Protein mediated glycolipid transfer is inhibited FROM sphingomyelin membranes but enhanced TO sphingomyelin containing raft like membranes

The mammalian glycolipid transfer protein, GLTP, catalyzes the transfer in vitro of glycolipids between membranes. In this study we have examined on one hand the effect of the variations in the donor vesicle composition and on the other hand the effects of variations in the acceptor vesicle composition on the GLTP-catalyzed transfer kinetics of galactosylceramide between bilayer vesicles. For this purpose a resonance energy transfer assay was used, the energy donor being anthrylvinyl-galactosylceramide and the energy acceptor DiO-C₁₆. First, we show that the transfer of anthrylvinyl-galactosylceramide from palmitoyl-oleoyl-phosphatidylcholine donor vesicles was faster than from dipalmitoyl-phosphatidylcholine vesicles, and that there is no transfer from palmitoyl-sphingomyelin vesicles regardless of the cholesterol amount. In this setup the acceptor vesicles were always 100% palmitoyl-oleoyl-phosphatidylcholine. We also showed that the transfer in general is faster from small highly curved vesicles compared to that from larger vesicles. Secondly, by varying the acceptor vesicle composition we showed that the transfer is faster to mixtures of sphingomyelin and cholesterol compared to mixtures of phosphatidylcholines and cholesterol. Based on these experiments we conclude that the GLTP mediated transfer of anthrylvinyl-galactosylceramide is sensitive to the matrix lipid composition and membrane bending. We postulate that a tightly packed membrane environment is most effective in preventing GLTP from accessing its substrates, and cholesterol is not required to protect the glycosphingolipid in the membrane from being transferred by GLTP. On the other hand GLTP can more easily transfer glycolipids to ‘lipid raft’ like membranes, suggesting that the protein could be involved in raft assembly.     

A fluorescence resonance energy transfer approach for monitoring protein-mediated glycolipid transfer between vesicle membranes

A lipid transfer protein, purified from bovine brain (23.7 kDa, 208 amino acids) and specific for glycolipids, has been used to develop a fluorescence resonance energy transfer assay (anthrylvinyl-labeled lipids; energy donors and perylenoyl-labeled lipids; energy acceptors) for monitoring the transfer of lipids between membranes. Small unilamellar vesicles composed of 1 mol% anthrylvinyl-galactosylceramide, 1.5 mol% perylenoyl-triglyceride, and 97.5% 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) served as donor membranes. Acceptor membranes were 100% POPC vesicles. Addition of glycolipid transfer protein to mixtures of donor and acceptor vesicles resulted in increasing emission intensity of anthrylvinyl-galactosylceramide and decreasing emission intensity of the nontransferable perylenoyl-triglyceride as a function of time. The behavior was consistent with anthrylvinyl-galactosylceramide being transferred from donor to acceptor vesicles. The anthrylvinyl and perylenoyl energy transfer pair offers advantages over frequently used energy transfer pairs such as NBD and rhodamine. The anthrylvinyl emission overlaps effectively the perylenoyl excitation spectrum and the fluorescence parameters of the anthrylvinyl fluorophore are nearly independent of the medium polarity. The nonpolar fluorophores are localized in the hydrophobic region of the bilayer thus producing minimal disturbance of the bilayer polar region. Our results indicate that this method is suitable for assay of lipid transfer proteins including mechanistic studies of transfer protein function.     

Probing for preferential interactions among sphingolipids in bilayer vesicles using the glycolipid transfer protein.

We have investigated the intervesicular transfer of galactosylceramide between unilamellar bilayer vesicles composed of differing sphingomyelin and phosphatidylcholine molar ratios. To monitor glycolipid transfer from donor to acceptor vesicles, we used a fluorescence resonance energy transfer assay involving anthrylvinyl-labeled galactosylceramide (AV-GalCer) and perylenoyl-labeled triglyceride. The transfer was mediated by glycolipid transfer protein (GLTP), purified from bovine brain and specific for glycolipids. The initial transfer rate and the total accessible pool of glycolipid in the donor vesicles were both measured. An increase in the sphingomyelin content of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) vesicles decreased the transfer rate in a nonlinear fashion. Decreased transfer rates were clearly evident at sphingomyelin mole fractions of 0.22 or higher. The pool of AV-GalCer available for GLTP-mediated transfer also was smaller in vesicles containing high sphingomyelin content. In contrast, AV-GalCer was more readily transferred from vesicles composed of POPC and different disaturated phosphatidylcholines. Our results show that GLTP acts as a sensitive probe for detecting interactions of glycosphingolipids with neighboring lipids and that the lateral mixing of glycolipids is probably affected by the matrix lipid composition. The compositionally driven changes in lipid interactions, sensed by GLTP, occur in membranes that are either macroscopically fluid-phase or gel/fluid-phase mixtures. Gaining insights into how changes in membrane sphingolipid composition alter accessibility to soluble proteins with affinity for membrane glycolipids is likely to help increase our understanding of how sphingolipid-enriched microdomains (i.e., "rafts" and caveolae) are formed and maintained in cells.     

Effects of bile salts on glucosylceramide containing membranes

The glycolipid transfer protein (GLTP) is capable of transporting glycolipids from a donor membrane, through the aqueous environment, to an acceptor membrane. The GLTP mediated glycolipid transfer from sphingomyelin membranes is very slow. In contrast, the transfer is fast from membranes composed of phosphatidylcholine. The lateral glycolipid membrane organization is known to be driven by their tendency to mix non-randomly with different membrane lipids. Consequently, the properties of the membrane lipids surrounding the glycolipids play an important role in the ability of GLTP to bind and transfer its substrates. Since GLTP transfer of glycolipids is almost nonexistent from sphingomyelin membranes, we have used this exceptionality to investigate if membrane intercalators can alter the membrane packing and induce glycolipid transfer. We found that the bile salts cholate, deoxycholate, taurocholate and taurodeoxycholate, cause glucosylceramide to become transferrable by GLTP. Other compounds, such as single chain lipids, ceramide and nonionic surfactants, that have membrane-perturbing effects, did not affect the transfer capability of GLTP. We speculate that the strong hydrogen bonding network formed in the interfacial region of glycosphingolipid-sphingomyelin membranes is disrupted by the membrane partition of the bile salts causing the glycosphingolipid to become transferrable.     

Charged membrane surfaces impede the protein-mediated transfer of glycosphingolipids between phospholipid bilayers

A lipid transfer protein that facilitates the transfer of glycolipids between donor and acceptor membranes has been investigated using a fluorescence resonance energy transfer assay. The glycolipid transfer protein (23-24 kDa, pI 9.0) catalyzes the high specificity transfer of lipids that have sugars beta-linked to either a ceramide or a diacylglycerol backbone, such as simple glycolipids and gangliosides, but not the transfer of phospholipids, cholesterol, or cholesterol esters. In this study, we examined the effect of different charged lipids on the rate of transfer of anthrylvinyl-labeled galactosylceramide (1 mol %) from a donor to acceptor vesicle population at neutral pH. Compared to neutral donor vesicle membranes, introduction of negatively charged lipid at 5 or 10 mol % into the donor vesicles significantly decreased the transfer rate. Introduction of the same amount of negative charge into the acceptor vesicle membrane did not impede the transfer rate as effectively. Also, positive charge in the donor vesicle membrane was not as effective at slowing the transfer rate as was negative charge in the donor vesicle. Increasing the ionic strength of the buffer with NaCl significantly reversed the charge effects. At neutral pH, the transfer protein (pI congruent with 9.0) is expected to be positively charged, which may promote association with the negatively charged donor membrane. Based on these and other experiments, we conclude that the transfer process follows first-order kinetics and that the off-rate of the transfer protein from the donor vesicle surface is the rate-limiting step in the transfer process.     

Membrane interaction and activity of the glycolipid transfer protein

In this study we have addressed the ability of the glycolipid transfer protein (GLTP) to transfer anthrylvinyl-galactosylceramide at different pH and sodium chloride concentrations, and the ability of three different mutants to transfer the fluorescently labeled galactosylceramide between donor and acceptor model membranes. We constructed single tryptophan mutants with site-directed mutagenesis where two of the three tryptophan (W) of wild-type human GLTP were substituted with phenylalanine (F) and named W85 GLTP (W96F and W142F), W96 GLTP (W85F and W142F) and W142 GLTP (W85F and W96F) accordingly. Wild-type GLTP and W96 GLTP were both able to transfer anthrylvinyl-galactosylceramide, but the two variants W85 GLTP and W142 GLTP did not show any glycolipid transfer activity, indicating that the tryptophan in position 96 is crucial for transfer activity. Tryptophan fluorescence emission showed a blue shift of the maximal emission wavelength upon interaction of glycolipid containing vesicle with wild-type GLTP and W96 GLTP, while no blue shift was recorded for the protein variants W85 GLTP and W142 GLTP. The quantum yield of tryptophan emission was highest for the W96 GLTP protein whereas W85 GLTP, W142 GLTP and wild-type GLTP showed a lower and almost similar quantum yield. The lifetime and anisotropy decay of the different tryptophan mutants also changed upon binding to vesicles containing galactosylceramide. Again wild-type GLTP and W96 GLTP showed similar behavior in the presence of vesicles containing glycolipids. Taken together, our data show that the W96 is involved not only in the activity of the protein but also in the interaction between the protein and glycolipid containing membranes.     

Membrane interaction and activity of the glycolipid transfer protein

In this study we have addressed the ability of the glycolipid transfer protein (GLTP) to transfer anthrylvinyl-galactosylceramide at different pH and sodium chloride concentrations, and the ability of three different mutants to transfer the fluorescently labeled galactosylceramide between donor and acceptor model membranes. We constructed single tryptophan mutants with site-directed mutagenesis where two of the three tryptophan (W) of wild-type human GLTP were substituted with phenylalanine (F) and named W85 GLTP (W96F and W142F), W96 GLTP (W85F and W142F) and W142 GLTP (W85F and W96F) accordingly. Wild-type GLTP and W96 GLTP were both able to transfer anthrylvinyl-galactosylceramide, but the two variants W85 GLTP and W142 GLTP did not show any glycolipid transfer activity, indicating that the tryptophan in position 96 is crucial for transfer activity. Tryptophan fluorescence emission showed a blue shift of the maximal emission wavelength upon interaction of glycolipid containing vesicle with wild-type GLTP and W96 GLTP, while no blue shift was recorded for the protein variants W85 GLTP and W142 GLTP. The quantum yield of tryptophan emission was highest for the W96 GLTP protein whereas W85 GLTP, W142 GLTP and wild-type GLTP showed a lower and almost similar quantum yield. The lifetime and anisotropy decay of the different tryptophan mutants also changed upon binding to vesicles containing galactosylceramide. Again wild-type GLTP and W96 GLTP showed similar behavior in the presence of vesicles containing glycolipids. Taken together, our data show that the W96 is involved not only in the activity of the protein but also in the interaction between the protein and glycolipid containing membranes.     

Molecular features of phospholipids that affect glycolipid transfer protein-mediated galactosylceramide transfer between vesicles

The glycolipid transfer protein (GLTP)-mediated movement of galactosylceramide from model membrane donor vesicles to acceptor vesicles is sensitive to the membrane environment surrounding the glycolipid. GLTP can catalyze the transfer of a fluorescently labeled GSL, anthrylvinyl-galactosylceramide (AV-GalCer), from vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and dipalmitoylphosphatidylcholine matrices, but not from vesicles prepared from N-palmitoylsphingomyelin, regardless of the cholesterol content of the vesicles. In this study, we have examined the structural features of sphingomyelin (SM) that are responsible for its inhibition of the rate of GLTP-catalyzed transfer of AV-GalCer. The rate of glycolipid transfer was enhanced when the N-palmitoyl chain of SM was replaced with an N-oleoyl chain. Analogs of N-palmitoyl-SM in which the 4,5-double bond of the long-chain base is reduced or the 3-hydroxy group is removed did not inhibit GLTP-catalyzed transfer of AV-GalCer. When the donor vesicles were prepared with phosphatidylcholines or ether-linked phosphatidylcholine analogs, the transfer rates of AV-GalCer increased with increasing degree of unsaturation. The rate of AV-GalCer transfer was strongly dependent on the unsaturation degree of the acyl and/or alkyl chains. For ester-linked PCs, the transfer rate increased in the order DPPC相似文献   

Molecular features of phospholipids that affect glycolipid transfer protein-mediated galactosylceramide transfer between vesicles

The glycolipid transfer protein (GLTP)-mediated movement of galactosylceramide from model membrane donor vesicles to acceptor vesicles is sensitive to the membrane environment surrounding the glycolipid. GLTP can catalyze the transfer of a fluorescently labeled GSL, anthrylvinyl-galactosylceramide (AV-GalCer), from vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and dipalmitoylphosphatidylcholine matrices, but not from vesicles prepared from N-palmitoylsphingomyelin, regardless of the cholesterol content of the vesicles. In this study, we have examined the structural features of sphingomyelin (SM) that are responsible for its inhibition of the rate of GLTP-catalyzed transfer of AV-GalCer. The rate of glycolipid transfer was enhanced when the N-palmitoyl chain of SM was replaced with an N-oleoyl chain. Analogs of N-palmitoyl-SM in which the 4,5-double bond of the long-chain base is reduced or the 3-hydroxy group is removed did not inhibit GLTP-catalyzed transfer of AV-GalCer. When the donor vesicles were prepared with phosphatidylcholines or ether-linked phosphatidylcholine analogs, the transfer rates of AV-GalCer increased with increasing degree of unsaturation. The rate of AV-GalCer transfer was strongly dependent on the unsaturation degree of the acyl and/or alkyl chains. For ester-linked PCs, the transfer rate increased in the order DPPC < POPC < DOPC, which have 0, 1, and 2 cis double bonds, respectively.     

Mechanism of cholesterol transfer from the Niemann-Pick type C2 protein to model membranes supports a role in lysosomal cholesterol transport

Cells acquire cholesterol either by de novo synthesis in the endoplasmic reticulum or by internalization of cholesterol-containing lipoproteins, particularly low density lipoprotein (LDL), via receptor-mediated endocytosis. The inherited disorder Niemann-Pick type C (NPC), in which abnormal LDL-cholesterol trafficking from the endo/lysosomal compartment leads to substantial cholesterol and glycolipid accumulation in lysosomes, is caused by defects in either of two genes that encode for proteins designated as NPC1 and NPC2. NPC2 is a small intralysosomal protein that has been characterized biochemically as a cholesterol binding protein. We determined the rate and mechanism by which NPC2 delivers cholesterol to model phospholipid membranes. A fluorescence dequenching assay was used to monitor the kinetics of cholesterol transfer from the protein to membranes. The endogenous tryptophan fluorescence of the NPC2 was quenched upon binding of cholesterol, and the subsequent addition of acceptor vesicles resulted in dequenching of the tryptophan signal, enabling the monitoring of cholesterol transfer to membranes. The rates of cholesterol transfer were evaluated as a function of acceptor vesicle concentration, acceptor vesicle phospholipid headgroup composition, and aqueous phase properties. The results suggest that NPC2 rapidly transports cholesterol to phospholipid vesicles via a collisional mechanism which involves a direct interaction with the acceptor membrane. Transfer of cholesterol to membranes is faster in an acidic environment and is greatly enhanced by the presence of the unique lysosomal/late endosomal phospholipid lyso-bisphosphatidic acid (LBPA) (also known as bismonoacylglycerol phosphate). Finally, we found that the rate of transfer of cholesterol from vesicles to NPC2 was dramatically increased by the presence of lyso-bisphosphatidic acid in the donor vesicles. These results support a role for the NPC2 protein in the egress of LDL derived cholesterol out of the endosomal/lysosomal compartment.     

Glycolipid transfer protein interaction with bilayer vesicles: modulation by changing lipid composition

Glycosphingolipids (GSLs) are important constituents of lipid rafts and caveolae, are essential for the normal development of cells, and are adhesion sites for various infectious agents. One strategy for modulating GSL composition in lipid rafts is to selectively transfer GSL to or from these putative membrane microdomains. Glycolipid transfer protein (GLTP) catalyzes selective intermembrane transfer of GSLs. To enable effective use of GLTP as a tool to modify the glycolipid content of membranes, it is imperative to understand how the membrane regulates GLTP action. In this study, GLTP partitioning to membranes was analyzed by monitoring the fluorescence resonance energy transfer from tryptophans and tyrosines of GLTP to N-(5-dimethyl-aminonaphthalene-1-sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phospho-ethanolamine present in bilayer vesicles. GLTP partitioned to POPC vesicles even when no GSL was present. GLTP interaction with model membranes was nonpenetrating, as assessed by protein-induced changes in lipid monolayer surface pressure, and nonperturbing in that neither membrane fluidity nor order were affected, as monitored by anisotropy of 1,6-diphenyl-1,3,5-hexatriene and 6-dodecanoyl-N,N-dimethyl-2-naphthylamine, even though the tryptophan anisotropy of GLTP increased in the presence of vesicles. Ionic strength, vesicle packing, and vesicle lipid composition affected GLTP partitioning to the membrane and led to the following conclusion: Conditions that increase the ratio of bound/unbound GLTP do not guarantee increased transfer activity, but conditions that decrease the ratio of bound/unbound GLTP always diminish transfer. A model of GLTP interaction with the membrane, based on the partitioning equilibrium data and consistent with the kinetics of GSL transfer, is presented and solved mathematically.     

Kinetics of ganglioside transfer between liposomal and synaptosomal membranes

The transfer of ganglioside GM1 from micelles to membranes and between different membrane populations has been examined by using a pyrene fatty acid derivative of the ganglioside. The transfer of gangliosides from micelles to membranes depends on the physical state as well as the molecular composition of the acceptor vesicles. At 30 degrees C, the transfer of micellar gangliosides to dipalmitoylphosphatidylcholine (DPPC) large unilameller vesicles (Tm = 41.3 degrees C) is characterized by a rate constant of 0.01 min-1; at 48 degrees C, however, the rate constant is 0.11 min-1. Below the phase transition temperature, the activation energy is 25 kcal/mol whereas above the phase transition it is 17 kcal/mol. Similar experiments performed with synaptic plasma membranes yielded a rate constant of 0.05 min-1 at 37 degrees C. The rate of transfer of ganglioside molecules, asymmetrically located on the outer layer of donor vesicles, to acceptor vesicles lacking ganglioside depends on the physical state of both the donor and acceptor vesicles. For the transfer of ganglioside from DPPC (donor) vesicles to dimyristoylphosphatidylcholine (DMPC) (acceptor) vesicles, the rates were essentially zero at 15 degrees C in which both vesicle populations were in the gel phase, 0.008 min-1 at 30 degrees C in which DPPC is in the gel phase and DMPC is in the fluid phase, and 0.031 min-1 at 48 degrees C in which both vesicle populations are in the fluid phase. The transfer of ganglioside from DPPC vesicles to synaptic plasma membranes was also dependent on the physical state of the donor vesicles and showed an inflection point at the phase transition temperature of DPPC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spontaneous cholesterol movement between lipid vesicles and monkey small intestinal brush border membrane

[14C]Cholesterol movement between egg phosphatidylcholine-cholesterol lipid vesicles and vesicles prepared from monkey small intestinal brush border membrane (BBMV) was studied in physiological buffer at 37 degrees C. The rate of cholesterol transfer from sonicated unilamellar vesicles (ULV) to BBMV follows apparently first-order kinetics. Intermembrane cholesterol movement was strikingly similar in both the directions. However, from BBMV to ULV, the transfer rate was three times faster than that of ULV to brush border membrane (BBM). Similarity in the rate constant was observed when cholesterol transfer was studied using either large multilamellar lipid vesicles or ULV as the donor and BBMV as the acceptor membrane. Rate constant was also the same when the acceptor membrane used was either intact BBMV or ULV prepared from BBM lipids. The rate of transfer of label was not affected even when the acceptor vesicle concentration was increased over fivefold, indicating the first-order nature of the reaction. Transfer of cholesterol from ULV to BBMV was accelerated by the presence of acetone, dimethyl sulfoxide (DMSO), deoxycholate, and papain. Partially purified nonspecific lipid-exchange protein increased the rate of cholesterol transfer by about threefold. Reduction in BBM cholesterol and phospholipid content was noted by DMSO, acetone, and deoxycholate, while papain caused a small depletion of membrane protein. Cholesterol transfer is temperature dependent with an activation energy of 31 kJ X mol-1, which is almost identical in the presence or absence of nonspecific lipid-exchange protein. The molecular mechanism of intermembrane cholesterol movement is discussed in view of the kinetic data obtained.     

Calcium/phosphate-induced immobilization of fluorescent phosphatidylserine in synthetic bilayer membranes: inhibition of lipid transfer between vesicles

Resonance energy transfer between 4-nitro-2,1,3-benzoxadiazole (NBD) acyl chain labeled phospholipid analogues and (lissamine) rhodamine B labeled phosphatidylethanolamine was used to monitor the rate of NBD-labeled lipid transfer between a variety of small unilamellar donor vesicles and dioleoylphosphatidylcholine (DOPC) acceptor vesicles. In the presence of appropriate concentrations of Ca2+ and phosphate, the transfer rate of NBD-phosphatidylserine (NBD-PS) from vesicles composed of lipid extracts from human red blood cells was reduced by approximately 10-fold, while the transfer rates of NBD-phosphatidylcholine, -ethanolamine, -glycerol, -N-succinylethanolamine, and -phosphatidic acid were essentially unaffected. A systematic evaluation of the lipid composition needed to facilitate the Ca2+/phosphate-induced inhibition of NBD-PS transfer revealed that the process was dependent upon the inclusion of both cholesterol and phosphatidylethanolamine (PE) in the donor vesicle population. Inhibition of NBD-PS transfer required the sequential addition of phosphate and Ca2+ to the vesicles, indicating that the combined interaction of Ca2+ and phosphate at the membrane surface was a prerequisite for inhibition to occur. Parallel experiments designed to determine the possible mechanism of this phenomenon showed that inhibition of NBD-PS transfer was not due to Ca2+-mediated phase separations or vesicle-vesicle fusion. However, the addition of Ca2+ and phosphate to vesicles composed of total red blood cell lipids or cholesterol/PE did result in their aggregation. On the other hand, aggregation per se did not seem to be responsible for the inhibition of transfer since NBD-PS-containing vesicles composed of DOPC or DOPC/DOPE also aggregated, although NBD-PS transfer was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteoliposome interaction with human erythrocyte membranes. Functional implantation of gamma-glutamyl transpeptidase

The transfer of detergent solubilized and purified gamma-glutamyl transpeptidase (gamma-GTase), of hog kidney cortex, from proteoliposomes into human erythrocyte ghost membranes has been studied. The transfer of gamma-glutamyl transpeptidase was observed upon incubation of gamma-GTase incorporated dipalmitoylphosphatidylcholine vesicles with erythrocyte ghost membranes at 37 degrees C for 12 h. The extent of transfer was dependent upon the fluidity of donor proteoliposomes, being more when dipalmitoylphosphatidylcholine proteoliposomes were used compared to dimyristoylphosphatidylcholine, and intermediate values were observed when binary mixtures of DMPC and DPPC were used. Moreover, the transfer of gamma-GTase was facilitated when rigid basic phospholipid proteoliposomes were used as donor. The transfer of gamma-GTase has been observed to be associated with the removal of intrinsic membrane proteins and lipids from erythrocytes, mainly acetylcholinesterase, sphingomyelin, and cholesterol. An enhancement in the fluorescence due to resonance energy transfer was observed when ghost membranes containing fluorescent donor probe were incubated with proteoliposomes containing fluorescent acceptor probe, indicating that fusion but not adsorption of vesicles occurs during the transfer process. However, the inability of entrapped [14C]-sucrose delivery from proteoliposomes into ghost membrane vesicle suggest that fusion per se is not primarily involved in the transfer process. It appears that the transfer of gamma-glutamyl transpeptidase occurs by a collisional transfer process resulting in intermembrane protein transfer. The gamma-glutamyl transpeptidase implanted ghost membranes exhibited the uptake of L-glutamate which was inhibited by serine-borate, an inhibitor of transpeptidase activity. In addition, the uptake of L-glutamate was inhibited by the dipeptide gamma-glutamyl-L-glutamate, thus supporting the proposed role of gamma-glutamyl transpeptidase in the uptake of amino acids in biological membranes.     

Transfer of cholesterol between phospholipid vesicles mediated by the steroidogenic acute regulatory protein (StAR)

The steroidogenic acute regulatory protein (StAR) mediates the acute stimulation of steroid synthesis by tropic hormones in steroidogenic cells. StAR interacts with the outer mitochondrial membrane and facilitates the rate-limiting transfer of cholesterol to the inner mitochondrial membrane where cytochrome P-450scc converts this cholesterol into pregnenolone. We tested the ability of N-62 StAR to transfer cholesterol from donor vesicles containing cholesterol but no cytochrome P-450scc to acceptor vesicles containing P-450scc but no cholesterol, using P-450scc activity as a reporter of the cholesterol content of synthetic phospholipid vesicles. N-62 StAR stimulated P-450scc activity in acceptor vesicles 5-10-fold following the addition of donor vesicles. Transfer of cholesterol to acceptor vesicles was rapid and sufficient to maintain a linear rate of pregnenolone synthesis for 10 min. The effect of N-62 StAR in stimulating P-450scc activity was specific for cholesterol transfer and was not due to vesicle fusion or P-450scc exchange between vesicles. Maximum stimulation of P-450scc activity in acceptor vesicles required preincubation of N-62 StAR with phospholipid vesicles prior to adding donor vesicles. The amount of N-62 StAR causing half-maximum stimulation of P-450scc activity in acceptor vesicles was 1.9 microm. Half-maximum stimulation required more than a 10-fold higher concentration of R182L N-62 StAR, a mutant associated with congenital lipoid adrenal hyperplasia. N-62 StAR-mediated transfer of cholesterol between vesicles showed low dependence on the cholesterol concentration in the donor vesicles. Thus StAR can transfer cholesterol between synthetic membranes without other protein components found in mitochondria.     

Effect of surface curvature on the rate of cholesterol transfer between lipid vesicles.

The effect of surface curvature on the spontaneous movement of cholesterol between membranes was investigated by measuring the rates of cholesterol transfer from donor vesicles of various sizes to a common acceptor vesicle. Donor vesicles of size in the range 40-240 nm were prepared by extruding multilamellar dispersions through polycarbonate filters of different pore sizes under pressure. The smallest donor vesicle and the acceptor vesicles were obtained by the normal sonication procedures. The rate of cholesterol transfer, as measured by the movement of [3H]cholesterol, decreases with increasing size of the donor vesicle in an almost linear fashion. The extrapolation of the results gave a half-time (t1/2) of 16-20 h of the desorption of cholesterol from a planar bilayer, and this can be considered as a reference value for most cellular membranes which are characterized by very low curvatures. Our earlier studies have shown that the t1/2 for cholesterol efflux is influenced by the presence of gangliosides and phosphatidylethanolamine, and the asymmetric distribution of these lipids in the plasma membrane could partially account for the large difference in the rates of cholesterol movement from the two sides of the plasma membrane. The small differences in rates arising from asymmetric distribution will be magnified by the longer t1/2 obtained here for membranes of low curvatures, so that the large difference in rates might be a coupled effect of lipid asymmetry and low curvature of the plasma membrane. This, in turn, may have a role in maintaining the large differences in cholesterol/phospholipid molar ratios observed between plasma membrane and intracellular membranes.     

Transverse location of the retinal chromophore of rhodopsin in rod outer segment disc membranes

Diffusion-enhanced fluorescence energy transfer was used to study the structure of photoreceptor membranes from bovine retinal rod outer segments. The fluorescent energy donor was Tb³⁺ chelated to dipicolinate and the acceptor was the 11-cis retinal chromophore of rhodopsin in vesicles made from disc membranes. The rapid-diffusion limit for energy transfer was attained in these experiments because of the long excited state lifetime of the terbium donor (~2 ms). Under these conditions, energy transfer is very sensitive to a, the distance of closest approach between the donor and acceptor (Thomas et al., 1978). Vesicles containing terbium dipicolinate in their inner aqueous space were prepared by sonicating disc membranes in the presence of this chelate and chromatographing this mixture on a gel filtration column. The sidedness of rhodopsin in these vesicles was the same as in native disc membranes. The transfer efficiency from terbium to retinal in this sample was 43%. For an R₀ value of 46.7 Å and an average vesicle diameter of 650 Å, this corresponds to an a value of 22 Å from the inner aqueous space of the vesicle. The distance of closest approach from the external aqueous space, determined by adding terbium dipicolinate to a suspension of already formed vesicles, was found to be 28 Å. These values of a show that the retinal chromophore is far from both aqueous surfaces of the disc membrane. Hence, the transverse location of the retinal chromophore is near the center of the hydrophobic core of the disc membrane. These findings suggest that conformational changes induced by photoisomerization are transmitted through a distance of at least 20 Å within rhodopsin to trigger subsequent events in visual excitation.     

An activation-collision mechanism for cholesterol transfer between membranes

We report the results of experiments which show that cholesterol transfer between membranes cannot proceed by aqueous diffusion, as widely held, but must involve a more complex mechanism. (a) The rate of transfer of [3H]cholesterol from red blood cells was found to vary inversely with the size of the acceptor particle (ghosts, vesicles of ghosts, liposomes, and plasma lipoproteins). (b) The transfer of [3H]cholesterol from red blood cells to ghosts was accelerated by the presence of plasma, even though the plasma competed with the ghosts as an acceptor. (c) The rate of transfer of [3H]cholesterol from red blood cells to ghosts decreased to zero with increasing dilution but was not simply second-order. (d) The cholesterol in retinal rod disc membranes is not at equilibrium with plasma lipoproteins in that disc cholesterol increased when the homogenates were incubated in vitro with plasma. (e) The kinetics of cholesterol transfer cannot be limited by unstirred layer effects since the transfer of lysolecithin in the same system was faster than that of cholesterol by 3 orders of magnitude. The simplest model compatible with all the data suggests a two-step pathway involving a first-order followed by a second-order process. The first step could be a unimolecular activation event, perhaps the movement of the sterol in the donor particle to a more exposed (hydrated) position. In the second step, the activated sterol would be transferred during transient collisions between donor and acceptor particles. When collision is not rate-limiting, the overall process would appear to be simply first-order, hence kinetically indistinguishable from the aqueous diffusion mechanism. The activation-collision model thus not only rationalizes our data but is also consistent with the simpler kinetics previously reported for the transfer of both membrane phospholipids and sterols.     

Point mutational analysis of the liganding site in human glycolipid transfer protein. Functionality of the complex

Mammalian glycolipid transfer proteins (GLTPs) facilitate the selective transfer of glycolipids between lipid vesicles in vitro. Recent structural determinations of the apo- and glycolipid-liganded forms of human GLTP have provided the first insights into the molecular architecture of the protein and its glycolipid binding site (Malinina, L., Malakhova, M. L., Brown, R. E., and Patel, D. J. (2004) Nature 430, 1048-1053). In the present study, we have evaluated the functional consequences of point mutation of the glycolipid liganding site of human GLTP within the context of a carrier-based mechanism of glycolipid intermembrane transfer. Different approaches were developed to rapidly and efficiently assess the uptake and release of glycolipid by GLTP. They included the use of glass-immobilized, glycolipid films to load GLTP with glycolipid and separation of GLTP/glycolipid complexes from vesicles containing glycolipid (galactosylceramide or lactosylceramide) or from monosialoganglioside dispersions by employing nickel-nitrilotriacetic acid-based affinity or gel filtration strategies. Point mutants of the sugar headgroup recognition center (Trp-96, Asp-48, Asn-52) and of the ceramide-accommodating hydrophobic tunnel (Phe-148, Phe-183, Leu-136) were analyzed for their ability to acquire and release glycolipid ligand. Two manifestations of point mutation within the liganding site were apparent: (i) impaired formation of the GLTP/glycolipid complex; (ii) impaired acquisition and release of bound glycolipid by GLTP. The results are consistent with a carrier-based mode of GLTP action to accomplish the intermembrane transfer of glycolipid. Also noteworthy was the inefficient release of glycolipid by wtGLTP into phosphatidylcholine acceptor vesicles, raising the possibility of a function other than intermembrane glycolipid transfer in vivo.