Acidic phospholipids strikingly potentiate sterol carrier protein 2 mediated intermembrane sterol transfer

A liposomal membrane model system was developed to examine the mechanism of spontaneous and protein-mediated intermembrane cholesterol transfer. Rat liver sterol carrier protein 2 (SCP2) and fatty acid binding protein (FABP, also called sterol carrier protein) both bind sterol. However, only SCP2 mediates sterol transfer. The exchange of sterol between small unilamellar vesicles (SUV) containing 35 mol % sterol was monitored with a recently developed assay [Nemecz, G., Fontaine, R. N., & Schroeder, F. (1988) Biochim. Biophys. Acta 943, 511-541], modified to continuous polarization measurement and not requiring separation of donor and acceptor membrane vesicles. As compared to spontaneous sterol exchange, 1.5 microM rat liver SCP2 enhanced the initial rate of sterol exchange between neutral zwwitterionic phosphatidylcholine SUV 2.3-fold. More important, the presence of acidic phospholipids (2.5-30 mol %) stimulated the SCP2-mediated increase in sterol transfer approximately 35-42-fold. Thus, acidic phospholipids strikingly potentiate the effect of SCP2 by 15-18 times as compared to SUV without negatively charged lipids. Rat liver FABP (up to 60 microM) was without effect on sterol transfer in either neutral zwitterionic or anionic phospholipid containing SUV. The potentiation of SCP2 action by acidic phospholipids was suppressed by high ionic strength, neomycin, and low pH. The results suggest that electrostatic interaction between SCP2 and negatively charged membranes may play an important role in the mechanism whereby SCP2 enhances intermembrane cholesterol transfer.     

The protein-mediated transfer of phosphatidylcholine between membranes. The effect of membrane lipid composition and ionic composition of the medium.

The phosphatidylcholine exchange protein from bovine liver catalyzes the transfer of phosphatidylcholine between rat liver mitochondria and sonicated liposomes. The effect of changes in the liposomal lipid composition and ionic composition of the medium on the transfer have been determined. In addition, it has been determined how these changes affected the electrophoretic mobility i.e. the surface charge of the membrane particles involved. Transfer was inhibited by the incorporation of negatively charged phosphatidic acid, phosphatidylserine, phosphatidylglycerol and phosphatidylinositol into the phosphatidylcholine-containing vesicles; zwitterionic phosphatidyl-ethanolamine had much less of an inhibitory effect while positively charged stearylamine stimulated. The cation Mg2+ and, to a lesser extent, K+ overcame the inhibitory effect exerted by phosphatidic acid, in that concentration range where these ions neutralized the negative surface charge most effectively. Under conditions where Mg2+ and K+ affected the membrane surface charge relatively little inhibition was observed. In measuring the protein-mediated transfer between a monolayer and vesicles consisting of only phosphatidylcholine, cations inhibited the transfer in the order La3+ greater than Mg2+ larger than or equal to Ca2+ greater than K+ = Na+. Inhibition was not related to the ionic strength, and very likely reflects an interference of these cations with an electrostatic interaction between the exchange protein and the polar head group of phosphatidylcholine.     

Purification and characterization of a phosphatidylinositol transfer protein from human platelets

We report the purification of a phospholipid transfer protein from human platelets. This protein preferentially transfers phosphatidylinositol, with phosphatidylcholine and phosphatidylglycerol being transferred to a lesser extent. Phosphatidylethanolamine is not transferred. Transfer activity is detected by measuring the transfer of radiolabeled phospholipids between two populations of small unilamellar vesicles. The protein was purified approximately 1000-fold over the platelet cytosol by chromatography on Sephadex G-75, sulfooxyethyl cellulose, and hydroxylapatite. The molecular weight of this protein appears to be 28 000 as determined by gel filtration chromatography. When the purified protein is analyzed on sodium dodecyl sulfate-polyacrylamide gels, two major components and several minor ones are observed. The molecular weight of the two major bands are 28 600 and 29 200. Isoelectric focusing of the platelet cytosol yielded phosphatidylinositol and phosphatidylcholine transfer activity at pH 5.6 and 5.9. The platelet phospholipid transfer protein is able to catalyze the transfer of phosphatidylinositol and phosphatidylcholine between vesicles and human platelet plasma membranes. One possible physiological role for this transfer protein is an involvement in the rapid turnover of inositol-containing lipids which occurs upon exposure of platelets to various stimuli.     

Stimulation of cholinephosphotransferase activity by phosphatidylcholine transfer protein. Regulation of membrane phospholipid synthesis by a cytosolic protein

The effect of rat liver phosphatidylcholine transfer protein on the incorporation of CDP-choline and dioleoylglycerol into phosphatidylcholine catalyzed by rat liver microsomal CDP-choline: 1,2-diacyl-sn-glycerol cholinephosphotransferase was studied. In the presence of phosphatidylcholine transfer protein, the incorporation of CDP-choline into phosphatidylcholine was markedly stimulated. Phosphatidylcholine transfer protein isolated from either rat or bovine liver was capable of this stimulatory effect; in contrast, phosphatidylinositol transfer protein from rat liver had no effect on phosphatidylcholine synthesis. Kinetic analysis showed that microsomal phosphatidylcholine synthesis increased 2.4-fold after 1 min and reached a maximum of approximately 10-fold within 10 min in the presence of phosphatidylcholine transfer protein; in the absence of this protein phosphatidylcholine synthesis stopped after 2-4 min. These results suggest that phosphatidylcholine transfer protein permits phosphatidylcholine synthesis to proceed further. With the addition of phospholipid vesicles, as an acceptor membrane in the reaction mixture, there was a significant amount of protein-mediated transfer of synthesized phosphatidylcholine to the vesicles. Measurable transfer of synthesized phosphatidylcholine to vesicles could only be detected after a lag of 2-4 min. The stimulation of cholinephosphotransferase could be nearly abolished by increasing the amount of added phospholipid vesicles; concurrently, a greater transfer to the vesicles was observed. These results describe a new property of phosphatidylcholine transfer protein which may be of physiological significance in the regulation of phosphatidylcholine synthesis in mammalian tissues.     

Phosphatidylinositol exchange protein. Effects of membrane structure on activity and evidence for a ping-pong mechanism.

Phosphatidylinositol exchange protein, purified from bovine cerebral cortex, catalyzes the transfer of phosphatidylinositol and, to a lesser extent, phosphatidylcholine between rat liver microsomes and egg phosphatidylcholine liposomes. Transfer activity is sensitive to pH, temperature, and the method of liposome preparation. Variation of the phospholipid composition of the liposomes produces vesicles for which the apparent Michaelis constant decreases with increasing molar proportions of phosphatidylinositol. Interaction of exchange protein with liposomes containing radioactively labeled phosphatidylcholine allows the isolation of a phospholipid-protein complex; dissociation of this complex occurs upon subsequent interaction with unlabeled liposomes. Changes in the concentration of the two membrane species, microsomes and liposomes, yield results which are interpreted in terms of a ping-pong kinetic mechanism for the protein-catalyzed, intermembrane transfer of phospholipids.     

An assay for the alpha-tocopherol binding protein mediated transfer of vitamin E between membranes

A model system consisting of donor membrane (egg lecithin liposomes) and acceptor membrane (human erythrocyte ghosts or rat liver mitochondria) were used to investigate the alpha-tocopherol binding protein (alpha TBP) mediated transfer of alpha-tocopherol. Liposomes containing RRR-[alpha-3H]tocopherol ([alpha-3H]T) were incubated with acceptor membrane at 37 degrees C for 0-45 min in the presence or absence of rat liver cytosol or a dialyzed 30-60% saturated ammonium sulfate precipitated fraction of rat liver cytosol (Fraction B). Erythrocyte ghosts and liver mitochondria were compared and found to behave similarly in the presence of Fraction B. alpha-Tocopherol transfer activity (alpha TTA) typically varied 0- to 27-fold greater than buffer blanks, depending upon type and concentration of protein preparation. Gel filtration of Fraction B yielded one alpha TTA peak (liver mitochondria as acceptor) with an estimated Mr of 39,000. [alpha-3H]T recovered from erythrocyte ghosts pellets by HPLC suggest that the [alpha-3H]T was transferred intact. alpha TTA of Fraction B in the presence of varying concentrations of erythrocyte ghosts and liposomal [alpha-3H]T followed saturation kinetics. Optimal concentrations gave alpha TTA responses directly proportional to rat liver cytosol concentration. alpha TTA was inhibited only 5% in the presence of a 32-fold excess of cold liposomal alpha-tocopheryl acetate suggesting that the free hydroxyl group on the chromanol ring of alpha-tocopherol is needed for transfer. Coefficient of variation of repeated measures of alpha TTA in rat liver cytosol was 2.9%. Thus, the intermembrane transfer phenomenon of alpha-tocopherol can be studied quantitatively and can be used to compare liver protein preparations exhibiting transfer activity.     

Activation of phosphorylating vesicles by net transfer of phosphatidyl choline by phospholipid transfer protein

Vesicles formed with phosphatidyl ethanolamine, phosphatidyl choline, cardiolipin, coupling factors and hydrophobic proteins from bovine heart mitochondria catalyzed a rapid³²P_i-ATP exchange. When phosphatidyl choline was deleted during the assembly of the vesicles, little³²P_i-ATP exchange was observed. Exchange activity was induced by incubating such deficient vesicles with phosphatidyl choline liposomes in the presence of a phosphatidyl choline transfer protein isolated from bovine heart. Transfer of [³²P] phosphatidyl choline was demonstrated by isolation of the activated vesicles by sucrose density centrifugation.     

Specificity of the phosphatidylcholine exchange protein from bovine liver

The phosphatidylcholine exchange protein from bovine liver stimulates the specific transfer of phosphatidylcholine (PC) from rat liver microsomes to mitochondria or phospholipid vesicles (Wirtz, K.W.A., Kamp, H.H., and van Deenen, L.L.M. (1972), Biochim. Biophys. Acta 274, 606). In the present study, it has been established which components of the PC molecule are essential to the specific interaction with the protein. Radiochemically labeled analogues of PC have been synthesized with modifications in the polar and apolar moiety, and their transfer was measured between donor and acceptor vesicles. Relative to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (egg yolk PC), transfer is inhibited or abolished when (a) the distance between phosphorus and nitrogen is decreased or increased and (b) a methyl group on the quaternary nitrogen is removed or substituted by an ethyl or propyl group. Transfer is much less affected when (a) the ester bonds are replaced by ether or carbon-carbon bonds, (b) the PC molecule contains two saturated fatty acids, and (c) the D stereoisomer is used. It is concluded that the protein has a binding site which interacts specifically with the phosphorylcholine head group and which cannot accommodate substantial configurational changes. Interaction with the apolar moiety of PC is less specific. However, lyso-PC is not transferred, suggesting that two hydrocarbon chains are required to stabilize the exchange protein-phospholipid complex. Interaction of [14C]PC-labeled exchange protein with vesicles of different phospholipid compositon has been analyzed by measuring the release of [14C]PC into these vesicles. Vesicles of egg PC or dimethylphosphatidylethanolamine function as acceptors, in contrast to vesicles of sphingomyelin or phosphatidylethanolamine.     

The non-specific lipid transfer protein (sterol carrier protein 2) from rat and bovine liver

The non-specific lipid transfer protein (nsL-TP) purified from rat and bovine liver accelerates the transfer of all common diacylglycerophospholipids, cholesterol as well as glycosphingolipids and gangliosides between membranes. These proteins have molecular weights in the order of 14 500 and are highly basic (isoelectric points between 8.5 and 9.5). The primary structure of nsL-TP from bovine liver has been elucidated yielding a single polypeptide chain of 121 aminoacid residues. The protein contains one cysteine residue, essential for transfer activity, a single tryptophan residue and lacks histidine, arginine and tyrosine residues. Rat liver nsL-TP was found to be identical to sterol carrier protein 2, stimulating the microsomal conversion of intermediates between lanosterol and cholesterol. Evidence was presented that nsL-TP binds cholesterol, suggesting that it acts as a carrier. On the other hand, failure to bind phospholipids disagrees with this proposed mode of action. A sensitive enzyme immunoassay was developed to determine levels of nsL-TP in rat tissues. By use of this assay, nsL-TP was found to be most prominently present in liver and intestinal mucosa (0.78 and 0.46 microgram nsL-TP per mg protein in 105 000 X g supernatant, respectively). Subfractionation studies showed that approx. 70% of nsL-TP was present in the membrane-free cytosol. However, application of an immunosorbent-purified antibody and protein A-linked gold particles to rat liver slices demonstrated a concentration of label over the peroxisomes. By way of immunoblotting it was shown that nsL-TP was absent from peroxisomes and that the immunoreactive material was a protein of mol. wt. 58 000. nsL-TP is capable of mediating net transfer of cholesterol to membranes, deficient in this lipid. Under such conditions of net transfer, nsL-TP stimulated the microsomal esterification of cholesterol and its conversion to pregnenolone by adrenal mitochondria. Levels of nsL-TP in Reuber H35 hepatoma cells was six per cent of that found in rat hepatocytes. This very low level of nsL-TP had no effect on de novo cholesterol biosynthesis and intracellular cholesterol esterification. These results raise doubts as to whether nsL-TP has a function in in situ cholesterol metabolism.     

Membrane properties modulate the activity of a phosphatidylinositol transfer protein from the yeast, Saccharomyces cerevisiae

A phospholipid transfer protein from yeast (Daum, G. and Paltauf, F. (1984) Biochim. Biophys. Acta 794, 385-391) was 2800-fold enriched by an improved procedure. The specificity of this transfer protein and the influence of membrane properties of acceptor vesicles (lipid composition, charge, fluidity) on the transfer activity were determined in vitro using pyrene-labeled phospholipids. The yeast transfer protein forms a complex with phosphatidylinositol or phosphatidylcholine, respectively, and transfers these two phospholipids between biological and/or artificial membranes. The transfer rate for phosphatidylinositol is 19-fold higher than for phosphatidylcholine as determined with 1:8 mixtures of phosphatidylinositol and phosphatidylcholine in donor and acceptor membrane vesicles. If acceptor membranes consist only of non-transferable phospholipids, e.g., phosphatidylethanolamine, a moderate but significant net transfer of phosphatidylcholine occurs. Phosphatidylcholine transfer is inhibited to a variable extent by negatively charged phospholipids and by fatty acids. Differences in the accessibility of the charged groups of lipids to the transfer protein might account for the different inhibitory effects, which occur in the order phosphatidylserine which is greater than phosphatidylglycerol which is greater than phosphatidylinositol which is greater than cardiolipin which is greater than phosphatidic acid which is greater than fatty acids. Although mitochondrial membranes contain high amounts of negatively charged phospholipids, they serve effectively as acceptor membranes, whereas transfer to vesicles prepared from total mitochondrial lipids is essentially zero. Ergosterol reduces the transfer rate, probably by decreasing membrane fluidity. This notion is supported by data obtained with dipalmitoyl phosphatidylcholine as acceptor vesicle component; in this case the transfer rate is significantly reduced below the phase transition temperature of the phospholipid.     

Kinetics of fluorescent-labeled phosphatidylcholine transfer between nonspecific lipid transfer protein and phospholipid vesicles

Recently, rat liver nonspecific lipid transfer protein (nsLTP) was shown to form a fluorescent complex when allowed to equilibrate with self-quenching vesicles prepared from the fluorescent phospholipid 1-palmitoyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4- yl)amino]dodecanoyl]phosphatidylcholine (P-C12-NBD-PC) [Nichols, J. W. (1987) J. Biol. Chem. 262, 14172-14177]. Investigation of the mechanism of complex formation was continued by studying the kinetics of transfer of P-C12-NBD-PC between nsLTP and phospholipid vesicles using a transfer assay based on resonance energy transfer between P-C12-NBD-PC and N-(lissamine rhodamine B sulfonyl)dioleoylphosphatidylethanolamine. The principles of mass action kinetics (which predict initial lipid transfer rates as a function of protein and vesicle concentration) were used to derive equations for two distinct mechanisms: lipid transfer by the diffusion of monomers through the aqueous phase and lipid transfer during nsLTP-membrane collisions. The results of these kinetics studies indicated that the model for neither mechanism alone adequately predicted the initial rates of formation and dissolution of the P-C12-NBD-PC-nsLTP complex. The initial rate kinetics for both processes were predicted best by a model in which monomer diffusion and collision-dependent transfer occur simultaneously. These data support the hypothesis that the phospholipid-nsLTP complex functions as an intermediate in the transfer of phospholipids between membranes.     

Phospholipid exchange activity in developing rat brain

Phospholipid exchange activity has been determined in the supernatant fraction of rat brain from birth through to maturity by measuring the protein-catalysed transfer of total and individual 32P-labelled phospholipids from microsomal membranes to mitochondria, and the transfer of [14C]phosphatidylcholine from liposomes to mitochondria. Transfer activity has also been compared in brain and liver supernatant. Overall phospholipid exchange activity in the brain increased only slightly with age. The activity at birth was 75% of the adult value. However, the transfer of individual phospholipids showed markedly different trends during postnatal brain development. The transfer of phosphatidylinositol (PI) and ethanolamine phospholipids increased postnatally to a maximum at 9 days of age, with lowest values in adult brain. Phosphatidylcholine (PC) transfer increased from 9 days to reach maximum values in the mature brain. The transfer of sphingomyelin was highest immediately after birth. PI transfer activity was higher in brain than liver, while PC and ethanolamine phospholipid transfer activity was higher in liver. The heterogeneity of phospholipid exchange proteins in central nervous system tissue is reflected in the developmental changes in exchange activity towards individual phospholipids. The various exchange proteins appear to have separate induction mechanisms. The presence of exchange-protein activity from birth in the rat indicates the functional importance of phospholipid transport during cell acquisition and membrane proliferation. Activity is not primarily associated with membrane formation such as the formation of the myelin sheath, and therefore is more likely to be involved in the process of phospholipid turnover.     

An in vitro ESR study of uncatalyzed rat liver protein-catalyzed spin-labeled phosphatidylcholine exchange

ESR spectrometry has been used to study fatty acid spin-labeled phosphatidylcholine exchange from single bilayer donor vesicles to various acceptor systems, such as intact or differently treated mitochondria, phospholipid multilamellar vesicles or single bilayer vesicles. This exchange is catalyzed by soluble non-specific rat liver protein, first investigated by Bloj and Zilversmit in 1977 (J. Biol. Chem. 252, 1613--1619). Non-catalyzed phosphatidylcholine exchange has also been studied. Full inhibition of both mechanisms occurs with lipid-depleted acceptor mitochondria, while N-ethylmaleimide-treated mitochondria behave as good acceptors during catalyzed exchange but are in no way effective during spontaneous exchange. Non-catalyzed exchange does not take place with phospholipase D-treated mitochondria as acceptors, while the pure catalyzed mechanism is inhibited by 28%. Neither multilamellar nor single bilayer phospholipid vesicles exchange spin-labeled phosphatidylcholine in the absence of protein, the former being a poorer acceptor system than the latter during catalyzed exchange, when this activity is 31 and 80%, respectively, of that of intact mitochondria. The hypothesis is made that the spontaneous mechanism is active among intact natural membranes and could be of some importance in vivo. Furthermore, the biomembrane protein moiety is assumed to be involved in the catalyzed exchange more as a phospholipid spacer than as a binder between the exchange protein and the membrane involved. Phospholipids, on the contrary, appear to be important for both functions.     

Phosphatidylinositol transfer protein from bovine brain: Substrate specificity and membrane binding properties

A recently developed fluorimetric transfer assay (Somerharju, P., Brockerhoff, H. and Wirtz, K.W.A. (1981) Biochim. Biophys. Acta 649, 521–528) has been applied to study the substrate specificity and membrane binding of the phosphatidylinositol-transfer protein from bovine brain. The substrate specificity was investigated by measuring the rate of transfer, either directly or indirectly, for a series of phosphatidylinositol analogues which included phosphatidic acid, phosphatidylglycerol as well as three lipids obtained from yeast phosphatidylinositol by partial periodate oxidation and subsequent borohydride reduction. Phosphatidylglycerol and the oxidation products of phosphatidylinositol were transferred at about one tenth of the rate observed for phosphatidylinositol while phosphatidic acid was not transferred. It is concluded that an intact inositol moiety favours the formation of the putative transfer protein-phosphatidylinositol complex. In addition to phosphatidylinositol, the transfer protein also transfers phosphatidylcholine. In order to obtain information on the possible occurrence of two sites of interaction, vesicles consisting of either pure 1-acyl-2-parinaroylphosphatidylinositol or 1-acyl-2-parinaroylphosphatidylcholine were titrated with the protein. Binding of labeled phospholipid to the protein was represented by an increase of lipid fluorescence and found to be much more efficient for phosphatidylinositol than for phosphatidylcholine. This is interpreted to indicate that the protein contains an endogenous phosphatidylinositol molecule which can be easily replaced by exogenous phosphatidylinositol but not by phosphatidylcholine, a lipid with a lower affinity for this protein. Thus the binding sites for the two phospholipids are mutually exclusive, i.e. phosphatidylinositol and phosphatidylcholine cannot be bound to the protein simultaneously. Finally, the effect of acidic phospholipids on the transfer protein activity was studied either by varying the content of phosphatidic acid in the acceptor vesicles or by adding vesicles of pure acidic phospholipids to the normal assay system. The latter vesicles consisted of either phosphatidic acid, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol or cardiolipin. In both instances the transfer protein activity was inhibited, obviously through the enhanced association of the protein with the negatively charged vesicles. These findings strongly suggest that relatively nonspecific ionic forces rather than specific protein-phospholipid headgroup interactions contribute to the association of the phosphatidylinositol-transfer protein with membranes.     

Phosphatidylinositol transfer protein from bovine brain. Substrate specificity and membrane binding properties

A recently developed fluorimetric transfer assay (Somerharju, P., Brockerhoff, H. and Wirtz, K.W.A. (1981) Biochim. Biophys. Acta 649, 521-528) has been applied to study the substrate specificity and membrane binding of the phosphatidylinositol-transfer protein from bovine brain. The substrate specificity was investigated by measuring the rate of transfer, either directly or indirectly, for a series of phosphatidylinositol analogues which included phosphatidic acid, phosphatidylglycerol as well as three lipids obtained from yeast phosphatidylinositol by partial periodate oxidation and subsequent borohydride reduction. Phosphatidylglycerol and the oxidation products of phosphatidylinositol were transferred at about one tenth of the rate observed for phosphatidylinositol while phosphatidic acid was not transferred. It is concluded that an intact inositol moiety favours the formation of the putative transfer protein-phosphatidylinositol complex. In addition to phosphatidylinositol, the transfer protein also transfers phosphatidylcholine. In order to obtain information on the possible occurrence of two sites of interaction, vesicles consisting of either pure 1-acyl-2-parinaroylphosphatidylinositol or 1-acyl-2-parinaroylphosphatidylcholine were titrated with the protein. Binding of labeled phospholipid to the protein was represented by an increase of lipid fluorescence and found to be much more efficient for phosphatidylinositol than for phosphatidylcholine. This is interpreted to indicate that the protein contains an endogenous phosphatidylinositol molecule which can be easily replaced by exogenous phosphatidylinositol but not by phosphatidylcholine, a lipid with a lower affinity for this protein. Thus the binding sites for the two phospholipids are mutually exclusive, i.e. phosphatidylinositol and phosphatidylcholine cannot be bound to the protein simultaneously. Finally, the effect of acidic phospholipids on the transfer protein activity was studied either by varying the content of phosphatidic acid in the acceptor vesicles or by adding vesicles of pure acidic phospholipids to the normal assay system. The latter vesicles consisted of either phosphatidic acid, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol or cardiolipin. In both instances the transfer protein activity was inhibited, obviously through the enhanced association of the protein with the negatively charged vesicles. These findings strongly suggest that relatively nonspecific ionic forces rather than specific protein-phospholipid headgroup interactions contribute to the association of the phosphatidylinositol-transfer protein with membranes.     

Bid, a widely expressed proapoptotic protein of the Bcl-2 family, displays lipid transfer activity

Bid is an abundant proapoptotic protein of the Bcl-2 family that is crucial for the induction of death receptor-mediated apoptosis in primary tissues such as liver. Bid action has been proposed to involve the relocation of its truncated form, tBid, to mitochondria to facilitate the release of apoptogenic cytochrome c. The mechanism of Bid relocation to mitochondria was unclear. We report here novel biochemical evidence indicating that Bid has lipid transfer activity between mitochondria and other intracellular membranes, thereby explaining its dynamic relocation to mitochondria. First, physiological concentrations of phospholipids such as phosphatidic acid and phosphatidylglycerol induced an accumulation of full-length Bid in mitochondria when incubated with light membranes enriched in endoplasmic reticulum. Secondly, native and recombinant Bid, as well as tBid, displayed lipid transfer activity under the same conditions and at the same nanomolar concentrations leading to mitochondrial relocation and release of cytochrome c. Thus, Bid is likely to be involved in the transport and recycling of mitochondrial phospholipids. We discuss how this new role of Bid may relate to its proapoptotic action.     

An in vitro ESR study of uncatalyzed and rat liver protein-catalyzed spin-labeled phosphatidylcholine exchange

ESR spectrometry has been used to study fatty acid spin-labeled phosphatidylcholine exchange from single bilayer donor vesicles to various acceptor systems, such as intact or differently treated mitochondria, phospholipid multilamellar vesicles or single bilayer vesicles. This exchange is catalyzed by soluble non-specific rat liver protein, first investigated by Bloj and Zilversmit in 1977 (J. Biol. Chem. 252, 1613–1619). Non-catalyzed phosphatidylcholine exchange has also been studied. Full inhibition of both mechanisms occurs with lipid-depleted acceptor mitochondria, while $\text{N-}\text{ethylmaleimide-treated}$ mitochondria behave as good acceptors during catalyzed exchange but are in no way effective during spontaneous exchange. Non-catalyzed exchange does not take place with phospholipase D-treated mitochondria as acceptors, while the pure catalyzed mechanism is inhibited by 28%. Neither multilamellar nor single bilayer phospholipid vesicles exchange spin-labeled phosphatidylcholine in the absence of protein, the former being a poorer acceptor system than the latter during catalyzed exchange, when this activity is 31 and 80%, respectively, of that of intact mitochondria. The hypothesis is made that the spontaneous mechanism is active among intact natural membranes and could be of some importance in vivo. Furthermore, the biomembrane protein moiety is assumed to be involved in the catalyzed exchange more as a phospholipid spacer than as a binder between the exchange protein and the membrane involved. Phospholipids, on the contrary, appear to be important for both functions.     

An essential requirement of cardiolipin for mitochondrial carnitine acylcarnitine translocase activity. Lipid requirement of carnitine acylcarnitine translocase

The phospholipid requirement for the optimal solubilization of carnitine acylcarnitine translocase from the inner membrane vesicles of rat liver mitochondria and for its reconstitution in liposomes was investigated. At the octylglucoside-solubilization step, the presence of cardiolipin proved superior to the other lipids tested. For reconstitution, a mixture having phosphatidylcholine, phosphatidylethanolamine and cardiolipin was found to be particularly effective. The requirement of cardiolipin at this step was met less effectively by other anionic phospholipids. Moreover, in intact mitochondria of rat liver and heart, the translocase activity was markedly inhibited by micromolar concentrations of doxorubicin, a specific cardiolipin-binding agent.     

Effect of N-ethylmaleimide on rat liver phosphatidylcholine-specific and non-specific transfer protein activities: its dependence on donor liposome composition

Phosphatidylcholine exchange between liposomes and mitochondria catalyzed by rat liver phosphatidylcholine transfer protein is strongly stimulated by N-ethylmaleimide (NEM) when PC/PI (molar ratio, 4:1) donor liposomes are used. In the presence of PC/PE or PC liposomes the exchange activity by this protein is unaffected. In the same experimental conditions, the activity of rat liver non-specific transfer protein is always stimulated by N-ethylmaleimide with all the types of liposomes tested in the order PC/PI greater than PC/PE greater than PC. Since the effect of NEM depends on the type of liposomes used and appears to be similar for both phospholipid transfer proteins, the possibility that their mode of action implies the formation of a ternary complex should be considered. As far as non-specific transfer protein is concerned, its interaction could vary depending on the nature of the exchanging membranes. Data are also presented indicating that when the two transfer proteins are together their activity is additive, therefore suggesting a specific role in phospholipid biomembrane assembly for each of them.     

Hydroxymethylglutaryl CoA reductase and the modulation of microsomal cholesterol content by the nonspecific lipid transfer protein

The influence of membrane cholesterol content on 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase, EC 1.1.1.34) in rat liver microsomes was investigated. Microsomes were enriched in cholesterol by incubation with egg phosphatidylcholine-cholesterol vesicles and the nonspecific lipid transfer protein from rat liver. By this method, the microsomal cholesterol content was 2.5-fold enhanced up to final concentrations of 140 nmol cholesterol per mg microsomal protein. In another experiment, microsomes isolated from rats fed a cholesterol-rich diet were depleted of cholesterol by incubation with egg phosphatidylcholine vesicles and the transfer protein. Both cholesterol enrichment and depletion had virtually no effect on the microsomal HMG-CoA reductase activity. In another set of experiments, normal rat liver microsomes were incubated with human serum, resulting in a rise of microsomal cholesterol content. This was reflected in an increase of acyl-CoA:cholesterol acyltransferase activity but failed to have an effect on HMG-CoA reductase.