Mitochondrial cholesterol import

All animal subcellular membranes require cholesterol, which influences membrane fluidity and permeability, fission and fusion processes, and membrane protein function. The distribution of cholesterol among subcellular membranes is highly heterogeneous and the cholesterol content of each membrane must be carefully regulated. Compared to other subcellular membranes, mitochondrial membranes are cholesterol-poor, particularly the inner mitochondrial membrane (IMM). As a result, steroidogenesis can be controlled through the delivery of cholesterol to the IMM, where it is converted to pregnenolone. The low basal levels of cholesterol also make mitochondria sensitive to changes in cholesterol content, which can have a relatively large impact on the biophysical and functional characteristics of mitochondrial membranes. Increased mitochondrial cholesterol levels have been observed in diverse pathological conditions including cancer, steatohepatitis, Alzheimer disease and Niemann-Pick Type C1-deficiency, and are associated with increased oxidative stress, impaired oxidative phosphorylation, and changes in the susceptibility to apoptosis, among other alterations in mitochondrial function. Mitochondria are not included in the vesicular trafficking network; therefore, cholesterol transport to mitochondria is mostly achieved through the activity of lipid transfer proteins at membrane contact sites or by cytosolic, diffusible lipid transfer proteins. Here we will give an overview of the main mechanisms involved in mitochondrial cholesterol import, focusing on the steroidogenic acute regulatory protein StAR/STARD1 and other members of the StAR-related lipid transfer (START) domain protein family, and we will discuss how changes in mitochondrial cholesterol levels can arise and affect mitochondrial function. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.     

Oxygen permeability of the lipid bilayer membrane made of calf lens lipids

The oxygen permeability coefficient across the membrane made of the total lipid extract from the plasma membrane of calf lens was estimated from the profile of the oxygen transport parameter (local oxygen diffusion-concentration product) and compared with those estimated for membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Profiles of the oxygen transport parameter were obtained by observing the collision of molecular oxygen with nitroxide radical spin labels placed at different depths in the membrane using the saturation-recovery EPR technique and were published by us earlier (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta. 1768 (2007) 1454-1465). At 35 °C, the estimated oxygen permeability coefficients were 51.3, 49.7, and 157.4 cm/s for lens lipid, POPC/Chol, and POPC membranes, respectively (compared with 53.3 cm/s for a water layer with the same thickness as a membrane). Membrane permeability significantly decreases at lower temperatures. In the lens lipid membrane, resistance to the oxygen transport is located in and near the polar headgroup region of the membrane to the depth of the ninth carbon, which is approximately where the steroid-ring structure of cholesterol reaches into the membrane. In the central region of the membrane, oxygen transport is enhanced, significantly exceeding that in bulk water. It is concluded that the high level of cholesterol in lens lipids is responsible for these unique membrane properties.     

Oxygen permeability of the lipid bilayer membrane made of calf lens lipids

The oxygen permeability coefficient across the membrane made of the total lipid extract from the plasma membrane of calf lens was estimated from the profile of the oxygen transport parameter (local oxygen diffusion-concentration product) and compared with those estimated for membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Profiles of the oxygen transport parameter were obtained by observing the collision of molecular oxygen with nitroxide radical spin labels placed at different depths in the membrane using the saturation-recovery EPR technique and were published by us earlier (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta. 1768 (2007) 1454-1465). At 35 degrees C, the estimated oxygen permeability coefficients were 51.3, 49.7, and 157.4 cm/s for lens lipid, POPC/Chol, and POPC membranes, respectively (compared with 53.3 cm/s for a water layer with the same thickness as a membrane). Membrane permeability significantly decreases at lower temperatures. In the lens lipid membrane, resistance to the oxygen transport is located in and near the polar headgroup region of the membrane to the depth of the ninth carbon, which is approximately where the steroid-ring structure of cholesterol reaches into the membrane. In the central region of the membrane, oxygen transport is enhanced, significantly exceeding that in bulk water. It is concluded that the high level of cholesterol in lens lipids is responsible for these unique membrane properties.     

Mitochondrial membranes modify mutant huntingtin aggregation

Huntington's disease (HD) is a neurodegenerative disease caused by the expansion of a polyglutamine (polyQ) tract near the N-terminus of the huntingtin (htt) protein. Expanded polyQ tracts are prone to aggregate into oligomers and insoluble fibrils. Mutant htt (mhtt) localizes to variety of organelles, including mitochondria. Specifically, mitochondrial defects, morphological alteration, and dysfunction are observed in HD. Mitochondrial lipids, cardiolipin (CL) in particular, are essential in mitochondria function and have the potential to directly interact with htt, altering its aggregation. Here, the impact of mitochondrial membranes on htt aggregation was investigated using a combination of mitochondrial membrane mimics and tissue-derived mitochondrial-enriched fractions. The impact of exposure of outer and inner mitochondrial membrane mimics (OMM and IMM respectively) to mhtt was explored. OMM and IMM reduced mhtt fibrillization, with IMM having a larger effect. The role of CL in mhtt aggregation was investigated using a simple PC system with varying molar ratios of CL. Lower molar ratios of CL (<5%) promoted fibrillization; however, increased CL content retarded fibrillization. As revealed by in situ AFM, mhtt aggregation and associated membrane morphological changes at the surface of OMM mimics was markedly different compared to IMM mimics. While globular deposits of mhtt with few fibrillar aggregates were observed on OMM, plateau-like domains were observed on IMM. A similar impact on htt aggregation was observed with exposure to purified mitochondrial-enriched fractions. Collectively, these observations suggest mitochondrial membranes heavily influence htt aggregation with implication for HD.     

Physical properties of the lipid bilayer membrane made of calf lens lipids: EPR spin labeling studies

The physical properties of a membrane derived from the total lipids of a calf lens were investigated using EPR spin labeling and were compared with the properties of membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in all three membranes. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are practically identical in lens lipid and POPC/Chol membranes, but differ drastically from profiles in pure POPC membranes. In both lens lipid and POPC/Chol membranes, the lipids are strongly immobilized at all depths, which is in contrast to the high fluidity of the POPC membrane. Hydrophobicity and oxygen transport parameter profiles in lens lipid and POPC/Chol membranes have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. At this position, hydrophobicity increases from the level of methanol to the level of hexane, and the oxygen transport parameter increases by a factor of 2-3. These profiles in POPC membranes are bell-shaped. It is concluded that the high level of cholesterol in lens lipids makes the membrane stable, immobile, and impermeable to both polar and nonpolar molecules.     

Physical properties of the lipid bilayer membrane made of calf lens lipids: EPR spin labeling studies

The physical properties of a membrane derived from the total lipids of a calf lens were investigated using EPR spin labeling and were compared with the properties of membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in all three membranes. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are practically identical in lens lipid and POPC/Chol membranes, but differ drastically from profiles in pure POPC membranes. In both lens lipid and POPC/Chol membranes, the lipids are strongly immobilized at all depths, which is in contrast to the high fluidity of the POPC membrane. Hydrophobicity and oxygen transport parameter profiles in lens lipid and POPC/Chol membranes have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. At this position, hydrophobicity increases from the level of methanol to the level of hexane, and the oxygen transport parameter increases by a factor of 2-3. These profiles in POPC membranes are bell-shaped. It is concluded that the high level of cholesterol in lens lipids makes the membrane stable, immobile, and impermeable to both polar and nonpolar molecules.     

Impact of cholesterol and Lumacaftor on the folding of CFTR helical hairpins

Cystic fibrosis (CF) is caused by mutations in the gene that codes for the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). Recent advances in CF treatment have included use of small-molecule drugs known as modulators, such as Lumacaftor (VX-809), but their detailed mechanism of action and interplay with the surrounding lipid membranes, including cholesterol, remain largely unknown. To examine these phenomena and guide future modulator development, we prepared a set of wild type (WT) and mutant helical hairpin constructs consisting of CFTR transmembrane (TM) segments 3 and 4 and the intervening extracellular loop (termed TM3/4 hairpins) that represent minimal membrane protein tertiary folding units. These hairpin variants, including CF-phenotypic loop mutants E217G and Q220R, and membrane-buried mutant V232D, were reconstituted into large unilamellar phosphatidylcholine (POPC) vesicles, and into corresponding vesicles containing 70 mol% POPC +30 mol% cholesterol, and studied by single-molecule FRET and circular dichroism experiments. We found that the presence of 30 mol% cholesterol induced an increase in helicity of all TM3/4 hairpins, suggesting an increase in bilayer cross-section and hence an increase in the depth of membrane insertion compared to pure POPC vesicles. Importantly, when we added the corrector VX-809, regardless of the presence or absence of cholesterol, all mutants displayed folding and helicity largely indistinguishable from the WT hairpin. Fluorescence spectroscopy measurements suggest that the corrector alters lipid packing and water accessibility. We propose a model whereby VX-809 shields the protein from the lipid environment in a mutant-independent manner such that the WT scaffold prevails. Such ‘normalization’ to WT conformation is consistent with the action of VX-809 as a protein-folding chaperone.     

The influence of cholesterol precursor – desmosterol – on artificial lipid membranes

The disorders in cholesterol biosynthesis pathway and various diseases manifest in the accumulation of cholesterol precursors in the human tissues and cellular membranes. In this paper the effect of desmosterol – one of cholesterol precursors – on model lipid membranes was studied. The investigations were performed for binary SM/desmo and POPC/desmo and ternary SM/POPC/desmo monolayers. Moreover, the experiments based on the gradual substitution of cholesterol by desmosterol in SM/POPC/chol = 1:1:1 system were done. The obtained results allowed one to conclude that desmosterol is of lower domains promoting and stabilizing properties and packs less tightly with the lipids in monolayers. Moreover, desmosterol probably could replace cholesterol in model membranes, but only at its low proportion in the system (2%), however, at a higher degree of cholesterol substitution a significant decrease of the monolayer stability and packing and alterations in the film morphology were detected. The results collected in this work together with those from previous experiments allowed one to analyze the effect of a double bond in the sterol side chain as well as its position in the ring system on membrane activity of the molecule and to verify Bloch hypothesis.     

Permeabilization of phospholipid bilayer membranes induced by gas-liquid flow in an airlift bubble column

The permeability of 5(6)-carboxyfluorescein (CF) through the phospholipid bilayer membranes was measured by using the system in which the CF-containing phospholipid vesicles (liposomes) were suspended in the gas-liquid flow in an external loop airlift bubble column. The airlift was operated at various superficial gas velocities UG up to 2.4 cm/s at 25 and 40 degrees C. The CF-containing liposomes composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) had the nominal diameters of 50, 100, and 200 nm. The 50- and 100-nm liposomes were stable at 40 degrees C for 5 h even at a high UG value of 2.4 cm/s based on the observed turbidity of the liposome suspension in the airlift. On the other hand, the 200-nm liposomes were stable at a low UG value of 1.4 cm/s, although a progressive decrease in size of the liposomes was implied at the high UG value of 2.4 cm/s. The permeability coefficient PCF of CF through the lipid membrane of the 100-nm liposomes was significantly increased with increasing UG at a high temperature of 40 degrees C, while at a low temperature of 25 degrees C the PCF value was little dependent on UG. As a typical result on the above liposomes, the PCF value (9.2 x 10(-11) cm/s) at 40 degrees C and UG = 2.4 cm/s in the airlift was more than 15 times larger than that at 25 degrees C in the static liquid corresponding to UG = 0. In addition, the dependence of the PCF value on UG at 40 degrees C became more significant with increasing the size of liposomes suspended. The results obtained revealed that the permeability of the liposome membranes could be regulated by suspending the liposomes in the gas-liquid flow in the airlift without modulating the membrane composition of liposomes.     

Effect of cholesterol on the lactosylceramide domains in phospholipid bilayers

Lactosylceramide (LacCer) in the plasma membranes of immune cells is an important lipid for signaling in innate immunity through the formation of LacCer-rich domains together with cholesterol (Cho). However, the properties of the LacCer domains formed in multicomponent membranes remain unclear. In this study, we examined the properties of the LacCer domains formed in Cho-containing 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) membranes by deuterium solid-state NMR and fluorescence lifetimes. The potent affinity of LacCer-LacCer (homophilic interaction) is known to induce a thermally stable gel phase in the unitary LacCer bilayer. In LacCer/Cho binary membranes, Cho gradually destabilized the LacCer gel phase to form the liquid-ordered phase by its potent order effect. In the LacCer/POPC binary systems without Cho, the ²H NMR spectra of 10′,10′-d₂-LacCer and 18′,18′,18′-d₃-LacCer probes revealed that LacCer was poorly miscible with POPC in the membranes and formed stable gel phases without being distributed in the liquid crystalline domain. The lamellar structure of the LacCer/POPC membrane was gradually disrupted at around 60°C, whereas the addition of Cho increased the thermal stability of the lamellarity. Furthermore, the area of the LacCer gel phase and its chain order were decreased in the LacCer/POPC/Cho ternary membranes, whereas the liquid-ordered domain, which was observed in the LacCer/Cho binary membrane, was not observed. Cho surrounding the LacCer gel domain liberated LacCer and facilitated forming the submicron to nano-scale small domains in the liquid crystalline domain of the LacCer/POPC/Cho membranes, as revealed by the fluorescence lifetimes of trans-parinaric acid and trans-parinaric acid-LacCer. Our findings on the membrane properties of the LacCer domains, particularly in the presence of Cho, would help elucidate the properties of the LacCer domains in biological membranes.     

tcBid promotes Ca(2+) signal propagation to the mitochondria: control of Ca(2+) permeation through the outer mitochondrial membrane

Calcium spikes established by IP(3) receptor-mediated Ca(2+) release from the endoplasmic reticulum (ER) are transmitted effectively to the mitochondria, utilizing local Ca(2+) interactions between closely associated subdomains of the ER and mitochondria. Since the outer mitochondrial membrane (OMM) has been thought to be freely permeable to Ca(2+), investigations have focused on IP(3)-driven Ca(2+) transport through the inner mitochondrial membrane (IMM). Here we demonstrate that selective permeabilization of the OMM by tcBid, a proapoptotic protein, results in an increase in the magnitude of the IP(3)-induced mitochondrial [Ca(2+)] signal. This effect of tcBid was due to promotion of activation of Ca(2+) uptake sites in the IMM and, in turn, to facilitation of mitochondrial Ca(2+) uptake. In contrast, tcBid failed to control the delivery of sustained and global Ca(2+) signals to the mitochondria. Thus, our data support a novel model that Ca(2+) permeability of the OMM at the ER- mitochondrial interface is an important determinant of local Ca(2+) signalling. Facilitation of Ca(2+) delivery to the mitochondria by tcBid may also support recruitment of mitochondria to the cell death machinery.     

Lipid raft components cholesterol and sphingomyelin increase H+/OH- permeability of phosphatidylcholine membranes

H+/OH- permeation through lipid bilayers occurs at anomalously high rates and the determinants of proton flux through membranes are poorly understood. Since all life depends on proton gradients, it is important to develop a greater understanding of proton leak phenomena. We have used stopped-flow fluorimetry to probe the influence of two lipid raft components, chol (cholesterol) and SM (sphingomyelin), on H+/OH- and water permeability. Increasing the concentrations of both lipids in POPC (palmitoyl-2-oleoyl phosphatidylcholine) liposomes decreased water permeability in a concentration-dependent manner, an effect that correlated with increased lipid order. Surprisingly, proton flux was increased by increasing the concentration of chol and SM. The chol effect was complex with molar concentrations of 17.9, 33 and 45.7% giving 2.8-fold (P<0.01), 2.2-fold (P<0.001) and 5.1-fold (P<0.001) increases in H+/OH- permeability from a baseline of 2.4x10(-2) cm/s. SM at 10 mole% effected a 2.8-fold increase (P<0.01), whereas 20 and 30 mole% enhanced permeability by 3.6-fold (P<0.05) and 4.1-fold respectively (P<0.05). Supplementing membranes containing chol with SM did not enhance H+/OH- permeability. Of interest was the finding that chol addition to soya-bean lipids decreased H+/OH- permeability, consistent with an earlier report [Ira and Krishnamoorthy (2001) J. Phys. Chem. B 105, 1484-1488]. We speculate that the presence of proton carriers in crude lipid extracts might contribute to this result. We conclude that (i) chol and SM specifically and independently increase rates of proton permeation in POPC bilayers, (ii) domains enriched in these lipids or domain interfaces may represent regions with high H+/OH- conductivity, (iii) H+/OH- fluxes are not governed by lipid order and (iv) chol can inhibit or promote H+/OH- permeability depending on the total lipid environment. Theories of proton permeation are discussed in the light of these results.     

Molecular characterization of the interaction of crotamine-derived nucleolar targeting peptides with lipid membranes

A novel class of cell-penetrating, nucleolar-targeting peptides (NrTPs), was recently developed from the rattlesnake venom toxin crotamine. Based on the intrinsic fluorescence of tyrosine or tryptophan residues, the partition of NrTPs and crotamine to membranes with variable lipid compositions was studied. Partition coefficient values (in the 10(2)-10(5) range) followed essentially the compositional trend POPC:POPG≤POPG相似文献   

Solid-state nuclear magnetic resonance relaxation studies of the interaction mechanism of antimicrobial peptides with phospholipid bilayer membranes

An 18-residue peptide, KWGAKIKIGAKIKIGAKI-NH(2) was designed to form amphiphilic beta-sheet structures when bound to lipid bilayers. The peptide possesses high antimicrobial activity when compared to naturally occurring linear antimicrobial peptides, most of which adopt an amphipathic alpha-helical conformation upon binding to the lipids. The perturbation of the bilayer by the peptide was studied by static (31)P and (2)H solid-state NMR spectroscopy using POPC and POPG/POPC (3/1) bilayer membranes with sn-1 chain perdeuterated POPC and POPG as the isotopic labels. (31)P NMR powder spectra exhibited two components for POPG/POPC bilayers upon addition of the peptide but only a slight change in the line shape for POPC bilayers, indicating that the peptide selectively disrupted the membrane structure consisting of POPG lipids. (2)H NMR powder spectra indicated a reduction in the lipid chain order for POPC bilayers and no significant change in the ordering for POPG/POPC bilayers upon association of the peptide with the bilayers, suggesting that the peptide acts as a surface peptide in POPG/POPC bilayers. Relaxation rates are more sensitive to the motions of the membranes over a large range of time scales. Longer (31)P longitudinal relaxation times for both POPG and POPC in the presence of the peptide indicated a direct interaction between the peptide and the POPG/POPC bilayer membranes. (31)P longitudinal relaxation studies also suggested that the peptide prefers to interact with the POPG phospholipids. However, inversion-recovery (2)H NMR spectroscopic experiments demonstrated a change in the relaxation rate of the lipid acyl chains for both the POPC membranes and the POPG/POPC membranes upon interaction with the peptide. Transverse relaxation studies indicated an increase in the spectral density of the collective membrane motion caused by the interaction between the peptide and the POPG/POPC membrane. The experimental results demonstrate significant dynamic changes in the membrane in the presence of the antimicrobial peptide and support a carpet mechanism for the disruption of the membranes by the antimicrobial peptide.     

Lipids, curvature stress, and the action of lipid prodrugs: free fatty acids and lysolipid enhancement of drug transport across liposomal membranes

Molecular shape and its impact on bilayer curvature stress are powerful concepts for describing the effects of lipids and fatty acids on fundamental membrane properties, such as passive permeability and derived properties like drug transport across liposomal membranes. We illustrate these relationships by studying the effects of fatty acids and lysolipids on the permeation of a potent anti-cancer drug, doxorubicin, across the bilayer of a liposome in which the drug is encapsulated. Using a simple fluorescence assay, we have systematically studied the passive permeation of doxorubicin across liposomal membranes in different lipid phases: the solid-ordered phase (DPPC bilayers), the liquid-disordered phase (POPC lipid bilayers), and the liquid-ordered phase induced by high levels of cholesterol (DOPC + cholesterol lipid bilayers). The effect of different free fatty acids (FA) and lysolipids (LL), separately and in combination, on permeability was assessed to elucidate the possible mechanism of phospholipase A₂-triggered release in cancer tissue of liposomal doxorubicin formulations. In all cases, FAs applied separately lead to significant enhancement of permeability, most pronounced in liquid-disordered bilayers and less pronounced in solid and solid-ordered bilayers. LLs applied separately had only a marginal effect on permeability. FA and LL applied in combination lead to a synergistic enhancement of permeability in solid bilayers, whereas in liquid-disordered bilayers, the combined effect suppressed the otherwise strong permeability enhancement due to the FAs.     

Impact of membrane-anchored fluorescent probes on the mechanical properties of lipid bilayers

Fluorescent probes are used in membrane biophysics studies to provide information about physical properties such as lipid packing, polarity and lipid diffusion or to visualize membrane domains. However, our understanding of the effects the dyes themselves may induce on the membrane structure and properties are sparse. As mechanical properties like bending elasticity were already shown to be highly sensitive to the addition of “impurities” into the membranes, we have investigated the impact of six different commonly used fluorescent membrane probes (LAURDAN, TR-DPPE, Rh-DPPE, DiIC18, Bodipy-PC and NBD-PC) on the bending elasticity of dye containing POPC GUVs as compared to single component POPC GUVs. Small changes in the membrane bending elasticity compared to single POPC bilayers are observed when 2 mol% of Rh-DPPE, Bodipy-PC or NBD-PC are added in POPC membranes. These binary membranes are showing non reproducible mechanical properties attributed to a photo-induced peroxidation processes that may be controlled by a reduction of the fluorescent dye concentration. For TR-DPPE, a measurable decrease of the bending elasticity is detected with reproducible bending elasticity measurements. This is a direct indication that this dye, when exposed to illumination by a microscope lamp and contrary to Rh-DPPE, does not induce chemical degradation. At last, LAURDAN and DiIC18 probes mixed with POPC do not significantly affect the bending elasticity of pure POPC bilayers, even at 2 mol%, suggesting these latter probes do not induce major perturbations on the structure of POPC bilayers.     

Changes of the fluidity of mitochondrial membranes induced by the permeability transition.

The dynamic properties of protein and lipid regions of mitochondrial membranes during the permeability transition (PT) process were studied by following the anisotropy changes of hematoporphyrin (HP) and 1,6-diphenyl-1,3,5-hexatriene (DPH), respectively. We show that opening of the PT pore is accompanied by a remarkable increase of mitochondrial membrane fluidity which is specifically localized to protein sites, while lipid domains are unaffected. The increased membrane fluidity is not related to the collapse of transmembrane potential that follows the PT, as demonstrated by a comparison between the anisotropy properties of permeabilized mitochondria and impermeable, depolarized organelles. Parameters such as osmotic swelling and temperature, which are shown to affect the mitochondrial membrane dynamics in the absence of permeability transition, cannot alone account for the pore dynamical properties. We suggest that the observed increase in fluidity is mainly due to a conformational change of pore-forming protein(s) during the "assembly" of the PT pore.     

HIV-1 Nef Perturbs Artificial Membranes: Investigation of the Contribution of the Myristoyl Anchor

Nef, an accessory protein from human immunodeficiency virus type 1, is critical for optimal viral replication and pathogenesis. Here, we analyzed the influence of full-length myristoylated and nonmyristoylated Nef on artificial lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). By means of cosedimentation assays, we found that neither nonmyristoylated nor myristoylated Nef stably binds to POPC unilamellar vesicles. Time-resolved ellipsometry rather indicates that the proteins perturb the assembly of POPC planar bilayers. This observation was corroborated by fluorescence and scanning force microscopy, suggesting that membrane disordering occurs upon interaction of full-length myristoylated and nonmyristoylated Nef with planar POPC membranes immobilized on SiO₂ surfaces resulting in loss of material from the surface. The membrane perturbations were further investigated by vesicle release experiments, demonstrating that the disordering results in defects through which the fluorophor carboxyfluorescein can pass. From these results, we conclude that Nef is capable of disordering and perturbing lipid membranes and that the myristoyl group is not the decisive determinant for the action of the protein on lipid membranes.     

Studies on incorporation of CoQ-homologs in mitochondrial membranes

In this work we have made a systematic study on the incorporation of different homologs of Coenzyme Q in mitochondrial membranes. We have used a diluted mitochondrial suspension constituting a biphasic system membrane/H2O in which exogenous Q will be distributed depending on its water solubility and on its affinity to the mitochondrial membrane. It was found that Ubiquinones are incorporated into mitochondria in different extents ranging from zero (Q1) to 10 fold (Q10) the concentration of endogenous Q per mg of mitochondrial protein. In Q-depleted mitochondria the extents of incorporation are greater for all the quinones. This study points out that there is a reflection between the isoprenoid units of each Q-homolog and its incorporation into mitochondrial membranes.