Membrane lipid biosynthesis in Acholeplasma laidlawii b: elongation of medium- and long-chain exogenous fatty acids in growing cells

The chain elongation of a wide variety of exogenous fatty acids and the subsequent incorporation of the chain elongation products into the total membrane lipids of Acholeplasma laidlawii B were systematically studied. Within each chemical class of fatty acids examined, the extent of chain elongation increased with increases in chain length, reached a maximum value, and then declined with further increases in chain length. Depending on chemical structure, exogenous fatty acids containing less than 6 to 9 carbon atoms or more than 15 to 18 carbon atoms were not substrates for the chain elongation system. The substrate specificity of this fatty acid elongation system was strikingly broad, and straight-chain, methyl isobranched, and methyl anteisobranched saturated fatty acids, as well as cis- and trans-monounsaturated, cis-cyclopropane, and cis-polyunsaturated fatty acids, underwent chain elongation in vivo. The extent of chain elongation and the average chain length of the primary elongation products correlated well with the physical properties (melting temperatures) of the exogenous fatty acid substrates. The specificity of fatty acid chain elongation in A. laidlawii B maintained the fluidity and physical state of the membrane lipids within a rather wide but definitely limited range. The fatty acid chain elongation system of this organism could be markedly influenced by the presence of a second exogenous fatty acid that was not itself a substrate for the chain elongation system but was incorporated directly into the membrane lipids. The presence of a relatively low-melting exogenous fatty acid increased both the extent of chain elongation and the average chain length of the elongation products generated, whereas the presence of a relatively high-melting fatty acid had the opposite effect. The extent of chain elongation and nature of the elongation products formed were not, however, dependent on the fluidity and physical state of the membrane lipids per se. The second exogenous fatty acid appeared instead to exert its characteristic effect by competing with the chain elongation substrate and elongation products for the stereospecific acylation of positions 1 and 2 of sn-glycerol-3-phosphate. The similar effects of alterations in environmental temperature, cholesterol content, and exposure to the antibiotic cerulenin on the fatty acid chain elongation and de novo biosynthetic activities suggested that the chain elongation system of this organism may be a component of the de novo biosynthetic system.     

Membrane lipid biosynthesis in Acholeplasma laidlawii B: Investigations into the in vivo regulation of the quantity and hydrocarbon chain lengths of de novo biosynthesized fatty acids in response to exogenously supplied fatty acids

The regulation of the nature and quantity of the fatty acids produced in vivo by Acholeplasma laidlawii B in the presence of various exogenous fatty acids has been investigated. In the presence of exogenous medium- or long-chain fatty acids, the organism appears to reduce the amounts of de novo biosynthesized fatty acids in its cellular lipid pool by two distinct mechanisms: an excretion of biosynthesized fatty acids to the growth medium as free fatty acids, and a reduction in total de novo biosynthetic output. These two mechanisms do not suffice to maintain constant total membrane lipid levels, but they do appear to significantly moderate the effect of exogenous fatty acids on the level of membrane lipid. In the presence of short-chain fatty acids, total membrane lipid levels are not elevated. Exogenous fatty acids can cause shifts in the average chain length of de novo biosynthesized fatty acids; the magnitudes and directions of these shifts can be correlated with the specificity of the exogenous species for esterification to the 1- or the 2-position of the glycerol moiety of membrane glycerolipids. As the various endogenously synthesized fatty acids differ in their positional specificity for glycerolipid esterification, we propose that the competition of an exogenous species with significant specificity for a particular position with the endogenously derived fatty acids specific for that position can selectively depress the synthesis of such endogenously derived species, thereby altering the overall product spectrum of de novo fatty acid biosynthesis in vivo.     

The biosynthetic incorporation of short-chain linear saturated fatty acids by Acholeplasma laidlawii B may suppress cell growth by perturbing membrane lipid polar headgroup distribution

Acholeplasma laidlawii B cells made fatty acid auxotrophic by growth in the presence of the biotin-binding agent avidin grow increasingly poorly at 37 degrees C when supplemented with single exogenous linear saturated fatty acids of decreasing hydrocarbon chain length. Interestingly, this progressive decrease in growth yields with decreasing hydrocarbon chain length is not observed when cells are cultured in the presence of other classes of exogenous fatty acids. Moreover, normal growth is observed is other types of fatty acids with equivalent or shorter hydrocarbon chain lengths, indicating that poor growth in the presence of short-chain linear saturated fatty acids cannot be due to a decrease in membrane lipid bilayer thickness per se. To understand the molecular basis of such growth inhibition, we determined the growth yields, membrane lipid fatty acid and polar headgroups compositions, and phase state and fluidity of the membrane lipids in cells progressively biosynthetically enriched in tridecanoic acid (13:0) or dodecanoic acid (12:0). The growth of fatty acid auxotrophic A. laidlawii B cells grown in the presence of binary combinations of an exogenous fatty acid which supports normal growth on its own and 13:0 or 12:0 revealed that growth inhibition is not observed until 13:0 and 12:0 biosynthetic incorporation levels reach about 90 and 60 mol %, respectively, after which growth is markedly inhibited. Differential scanning calorimetric analyses of membranes from cells maximally enriched in 13:0 indicate that the lipid gel/liquid-crystalline phase transition temperature is unexpectedly high but that at the growth temperature of 37 degrees C, the membrane lipid bilayer is almost exclusively in the liquid-crystalline state but is certainly not excessively fluid. However, high levels of 13:0 incorporation produce a greatly elevated level of the high melting, reversed nonlamellar phase-preferring lipid component monoglucosyl diacylglycerol, and greatly reduced levels of all other membrane lipid components. This marked elevation of monoglucosyl diacylglycerol levels can be rationalized as a regulatory response which maintains the lamellar/nonlamellar phase-forming propensity of the total membrane lipid mixture relatively constant in the face of the biosynthetic incorporation of increasing quantities of short-chain saturated fatty acids, which favor the lamellar phase. However, this lipid biosynthetic response produces a marked decline in the levels of anionic phospholipid and phosphoglycolipid which are probably required to maintain the minimal negative surface charge density of the lipid bilayer, which we suggest is responsible for the observed growth inhibition. This work shows that the lipid biosynthetic regulatory mechanisms present in this organism may sometimes operate at cross purposes such that it is not possible to simultaneously optimize all of the biologically relevant physical properties of the membrane lipid bilayer.     

Membrane lipid metabolism in Acholeplasma laidlawii A EF 22. Influence of cholesterol and temperature shift-down on incorporation of fatty acids and synthesis of membrane lipid species

1. Membrane lipid metabolism in Acholeplasma laidlowii A EF 22 has been studied under different conditions by applying three different techniques for changing membrane viscosity: fatty acid and cholesterol supplementation and temperature changes. 2. The molar relationship between the two dominating membrane lipids, monoglucosyldiglyceride and diglucosyldiglyceride, is to a large extent determined by membrane viscosity properties. This is shown by the varying metabolic responses occurring during incorporation of different fatty acids with and without cholesterol and by temperature shift-down experiments. Higher viscosity in membranes stimulates synthesis of monoglucosyldiglyceride at the expense of diglucosyldiglyceride. Synthesis of phospho and phosphoglucolipids is affected as well. 3. Temperature shift-down from 37 degrees C to 17 degrees C results in an immediate synthesis of monoglucosyldiglyceride accompanied by an increased incorporation of unsaturated fatty acids into this lipid. Synthesis of the other membrane lipid species (containing more unsaturated fatty acids) lags behind temporarily. 4. Incorporation from an equimolar mixture of palmitic and oleic acids together with cholesterol yields greater amounts of oleic acid in membrane lipids than incorporation in the absence of cholesterol, indicating that incorporation is viscosity dependent. 5. Studies of precursor relationships reveal that all main lipids have an active turnover which differs depending on membrane composition and conditions. Furthermore, this turnover proceeds with different intra-lipid pools. 6. Isolated membranes contain no detectable lipolytic enzymes capable of hydrolyzing membrane phospho or glycolipids. It is suggested that lipid turnover is partly mediated by enzymatic interlipid conversions, thus not allowing intermediates to accumulate.     

Membrane composition and virus susceptibility of Acholeplasma laidlawii.

The membrane composition of 11 strains of Acholeplasma laidlawii, including three strains persistently infected with mycoplasmaviruses MVL51, MVL2, and MVL3, was studied and correlated with mycoplasmavirus sensitivity. Membranes of the strains had similiar sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns, and all strains were inhibited by an antiserum produced against membranes from one of the strains. The amounts of integral membrane proteins solubilized by the nonionic detergent Tween 20 differed considerably. Therefore, characteristic crossed immunoelectrophoresis patterns were obtained for each strain. Strains persistently infected with MVL2 and MVL3 were notably different from the noninfected host. The ability to propagate any of the viruses was not correlated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis or crossed immunoelectrophoresis patterns. The persistently infected strains had a characteristic lipid composition. MVL51-resistant strains, including a resistant clone selected from a sensitive strain, were characterized by a large monoglucosyldiglyceride/diglucosyldiglyceride ratio and trace amounts of diphosphatidylglyceol (as opposed to the sensitive strains). Differences in lipid composition in A. laidlawii seem to affect the relationship between cells and viruses.     

Membrane potential, lipid regulation and adenylate energy charge in acyl chain modified Acholeplasma laidlawii

In Acholeplasma laidlawii variations induced in the transmembrane electrical potential have been shown to affect the membrane lipid composition. Particularly the molar ratio between the predominant glucolipids, monoglucosyldiacylglycerol and diglucosyldiacylglycerol, decreases upon hyperpolarization and increases upon depolarization (Clementz et al. (1986) Biochemistry 25, 823-830). Upon variation of the degree of membrane fatty acyl chain unsaturation, known to affect the passive permeability for a number of small molecules, there was no significant correlation between acyl chain composition and the magnitude of the electrical potential. Hyperpolarization by valinomycin decreased the glucolipid ratio for all kinds of membranes, but the size of the decrease was not correlated to the acyl chain composition. However, a clear relationship, independent of acyl chain composition, was found between the extent of hyperpolarization and the size of the decrease in the glucolipid ratio. The adenylate energy charge value (Ec) of the cells was affected by the acyl chain composition, although not exclusively by the proportion of unsaturation. Furthermore, a larger hyperpolarization upon valinomycin addition was accompanied by a stronger reduction in Ec.     

Lipid compositional manipulation in Acholeplasma laidlawii B. Effect of exogenous fatty acids on fatty acid composition and cell growth when endogenous fatty acid production is inhibited

A variety of potential inhibitors of de novo fatty acid biosynthesis have been tested for activity in Acholeplasma laidlawii B. Two compounds, avidin and N,N-dimethyl-4-oxo-2trans-dodecenamide (CM-55), an antimicrobial fatty amide, strongly inhibit de novo biosynthesis without nonspecific toxic effects at moderate dosages. Avidin is the more potent inhibitor, abolishing de novo fatty acid synthesis and greatly reducing the chain elongation of exogenous fatty acids at level of 25 U/l. CM-55 gives complete inhibition of de novo biosynthesis only at low temperatures and inhibits exogenous fatty acid elongation to a variable extent. However, CM-55 is still a more potent antilipogenic agent in this organism than is the fungal antibiotic cerulenin. Cells cultured with avidin grow only when one or more exogenous medium- or long-chain fatty acids are added to the growth medium. The extent of cell growth under these conditions depends primarily on the physical properties of the exogenous fatty acid(s). In general, fatty acids giving diacylglycerolipids of very high or very low fluidity are unsuitable growth substrates, while those whose diacylglycerol derivatives are of intermediate fluidity support fair to good cell growth.     

Osmotic behaviour of Acholeplasma laidlawii B cells with membrane lipids in liquid-crystalline and gel state.

The osmotic behaviour of Acholeplasma laidlawii B cells was investigated with combined spectrophotometric and enzymatic measurements. The conclusion could be drawn that this osmotic behaviour depends largely on the physical state of the membrane lipids. When part of the membrane lipids is in the liquid-crystalline phase the cell is able to swell and behaves as a good osmometer. However, when the membrane lipid is in the gel phase, the cell is unable to swell and the change in absorbance of the cell suspension is then completely due to lysis.     

Lateral organization in Acholeplasma laidlawii lipid bilayer models containing endogenous pyrene probes.

In membranes of the small prokaryote Acholeplasma laidlawii bilayer- and nonbilayer-prone glycolipids are major species, similar to chloroplast membranes. Enzymes of the glucolipid pathway keep certain important packing properties of the bilayer in vivo, visualized especially as a monolayer curvature stress ('spontaneous curvature'). Two key enzymes depend in a cooperative fashion on substantial amounts of the endogenous anionic lipid phosphatidylglycerol (PG) for activity. The lateral organization of five unsaturated A. laidlawii lipids was analyzed in liposome model bilayers with the use of endogenously produced pyrene-lipid probes, and extensive experimental designs. Of all lipids analyzed, PG especially promoted interactions with the precursor diacylglycerol (DAG), as revealed from pyrene excimer ratio (Ie/Im) responses. Significant interactions were also recorded within the major nonbilayer-prone monoglucosylDAG (MGlcDAG) lipids. The anionic precursor phosphatidic acid (PA) was without effects. Hence, a heterogeneous lateral lipid organization was present in these liquid-crystalline bilayers. The MGlcDAG synthase when binding at the PG bilayer interface, decreased acyl chain ordering (increase of membrane free volume) according to a bis-pyrene-lipid probe, but the enzyme did not influence the bulk lateral lipid organization as recorded from DAG or PG probes. It is concluded that the concentration of the substrate DAG by PG is beneficial for the MGlcDAG synthase, but that binding in a proper orientation/conformation seems most important for activity.     

Influence of lipid composition on the orientational order in Acholeplasma laidlawii strain B membranes: a deuterium NMR study.

2H NMR techniques have recently been developed to determine the complete orientational order profile of lipid bilayers employing lipids containing perdeuteriated palmitic acid [Lafleur, M., Fine, B., Sternin, E., Cullis, P.R., & Bloom, M. (1989) Biophys. J. 56, 1037-1041]. In this work, these techniques have been applied to study order profiles in intact membranes derived from Acholeplasma laidlawii strain B. It is shown that complete orientational order profiles can be readily obtained from the intact membranes of A. laidlawii B grown on equimolar amounts of perdeuteriated palmitic acid and a nondeuteriated fatty acid of varying length and unsaturation. By variation of the fatty acid composition employing mixtures of perdeuteriated palmitic acid with myristic, elaidic, oleic, or linoleic acid, a range of hydrocarbon order compatible with high rates and extents of cell growth has been obtained where the average order parameter, mean value of S, varies over the range 0.140-0.176. This same variation in order is seen for liposomes derived from total lipids extracted from these intact membranes. 2H NMR studies on liposomes composed of individual species of the extracted lipids indicate that modulation of the membrane lipid headgroup composition has the potential to play an important role in maintaining the membrane order within this range.     

19F-nuclear magnetic resonance study of glycerolipid fatty acyl chain order in Acholeplasma laidlawii B membranes

The technique of 19F-nuclear magnetic resonance (19F-NMR) spectroscopy offers a number of advantages for studies of lipid fatty acyl chain orientation and dynamics in biomembranes. However, the geminal difluoromethylene fatty acid probes usually employed in such studies appreciably perturb the organization of lipid bilayers. We have thus synthesized a series of specifically monofluorinated palmitic acids and carried out biophysical, biochemical, and physiological studies establishing their suitability as relatively non-perturbing probes of lipid hydrocarbon chain organization. These 19F-NMR probes were then used to determine the fatty acyl chain order profiles of Acholeplasma laidlawii B membranes highly enriched in a variety of different exogenous fatty acids, particularly those containing a methyl branch or a trans-double bond.     

Phospholipid scramblase-3 regulates cardiolipin de novo biosynthesis and its resynthesis in growing HeLa cells

PLS3 (phospholipid scramblase-3) is a new member of the family of phospholipid scramblases and transports CL (cardiolipin) from the inner to the outer mitochondrial membrane. In the present paper we examined whether changing the levels of functional PLS3 in HeLa cells altered de novo CL biosynthesis and its resynthesis. HeLa cells overexpressing PLS3 or expressing a disrupted PLS3 (F258V) or control were incubated with [1,3-3H]glycerol and radioactivity incorporated into CL was determined. CL biosynthesis from [1,3-3H]glycerol was increased 1.8-fold in PLS3 cells and 2.1-fold in F258V cells compared with control. This was due to a 64% (P<0.05) and 2.6-fold (P<0.05) elevation in CL synthase activity in PLS3 and F258V cells respectively, compared with control, and not due to changes in phosphatidylglycerolphosphate synthase activity. The increase in CL synthase activity in these cells was due to an increase in its mRNA expression. In contrast, resynthesis of CL from [1-14C]linoleic acid was reduced 52% (P<0.05) in PLS3 and 45% (P<0.05) in F258V cells compared with control and this was due to a reduction in mitochondrial monolysocardiolipin acyltransferase activity. Although protein levels of mitochondrial monolysocardiolipin acyltransferase were unaltered, activity and mRNA expression of endoplasmic reticulum monolysocardiolipin acyltransferase was upregulated in PLS3 and F258V cells compared with controls. These data indicate that the CL resynthesis in HeLa cells is sensitive to the mitochondrial localization of CL and not the level of the reacylating enzymes. Alterations in functional PLS3 levels in PLS3 or F258V cells did not affect the mitochondrial decarboxylation of phosphatidylserine to phosphatidylethanolamine indicating that the biosynthetic changes to CL were specific for this mitochondrial phospholipid. We hypothesize that the cardiolipin resynthesis machinery in the cell 'senses' altered levels of CL on mitochondrial membranes and that de novo CL biosynthesis is up-regulated in HeLa cells as a compensatory mechanism in response to altered movement of mitochondrial CL. The results identify PLS3 as a novel regulator of CL de novo biosynthesis and its resynthesis.