in contact with the external environment, the exoplasmic leaflet, and that which faces in and is in contact with the cellular contents, the cytoplasmic leaflet. As we shall see, these two aspects of the lipid bilayer are quite distinct. 

Now it would make no sense at all to have the insnhn receptor turned end for end in a biological membrane. That would put the insulin recognition site on the inside on the cell, where it would be useless, and the intracellular domain with its kinase activity on the ontside, where it would be equally useless. It follows that aU insulin receptors have the same, specific orientation with respect to the bilayer leaflets, with the receptor site adjacent to the exoplasmic leaflet and the kinase site adjacent to the cytoplasmic leaflet. 

Plasma membrane lipids are asymmetrically distributed between the two monolayers of the bilayer, although the asymmetry, unlike that of membrane proteins, is not absolute. In the plasma membrane of the erythrocyte, for example, choline-containing lipids (phosphatidylcholine and sphingomyelin) are typically found in the outer (extracellular or exoplasmic) leaflet (Fig. 11-5), whereas phosphatidylserine, phosphatidyl-ethanolamine, and the phosphatidylinositols are much more common in the inner (cytoplasmic) leaflet. Changes in the distribution of lipids between plasma membrane leaflets have biological consequences. For example, only when the phosphatidylserine in the plasma membrane moves into the outer leaflet is a platelet able to play its role in formation of a blood clot. For many other cells types, phosphatidylserine exposure on the outer surface marks a cell for destruction by programmed cell death. 

P. H. M. Lommerse, G. A. Blab, L. Cognet, G. S. Harms, E. B. Snaar-Jagalska, H. P. Spaink and T. Schmidt, Single-molecule imaging of lipid-anchored proteins reveals domains in the cytoplasmic leaflet of the cell membrane, Biophys. J. 86, 609 (2004). 

An asymmetric lipid distribution between the exoplasmic and cytoplasmic leaflets of plasma membranes is typical (Devaux, 1991). Por example, it is well documented that highly unsaturated species of PE and PS are found primarily on the inner leaflet of many membranes. This has been reported for human erythrocytes (Knapp et al., 1994), murine synaptosomal plasma membranes (Pontaine et al., 1980), and human lymphocytes (Bougnoux et al., 1985), among others. It has also been shown that polyunsaturated fatty acids, including DHA, are found in higher concentrations in the aminophospholipids in the inner, cytoplasmic leaflet (Crinier et al., 1990 Hullin et al., 1991). As a result, the cytoplasmic leaflet of erythrocytes is more fluid than the exoplasmic leaflet (Morrot et al., 1986). The addition of polyunsaturated fatty acids to membranes have been shown to translocate cholesterol to the outer leaflet, where its efflux from membranes is enhanced (Dusserre et al., 1995). 

A tentative alignment of the M2 sequence with the densities in the cytoplasmic leaflet suggests that a line of small polar (serine or threonine) residues would lie almost parallel to the axis of the pore when the channel opens (Fig. 11.7), an orientation that should stabilize the passing ions by providing an environment of high polarizability. The threonine residue at the point of maximum constriction (Tor-pedo aT244), when substituted by other residues of different volume, has a pronounced effect on ion flow, as if it were at the narrowest part of the open pore (3 3). The diameter of this most constricted portion of the channel, based on permeability measurements made with small uncharged molecules of different size, is about 10 A (34, 35). This value is similar to that indicated by the structural results. 

Figure 11.7. Transient configuration of M2 helices around the open pore, (a) A barrel of a-helical segments, having a pronounced twist, forms in the cytoplasmic leaflet of the bilayer, constricting the pore maximally at the cytoplasmic membrane surface. The bend in the rods is at the same level as for the closed pore, but instead of pointing inward has rotated over to the side, (b) Schematic representation of the most distant three rods. A tentative alignment of the amino acid sequence with the densities suggests that a line of polar residues (serines and threonine see Fig. 11.3) should be facing the open pore. [From Unwin (31).]...

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