Transbilayer distribution and mobility of phosphatidylinositol in human red blood cells.

The present studies describe the distribution of phosphatidylinositol (PI) within the membrane bilayer of the human red blood cell (RBC) as well as its transbilayer mobility. The membrane bilayer distribution was determined by measuring the hydrolysis of PI in the exterior leaflet of the RBC membrane using a PI-specific phospholipase C and by extraction of PI from the exterior leaflet using bovine serum albumin. The transbilayer mobility of PI was measured by following the fate of radiolabeled PI which was first incorporated into the outer leaflet of the RBC membrane. Our results indicate that PI is asymmetrically distributed in the membrane, with approximately 80% located in the inner and 20% in the outer leaflet of the bilayer. The rate of transbilayer mobility of PI is similar to that for certain molecular species of phosphatidylcholine and much slower than that reported for the aminophospholipids in the RBC membrane.

The present studies describe the distribution of phosphatidylinositol (PI) within the membrane bilayer of the human red blood cell (RBC) as well as its transbilayer mobility.
The membrane bilayer distribution was determined by measuring the hydrolysis of PI in the exterior leaflet of the RBC membrane using a PI-specific phospholipase C and by extraction of PI from the exterior leaflet using bovine serum albumin. The transbilayer mobility of PI was measured by following the fate of radiolabeled PI which was first incorporated into the outer leaflet of the RBC membrane.
Our results indicate that PI is asymmetrically distributed in the membrane, with approximately 80% located in the inner and 20% in the outer leaflet of the bilayer. The rate of transbilayer mobility of PI is similar to that for certain molecular species of phosphatidylcholine and much slower than that reported for the aminophospholipids in the RBC membrane.
The major phospholipid classes of the human red blood cell (RBC)' membrane, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin, account for more than 94% of total membrane phospholipid. These phospholipids are distributed asymmetrically over the two halves of the lipid bilayer. The aminophospholipids PE and PS are largely confined to the inner leaflet of the bilayer, whereas the choline-containing phospholipids PC and sphingomyelin are predominantly localized in the outer leaflet (Roelofsen, 1982). This asymmetry appears to be generated and maintained by an ATP-dependent translocation of aminophospholipids from outer to inner leaflet (Seigneuret and Devaux, 1984;Daleke and Huestis, 1985;Connor and Schroit, 1988;Middelkoop et al., 1988), and by interaction of aminophospholipids with RBC membrane skeletal proteins (Haest, 1982;Tilley et al., 1986;Middelkoop et al., 1988). The outside-inside translocation rate is rapid for PS and PE, with half-times (&) of less than 10 min and approximately 50 min, respectively. In contrast, translocation of PC across the bilayer occurs at a slower rate (tlh = 3-26 h) and seems not to involve a specific translocase protein (van Meer and Op den Kamp, 1982;Middelkoop et al., 1986;Morrot et al., 1989).
The phosphoinositides, phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylino-sitol4,5-bisphosphate (PIP2), account for 3-4% of total RBC membrane phospholipid. Despite representing a minor membrane component in most cells, phosphoinositides are metabolically very active and often play an important role in transmembrane signal transduction (reviewed by Majerus et al., 1986). In addition, RBC phosphoinositides may be involved in maintaining RBC membrane structure and function (Ferrell and Huestis, 1984;Anderson and Marchesi, 1985). Although it is believed that the phosphoinositides are confined to the cytoplasmic side of the red cell membrane, in proximity to the enzymes and substrates that interconvert these lipid classes (Mack and Palmer, 1988;Ling et al., 1989), neither their organization within the bilayer nor their potential transbilayer mobility has been investigated in human erythrocytes. To determine the transbilayer distribution and mobility of PI in human RBC, we measured the accessibility of endogenous as well as newly introduced PI to the action of extracellular PI-specific phospholipase C (PI-PLC) and to back-extraction by bovine serum albumin (BSA). Lipids were separated by two-dimensional thin layer chromatography as described before (Biitikofer et al., 1989). Lipid phos-phorus was determined according to Bartlett (1959 (phosphoryl) [3H]inositol. Even if we assume that all radiolabel was released as free inositol and subsequently entered the cell, it could not have been used for resynthesis of PI at the inner leaflet of the membrane because mature RBC membranes are unable to synthesize PI from its precursors, inositol and CDP-diglyceride or phosphatidate (Percy et al., 1973;Shohet and Nathan, 1970). Therefore, even if spontaneous hydrolysis of [3H]PI had occurred, it would not have affected our results.
The metabolic fate of the ["HIP1 introduced into RBC was determined by analyzing the different RBC phosphoinositide classes (PI, PIP, PIP*). At the beginning of incubation, almost all the radiolabel was recovered in the PI fraction (Fig. 3A). After 25 h of incubation, the relative amount of radioactivity in PI decreased to about 85% of label in the phosphoinositides (Fig. 3A) and the radiolabel was detected in PIP (6%) and PIP, (9%) (Fig. 3B). This process seemed to reverse slightly after prolonged incubation (Fig. 3, A and B Mohandas et al., 1982;Morrot et al., 1989), PI can be extracted from membranes with BSA. Thus, the amount of endogenous PI located in the exterior leaflet of the RBC membrane detected by PI-PLC was confirmed by BSA extraction of intact RBC (i.e. 18% of PI). The extractability of PI from RBC membranes by BSA suggests that it may be available for exchange between membranes and plasma in uiuo.
To determine the transbilayer mobility of PI in human RBC, we incorporated trace amounts of [3H]PI into RBC and followed its transbilayer distribution by measuring its accessibility to hydrolysis by extracellular PI-PLC and to backextraction by BSA. Between 85 and 95% of incorporated [3H] PI could be hydrolyzed from labeled RBC by PI-PLC or was removed by BSA (Figs. 1B and 2). These results indicate that after the initial labeling procedure most of the exogenously added PI was located in the outer leaflet of the membrane bilayer. The [3H]PI not hydrolyzed by PI-PLC likely represents the same pool of radiolabeled PI that was not extracted by BSA which suggests that a small amount of exogenously added PI translocated from outer to inner leaflet of the membrane bilayer during the labeling procedure. The small increase in the amount of [3H]PI removed from RBC during prolonged incubation with BSA ( Fig. 1B) is consistent with a limited redistribution of cytoplasmic label to the outer half of the membrane bilayer.
The accessibility of incorporated [3H]PI to hydrolysis by PI-PLC decreased to approximately 22% of the total after 48 h (Fig. 2), indicating that approximately 78% of the radiolabeled PI had been translocated from outer to inner leaflet of the membrane bilayer. The kinases and hydrolases that phosphorylate and dephosphorylate phosphoinositides within the RBC membrane are located intracellularly, either in the cytosol or in association with the membrane (Mack and Palmer, 1988;Ling et al., 1989). Our detection of incorporated radiolabel in the polyphosphoinsitides (PIP and PIPJ (Fig. 3) is additional evidence that part of the [3H]PI had translocated to the inner leaflet of the RBC membrane and became accessible to phosphorylation. The low level of redistribution of the radiolabel into PIP and PIP2 is not surprising since recent reports indicate that only a fraction of the phosphoinositide classes participate in a pool of rapid metabolic turnover (Muller et al., 1986;King et al., 1987King et al., , 1989. Since the distribution of tracer [3H]PI in the inner leaflet of the RBC membrane bilayer at equilibrium (78%) was similar to that of endogenous PI in the inner leaflet (76-82%), this marker was used to determine the transbilayer movement of endogenous PI. To calculate the transbilayer mobility of PI in RBC, we analyzed our data using a two-pool closed model system. The mathematical equation describing the transbilayer movement of PI from the outer (pool A) to the inner leaflet (pool B) was derived from the standard formulas of compartmental analysis (Shipley and Clark, 1972). In the case where all the radiolabel starts out in pool A, and considering the bilayer distribution of endogenous PI as well as the distribution of [3H]PI at equilibrium, the fraction of label at time t in pool A (QA,) relative to the fraction exchangeable label (QA,) in the outer leaflet can be given by the equation: QAI = 0.7f&-Q.W4 + 0.22

Q
(1) 47 The graphs obtained from this mathematical model fit our experimental data for transbilayer movement of [3H]PI from outer to inner leaflet of the RBC membrane bilayer (Fig. 2, solid line). The calculated half-time for t3H]P1 to reach equilibrium between the two leaflets of the membrane is 173 min.
Our results indicate that the bilayer asymmetry of PI in human RBC is similar to that reported for PE (Roelofsen, 1982). However, PE is actively transported across the membrane bilayer by a putative aminophospholipid translocase (Seigneuret and Devaux, 1984;Daleke and Huestis, 1985;Zachowski et al., 1986;Connor and Schroit, 1988) with a halftime for translocation of approximately 50 min. In contrast, the rate of translocation of exogenously added PI is slow, with a halftime in the order of 3 h. This rate is comparable to that for translocation of certain PC molecular species across the RBC membrane bilayer (van Meer and Op den Kamp, 1982;Middelkoop et al., 1986;Morrot et al., 1989). The slow rate of translocation of PI across the membrane bilayer suggests that, like PC, PI is not actively transported across the RBC membrane bilayer. Therefore, in contrast to the aminophospholipids (Seigneuret and Devaux, 1984;Daleke and Huestis, 1985;Zachowski et al., 1986;Connor and Schroit, 1988), the asymmetric distribution of PI can not be explained by a fast and active transport across the membrane bilayer. It has been reported that PI is tightly bound to glycophorin (Yeagle and Kelsey, 1989) and that the state of phosphorylation of PI regulates the affinity of glycophorin for protein 4.1, a membrane skeletal protein (Anderson and Marchesi, 1985). It is possible that such interactions, at the cytoplasmic side of the membrane, prevent the equilibration of PI between the two leaflets of the membrane bilayer and help maintain the asymmetric distribution of PI in the human RBC.