Nanodiscs bounded by styrene-maleic acid allow trans-cis isomerization of enclosed photoswitches of azobenzene labeled lipids.

Styrene-and-maleic acid (SMA) copolymers behave as amphipathic belts encircling lipids in the form of nanodiscs. It is unclear to what extent the SMA belt affects the order and dynamics of the enclosed lipids. We aimed to obtain insight into this by making use of synthetic azobenzene-labeled phospholipids incorporated into di-16:0 PC nanodiscs. Azobenzene lipids undergo geometric isomerization upon exposure to light at 365 nm, resulting in the formation of cis-isomers that possess a larger cross-sectional area than the trans-isomers. The influence of the lipid properties on the kinetics and extent of isomerization of the azobenzene groups was first tested in large unilamellar vesicles constituted by lipid mixtures with different packing properties of the acyl chains. Fastest isomerization kinetics were found when azolipids were present in membranes supplemented with lysolipids and slowest in those supplemented with di-unsaturated lipids, suggesting that the isomerization rate is sensitive to the lateral pressure profile in the lipid bilayer and hence may be considered a convenient tool to monitor packing properties of lipids enclosed in nanodiscs. When azolipids were incorporated in SMA-bounded nanodiscs, azolipid isomerization was found to take place readily, indicating that SMA polymers behave as rather flexible belts and allow expansion of the enclosed lipid material.

Thermotropic properties of phosphatidylcholine nanodiscs bounded by styrene-maleic acid copolymers.

Styrene-maleic acid copolymers (SMA) have been gaining interest in the field of membrane research due to their ability to solubilize membranes into nanodics. The SMA molecules act as an amphipathic belt that surrounds the nanodiscs, whereby the hydrophobic styrene moieties can insert in between the lipid acyl chains. Here we used SMA variants with different styrene-to-maleic acid ratio (i.e. 2:1, 3:1 and 4:1) to investigate how lipid packing in the nanodiscs is affected by the presence of the polymers and how it depends on polymer composition. This was done by analyzing the thermotropic properties of a series of saturated phosphatidylcholines in nanodiscs using laurdan fluorescence and differential scanning calorimetry. In all cases it was found that the temperature of the main phase transition (Tm) of the lipids in the nanodiscs is downshifted and that its cooperativity is strongly reduced as compared to the situation in vesicles. These effects were least pronounced for lipids in nanodiscs bounded by SMA 2:1. Unexpected trends were observed for the calorimetric enthalpy of the transition, suggesting that the polymer itself contributes, possibly by rearranging around the nanodiscs when the lipids adopt the fluid phase. Finally, distinct differences in morphology were observed for nanodiscs at relatively high polymer concentrations, depending on the SMA variant used. Overall, the results suggest that the extent of preservation of native thermodynamic properties of the lipids as well as the stability of the nanodiscs at high polymer concentrations is better for SMA 2:1 than for the other SMA variants.

The phosphatidylcholine to phosphatidylethanolamine ratio of Saccharomyces cerevisiae varies with the growth phase.

This study compares the effect of the growth phase on the phospholipid composition and the activity of several phospholipid biosynthetic enzymes in a wild-type yeast grown in fermentable (glucose) and non-fermentable (lactate) semi-synthetic and complete synthetic media. Several distinct differences as well as similarities were found. The cellular phosphatidylcholine: phosphatidylethanolamine (PC:PE) ratio was found to vary with the growth phase, with increases in PC levels at the expense of PE during the transition to stationary phase. The variation was most pronounced in semi-synthetic lactate medium, which is routinely used for the isolation of mitochondria, where the PC:PE ratio changed from 0.9 to 2.2 during this transition. Similar growth phase-dependent changes in PC and PE content were observed in isolated organelles such as mitochondria, mitochondria-associated membranes and microsomes. Phosphatidylinositol (PI) levels were much higher in cells grown on lactate compared to cells grown on glucose (20% vs. 5-10%). Irrespective of the medium, PI levels increased upon entering stationary phase. The activities of the phospholipid biosynthetic enzymes phosphatidylserine synthase and the phospholipid-N-methyltransferases were found to be maximal at the end of logarithmic growth and to decrease upon entering stationary phase in all media. Cells grown on lactate displayed a significantly higher phospholipid:protein ratio than cells grown on glucose. The results are discussed in terms of regulation of phospholipid biosynthesis and membrane biogenesis in response to growth phase and carbon source.

Transbilayer movement of phosphatidylcholine in the mitochondrial outer membrane of Saccharomyces cerevisiae is rapid and bidirectional.

The process of transmembrane movement of phosphatidylcholine (PC) across the outer membrane of mitochondria was investigated in vitro in mitochondrial outer membrane vesicles (OMV) from the yeast Saccharomyces cerevisiae. Phosphatidylcholine-transfer protein (PC-TP) was used to extract radiolabeled PC from OMV, with small unilamellar vesicles serving as acceptor system. Endogenously radiolabeled PC synthesized either via the CDP-choline pathway or via methylation of phosphatidylethanolamine can be extracted completely from the OMV with a t(1/2) of 1 min or less at 30 degrees C. The size of the pool of PC in OMV available for exchange by PC-TP is not affected by pretreatment of the OMV with proteinase K or sulfhydryl reagents. In the reverse experiment where radiolabeled PC was introduced into the OMV, similar characteristics for the exchange were found. The accessibility of labeled PC to externally added phospholipase A(2) was used as a measure for its transmembrane distribution. It was found that PC is not exclusively located in the outer leaflet of the OMV. Only 30-35% can be degraded in intact OMV by phospholipase A(2), irrespective of whether the PC is introduced by PC-TP or endogenously synthesized via either of the pathways of biosynthesis. The results demonstrate the occurrence of rapid bidirectional transbilayer movement of both endogenous and in vitro introduced PC in OMV. Furthermore, there appears to be no preference for mitochondrial import of PC synthesized by either of the pathways in vivo.

Isolation and characterization of highly purified mitochondrial outer membranes of the yeast Saccharomyces cerevisiae (method).

Mitochondrial outer membrane vesicles (OMV) from the yeast Saccharomyces cerevisiae were prepared by osmotic swelling and mechanical disruption of mitochondria that had been isolated at pH 6.0 and purified by density gradient centrifugation. The OMV were obtained in a yield of 1% (protein/protein) with respect to the mitochondria. The OMV were shown to be essentially free of mitochondrial inner membrane protein markers, while contamination with endoplasmic reticulum was around 5% (protein-based). The very low phosphatidylserine synthase activity present in the OMV preparation indicated that contamination with mitochondria-associated membranes (MAM) was negligible. The resistance of the outer membrane protein Tom40 to digestion by trypsin demonstrated the sealed nature and right-side out orientation of the OMV. Analysis of the phospholipid composition revealed that the contents of phosphatidylcholine and phosphatidylinositol are higher and the content of phosphatidylethanolamine is lower in the mitochondrial outer membrane as compared to whole mitochondria. Cardiolipin is largely depleted in the OMV.

Non-bilayer lipids are required for efficient protein transport across the plasma membrane of Escherichia coli.

The construction of a mutant Escherichia coli strain which cannot synthesize phosphatidylethanolamine provides a tool to study the involvement of non-bilayer lipids in membrane function. This strain produces phosphatidylglycerol and cardiolipin (CL) as major membrane constituents and requires millimolar concentrations of divalent cations for growth. In this strain, the lipid phase behaviour is tightly regulated by adjustment of the level of CL which favours a nonbilayer organization in the presence of specific divalent cations. We have used an in vitro system of inverted membrane vesicles to study the involvement of non-bilayer lipids in protein translocation in the secretion pathway. In this system, protein translocation is very low in the absence of divalent cations but can be enhanced by inclusion of Mg2+, Ca2+ or Sr2+ but not by Ba2+ which is unable to sustain growth of the mutant strain and cannot induce a non-bilayer phase in E. coli CL dispersions. Alternatively, translocation in cation depleted vesicles could be increased by incorporation of the non-bilayer lipid DOPE (18:1) but not by DMPE (14:0) or DOPC (18:1), both of which are bilayer lipids under physiological conditions. We conclude that non-bilayer lipids are essential for efficient protein transport across the plasma membrane of E. coli.

Regulation of lipid polymorphism is essential for the viability of phosphatidylethanolamine-deficient Escherichia coli cells.

Escherichia coli strain AD93 is unable to synthesize the nonbilayer lipid phosphatidylethanolamine and requires high concentrations of specific divalent cations for growth. Previous studies suggested that in this strain, cardiolipin in combination with divalent cations functionally replaces phosphatidylethanolamine, reflecting polymorphic regulation of membrane lipid composition. However, it is also possible that divalent cations are required for regulation of lipid packing or membrane surface potential. 2H NMR was employed to measure the effect of different divalent cations on lipid packing in aqueous dispersions of lipid extracts isolated from AD93 and the wild type parental strain W3899, which were grown with [11,11-2H2]oleic acid. The results indicate that a range of acyl chain order is compatible with growth and that Ba2+, which cannot support growth of AD93, can increase chain packing to the wild type level. By means of microelectrophoresis, it was shown that the growth-promoting cations and Ba2+ have a strong and comparable ability to screen the surface charge of large unilamellar vesicles prepared from AD93 lipid extracts. Therefore, it is unlikely that the growth-promoting capacity of divalent cations is primarily due to their effect on lipid packing or their potency to decrease the surface potential. Furthermore, the addition of small amounts of Ba2+ to a AD93 lipid dispersion with excess Mg2+ diminished HII phase formation. This observation can explain the growth arrest in AD93 cultures upon the addition of Ba2+ and further supports the conclusion that the cation requirement of this strain arises mainly from polymorphic regulation of lipid composition.

Effect of divalent cations on lipid organization of cardiolipin isolated from Escherichia coli strain AH930.

Escherichia coli strain AH930 is a lipid biosynthetic mutant, which is unable to synthesize phosphatidylethanolamine. Instead it produces large amounts of phosphatidylglycerol and cardiolipin and has an absolute requirement for certain divalent cations. Cardiolipin was isolated from this mutant strain and its interaction with divalent cations was studied by various biophysical techniques. Monolayer measurements showed that the cations decrease the molecular surface area of cardiolipin in the order Ca2+ approximately Mg2+ > Sr2+ > Ba2+. 31P-NMR and X-ray diffraction measurements demonstrated a comparable sequence for the ability of the cations to promote HII phase formation in dispersions of the E. coli cardiolipin: Ca2+ and Mg2+ induced HII phase formation at 50 degrees C, Sr2+ at 75 degrees C, while Ba2+ was found to be unable to promote HII phase formation in the temperature range measured. Furthermore, all divalent cations were found to increase the temperature at which the transition to the liquid-crystalline phase takes place, which was below 5 degrees C for the lipid in the absence of divalent cations. In the presence of Sr2+, Mg2+ and Ba2+ and at 25 degrees C two lamellar phases were observed, one corresponding to a liquid-crystalline phase, the other to either a gel or a crystalline phase. In the presence of Ca2+ at 25 degrees C and even at 45 degrees C no evidence for a liquid-crystalline phase was obtained and only a crystalline phase could be observed. The ability of the different cations to promote HII phase formation in the isolated E. coli cardiolipin was found to correlate with their ability to support growth of the mutant strain (De Chavigny, A., Heacock, P.N., Dowhan, W. (1991) J. Biol. Chem. 266, 5323-5332), suggesting that cardiolipin with divalent cations can replace the role of phosphatidylethanolamine in the mutant strain, and that this role involves the preference of these lipids for organization in non-bilayer lipid structures.

Acidic interaction of the colicin A pore-forming domain with model membranes of Escherichia coli lipids results in a large perturbation of acyl chain order and stabilization of the bilayer.

2H and 31P NMR techniques were used to study the effects on acyl chain order and lipid organization of the well-characterized pore-forming domain of colicin A (20-kDa thermolytic fragment of colicin A) upon insertion in model membrane systems derived from the Escherichia coli fatty acid auxotrophic strain K 1059, which was grown in the presence of [11,11-2H2]-labeled oleic acid. Addition of the protein to dispersions of the E. coli total lipid extract, in a 1/70 molar ratio of peptide to lipids, resulted in a large pH-dependent decrease in quadrupolar splitting of the 2H NMR spectra. The decrease of the quadrupolar splitting obtained at the various pH values was correlated with the pH dependence of the insertion of the protein in monolayer films using the same E. coli lipid extracts. The pK governing the perturbing effects on the order of the fatty acyl chains was around 5, in agreement with the values of the pH-dependent conformational changes of the pore-forming domain of colicin A required for membrane insertion as reported by van der Goot et al. [(1991) Nature 354, 408-410]. 31P NMR measurements show that the bilayer organization remains intact upon addition of the protein to dispersions of lipid extract. Surprisingly, 31P NMR measurements as a function of temperature indicate that the pore-forming domain of colicin A even stabilizes bilayer lipid structure at pH 4. Both the large effect of the protein on acyl chain order and its bilayer-stabilizing activity are indicative of a surface localization of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)