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J Biol Chem, Vol. 275, Issue 13, 9296-9302, March 31, 2000

The Nonbilayer/Bilayer Lipid Balance in Membranes
REGULATORY ENZYME IN ACHOLEPLASMA LAIDLAWII IS STIMULATED BY METABOLIC PHOSPHATES, ACTIVATOR PHOSPHOLIPIDS, AND DOUBLE-STRANDED DNA^*

Susanne Vikström, Lu Li, and Åke Wieslander

From the Department of Biochemistry, Umeå University, 901 87 Umeå, Sweden

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In membranes of Acholeplasma laidlawii a single glucosyltransferase step between the major, nonbilayer-prone monoglucosyl-diacylglycerol (MGlcDAG) and the bilayer-forming diglucosyl-diacylglycerol (DGlcDAG) is important for maintenance of lipid phase equilibria and curvature packing stress. This DGlcDAG synthase is activated in a cooperative fashion by phosphatidylglycerol (PG), but in vivo PG amounts are not enough for efficient DGlcDAG synthesis. In vitro, phospholipids with an sn-glycero-3-phosphate backbone, and no positive head group charge, functioned as activators. Different metabolic, soluble phosphates could supplement PG for activation, depending on type, amount, and valency. Especially efficient were the glycolytic intermediates fructose 1,6-bisphosphate and ATP, active at cellular concentrations on the DGlcDAG but not on the preceding MGlcDAG synthase. Potencies of different phosphatidylinositol (foreign lipid) derivatives differed with numbers and positions of their phosphate moieties. A selective stimulation of the DGlcDAG, but not the MGlcDAG synthase, by minor amounts of double-stranded DNA was additive to the best phospholipid activators. These results support two types of activator sites on the enzyme: (i) lipid-phosphate ones close to the membrane interphase, and (ii) soluble (or particulate)-phosphate ones further out from the surface. Thereby, the nonbilayer (MGlcDAG) to bilayer (DGlcDAG) lipid balance may be integrated with the metabolic status of the cell and potentially also to membrane and cell division.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The "melted," i.e. liquid-crystalline (L), state of the lipids in biological membranes is essential for many membrane-associated processes. The types of polar lipid species are also important. In the small cell wall-less prokaryote Acholeplasma laidlawii an extensive metabolic regulation in the amounts of the major phospholipids and glucolipids occurs under a variety of conditions (e.g. see Refs. 1 and 2). This serves to maintain (i) a certain surface charge density, (ii) phase equilibria close to a potential bilayer to nonbilayer transition, and (iii) a nearly constant spontaneous curvature for the lipid bilayer. However, A. laidlawii cannot efficiently regulate the melting temperature of the lipids (3). The properties (i-iii) are believed to be important for the functional characteristics of many membrane proteins and a readiness for local structural transformations in/of the membrane (4-7). Similar principles also apply to membrane lipid composition in Escherichia coli (8, 9) and probably many bacteria. Starting from the common precursor phosphatidic acid (PA),¹ the pathways of the syntheses in vivo of the major A. laidlawii lipids (in bold) are tentatively as shown in Scheme 1. 

The two major glucolipids, the nonbilayer-prone monoglucosyldiacylglycerol (MGlcDAG) and the bilayer-forming diglucosyldiacylglycerol (DGlcDAG), are key lipids for regulation of the packing properties (ii and iii above) under many conditions. This is mainly accomplished by a metabolic altering in the relative amounts of the two lipids. The two membrane-bound glucolipid-synthesizing enzymes (glucosyltransferases) must sense bilayer packing properties directly or get specific communicating signals, which activate the genes encoding these enzyme, or modify activities of the enzymes. The MGlcDAG synthase is dependent upon a lipid environment where a critical fraction of anionic amphiphiles must be present in substantial amounts (10, 11). A conformational change and activation of the enzyme is mediated by these anionic lipids (12), and it seems to be the main site for the lipid surface charge regulation, balancing the phospholipid and glucolipid pathways. PG is particularly important because the consecutively acting DGlcDAG synthase is only active, in a cooperative fashion, in the presence of substantial amounts of an activator lipid, especially PG. The nonbilayer/bilayer lipid balance and curvature are more likely regulated by the synthesis of DGlcDAG because this step is strongly stimulated by certain nonbilayer-prone additives in vitro (13, 14) but also by "patching" of the activator PG (15).

A potential coupling between the bilayer packing properties brought by the MGlcDAG/DGlcDAG balance, and cell metabolism, is anticipated from a correlation between the glucolipid ratio and cellular energetics, i.e. the transmembrane electrical potential, the cellular K⁺ and ATP content, and glucose consumption, respectively (16, 17). Nucleotide phosphates also affect the two glucosyltransferases differently in vitro (18), and certain soluble phosphates stimulated the purified DGlcDAG synthase (14). Important phosphate molecules in Mycoplasmas are, in addition to RNA, DNA, polyphosphates, and phospholipids (19), also fructose 1,6-bisphosphate (FBP) and ATP (20). Amounts of the latter two are also strongly correlated to the extent of glucose consumption (glycolysis) in related Gram-positive bacteria (21, 22). Furthermore, FBP is an important gene regulator for metabolic responses in such bacteria (23) and an activator for the lactate dehydrogenase in A. laidlawii (24).

We report here for the purified DGlcDAG synthase that there is strong support for a coupling between bilayer packing properties and the general metabolic status of the cell. Several important phosphate-containing metabolites stimulated the DGlcDAG synthase activity substantially and additive to PG. The preceding MGlcDAG synthase was indifferent to these phosphate compounds.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Strain and Growth Conditions-- For enzyme purification A. laidlawii strain A-EF22 was cultivated in an oleic acid (18:1c)-supplemented medium (13). For variation in lipid acyl chain length (C_n) and unsaturation (% UFA), A. laidlawii was grown with one saturated (8, 12, 14, 16, 18, 20, or 22 carbons chain length) plus one monounsaturated fatty acid (14, 16, 18, 20, or 22 carbons chain length), with five or six combinations within each pair and a total concentration of 150 µM (25). Incorporation into the membrane lipids was monitored by added ¹⁴C- and ³H-labeled fatty acids. Extraction, separation by TLC, and quantitation of the lipids by liquid scintillation counting and gas-liquid chromatography was performed as described (25).

Lipids and Other Material-- MGlcDAG was prepared from A. laidlawii grown in the presence of oleic acid, which gives polar lipids with more than 90% (mol/mol) 18:1c acyl chains (13). Synthetic rac-1,2-dioleoylglycerol (1,2-DOG) and 1,3-dioleoylglycerol (1,3-DOG) were purchased from Larodan (Sweden). 1,2-Dioleoylphosphatidic acid (DOPA), 1,2-dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoylphosphatidylserine (DOPS), 1,2-dioleoyl-phosphatidylcholine (DOPC), 1,2-dioleoyl-phosphatidylinositol (DOPI), and bovine heart diphosphatidylglycerol (DPG) were purchased from Avanti Polar Lipids. Immobilized proteinase K was purchased from Merck. Fructose 1,6-bisphosphate, phosphoenolpyruvate, ADP, ATP, GTP, CTP, UDP, UTP, and tetrasodium pyrophosphate were from Roche Molecular Biochemicals. RNA, phosphatidylinositol 4-monophosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP₂), sn-glycero-3-phosphate (G-3-P), and sn-glycero-1-phosphate (G-1-P; racemic, also containing some G-3-P) were purchased from Sigma. Sodium-meta-vanadate was purchased from Riedel-de Haën (Germany). Sodium molybdate was purchased from KEBO (Sweden). DNA was prepared from A. laidlawii using the Rapid Prep^TM Kit (Amersham Pharmacia Biotech). Control template  DNA was purchased from Perkin-Elmer (PCR Reagent Kit).

Purification of Glucosyltransferases-- DGlcDAG synthase was purified from detergent-solubilized A. laidlawii cells by ion exchange, hydroxyapatite, and dye ligand affinity chromatography according to Ref. 14. Homogenous and detergent-free MGlcDAG synthase was purified according to (12).

Assay for Glucosyltransferase Activity-- Assays in mixed micelles were technically performed as described before (11, 14). The CHAPS assay buffer contained 18.8 mM CHAPS, 100 mM Tris maleate, pH 8, and 5-20 mM MgCl₂. Standard lipid concentration used was 11.2 mM (0.3 mM substrate, 7.5 mM (25 mol %) lipid activator, and 3.4 mM matrix lipids (DOPC, if not otherwise stated)). The procedure was modified, however; the assay time used was 30 min, and 4 µl of enzyme solution (0.08 µg of protein) was added to 86 µl of micellar solution. Reactions were started by addition of 10 µl of UDP-[¹⁴C]glucose to give a concentration of 1 mM in a final volume of 100 µl of assay buffer. For assays with PI, PIP, and PIP₂ the assay volume was reduced to 50 µl, and the lipid composition was 6 mM DOPG (20 mol %) and 4.9 mM matrix lipids. After 30 min at 28 °C, reactions were stopped with 375 µl of methanol/chloroform, 2:1 (v/v), and the lipids, including newly synthesized MGlcDAG or DGlcDAG, were extracted and separated by TLC as described (10) and then quantified using electronic autoradiography (Packard Instant Imager).

Limited Proteolytic Digestion of DGlcDAG Synthase-- DGlcDAG synthase (2.3 µg, 15 µl) was incubated with lipid mixed micelles at a final concentration of 10 mM lipids, 110 mM Tris maleate, 20 mM MgCl₂, 20 mM CHAPS, pH 8, for 30 min on ice. In the samples with 100 or 800 mM phosphate, no magnesium was present. Immobilized proteinase K (0.01 unit) was then added to the mixed micelles and gently shaken at 28 °C for 20 min. Proteolysis was terminated by removing the immobilized proteinase using centrifugation at 2,000 × g. Supernatants were analyzed by SDS-polyacrylamide gel electrophoresis, using 15% acrylamide in the resolving gel (26), and proteins were visualized by silver staining.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

DGlcDAG and PG Amounts in Vivo and in Vitro-- The presence of substantial amounts of activator PG for DGlcDAG synthesis is crucial in vitro, both with crude cellular proteins and the purified enzyme (13, 14). In vivo there is a correlation between these two lipids; Fig. 1A shows the PG and DGlcDAG amounts in many different A. laidlawii cultures, and Fig. 1B shows the DGlcDAG synthesis in vitro. The lower PG amounts in vivo (Fig. 1A) should not be sufficient for achieving a strong DGlcDAG synthesis in vitro, according to Fig. 1B. Furthermore, the substantial stimulation at high PG concentration caused by nonbilayer-prone molecules, like 1,3-DOG (Fig. 1B), is much less pronounced at lower PG amounts (i.e. 10-15 mol %) (13).

View larger version (24K): [in this window] [in a new window]   Fig. 1.   DGlcDAG and activator PG in vivo and in vitro. A, correlation between PG and DGlcDAG amounts in A. laidlawii membranes. Cells were grown with a variety of fatty acids (see "Experimental Procedures"), yielding large differences in lipid chain length (Cn) and monounsaturation (% UFA). Open circles, average lipid amounts of PG and DGlcDAG with Cn (1) <15, (2) 15-16, (3) 16-17, (4) 17-18, (5) 18-19, and (6) >19. Filled circles, average amounts for the two lipids with % UFA (A) <20, (B) 20-40, (C) 40-60, (D) 60-80, and (E) >80 mol %. Regression lines are shown, and correlation coefficients were 0.95 and 0.97, respectively. Lipids decreasing concomitantly are MGlcDAG and the phosphoglucolipids. Part of the data (PG) is from Ref. 25. B, DGlcDAG enzymatic synthesis in vitro with DOPG as activator in (open circles) mixed micelles (14), and (filled circles) liposome models (13). Dashed arrows, stimulation by addition of nonbilayer-prone 1,3-DOG (11.5 and 15 mol %, open and filled squares). Values are normalized relative to the activity rate (V, nmol/h) with 25 mol % PG present.

These data indicate that the amounts of the "essential" activator PG may not be sufficient to achieve enough DGlcDAG synthesis at in vivo conditions. Therefore, several cellular phosphate-containing molecules, where the phosphate moieties are located in similar carbon molecular environments (see Fig. 2), were analyzed for their ability to support activation of the DGlcDAG synthase by PG.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Schematic structures of phosphate-containing metabolites used. A, PA is the structural base for the phospholipids. The backbone of PA (and the other phospholipids) is sn-glycero-3-phosphate (G-3-P), whereas G-1-P is the polar head group of PG. B, Pi; C, PEP; D, FBP; E, ATP; F, DNA (RNA). A circled P corresponds to a phosphate moiety.

Activation of DGlcDAG Synthase by Phosphate and Mg²⁺ Ions-- The DGlcDAG synthase can bind to a calcium phosphate matrix and subsequently be released by 300-400 mM phosphate (14). Many glucosyltransferases have a requirement for Mg²⁺ as a cofactor (27). The influence of phosphates and magnesium ions on the DGlcDAG synthase was analyzed at an activator PG concentration yielding a fairly low enzyme activity (25 mol %, cf. Fig. 1B). For the DGlcDAG synthase the dependence of Mg²⁺ peaked at ~10 mM Mg²⁺ (Fig. 3A); some Mg²⁺ may still be bound to the enzyme after the purification procedure. The inhibition by Mg²⁺ at higher concentrations might be due to interaction with the soluble substrate UDP-Glc (1 mM). Inorganic phosphate stimulated the enzyme activity 5-fold at 20 mM with only 5 mM Mg²⁺ present (Fig. 3A). sn-Glycero-1-phosphate (G-1-P; also containing some G-3-P) had a stimulating effect on the DGlcDAG synthesis activity above 15 mM but lower than for the free phosphate (Fig. 3A). The activity maximum appeared at 40 mM; far above a physiological concentration. On the other hand, G-3-P strongly inhibited the enzyme (Fig. 3A). G-3-P is the backbone of PG (and the other phospholipids used, Fig. 2), and G-1-P is identical to the PG head group. With no Mg²⁺ added, inorganic phosphate increased the enzyme activity about 16 times at 20 mM phosphate (Fig. 3B); with Mg²⁺ present the DGlcDAG maximum activity decreased with increasing concentrations of Mg²⁺ (Fig. 3B).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effects of phosphate and Mg2+ ions on the DGlcDAG synthase activity. The DGlcDAG activity (V, nmol/h) was measured in CHAPS mixed micelles (25 mol % DOPG activator) as a function of potassium phosphate, Mg2+, sn-glycero-1- or -3-phosphate (A); phosphate plus Mg2+ (B). Curves are normalized relative to their common point where no ions were added.

Hence, these experiments show that, at corresponding concentrations and low PG activator, certain soluble phosphates stimulated DGlcDAG synthase activity substantially and more than Mg²⁺. Most important, the soluble G-3-P analog of the PG backbone inhibited the enzyme strongly.

Important Cell Metabolites Influence the Nonbilayer/Bilayer Lipid Balance-- The substantial stimulation of DGlcDAG synthesis with small soluble phosphates (above) prompted a screening of other important phosphorylated cell metabolites. For this, the Mg²⁺ and DOPG concentrations were maintained at a low but physiological and responsive levels. Of ADP, ATP, CTP, GTP, UDP, and UTP at physiological concentrations (0.5 mM), especially ATP stimulated the DGlcDAG synthase, whereas the MGlcDAG synthase was more indifferent (data not shown). Several key phosphorylated intermediates in the glycolysis were also tested. Fructose 1,6-bisphosphate (FBP) increased the DGlcDAG synthase activity, with a sharp rise up to 1 mM FBP. The activity maximum corresponded to an 18-fold stimulation (Fig. 4); concentrations of FBP higher than ~1 mM yielded lower activity but a stimulation still persisted at 2 mM FBP (Fig. 4). In contrast, activity of the preceding MGlcDAG synthase remained the same in the presence of increasing FBP (Fig. 4). Several enzymatic reactions in A. laidlawii, including the synthesis of FBP from fructose 6-phosphate is dependent on pyrophosphate (PP_i) (28). The DGlcDAG synthesis increased ~5-fold when 0.5 mM PP_i was present; for the preceding MGlcDAG synthesis no effect (or a slight decrease) was observed (data not shown). The glycolytic intermediate phosphoenolpyruvate (PEP), which often has a regulatory role in cell metabolism, was also analyzed at physiological concentrations. The DGlcDAG activity increased up to 0.5 mM PEP and an enhancement of the enzyme activity 4× was observed (Fig. 4). However, for the MGlcDAG synthase PEP had essentially no influence on the activity (Fig. 4).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   The influence of important cellular metabolites on the DGlcDAG and MGlcDAG syntheses. The MGlcDAG (open symbols) and DGlcDAG synthases activity (filled symbols) were measured in CHAPS mixed micelles (25 mol DOPG % activator, 5 mM Mg2+). FBP, PEP, and ATP concentrations were increased stepwise. Curves are normalized relative to the common point where no metabolites were added. The single curve shown for MGlcDAG synthesis is valid for the three different additives.

A. laidlawii obtains its ATP demand from substrate level phosphorylation only (28). For the DGlcDAG synthase, enzyme activity increased up to an ATP concentration of 0.75 mM, yielding a 12-fold stimulation (Fig. 4). Concentrations higher than 1.5 mM inhibited the enzyme. The MGlcDAG synthase activity was not altered by the ATP concentration range examined. The latter coincides with the range of ATP concentration recorded in A. laidlawii cells (17, 29), related bacteria (21, 30), and slow growing E. coli (31). The MGlcDAG synthase is strongly dependent on Mg²⁺ ions, but no inhibition was observed at raised ATP concentrations. Hence, the inhibition of the DGlcDAG synthase at the higher ATP concentrations is probably not a chelating effect of ATP on the Mg²⁺ ions. At 10 mM ATP with 20 mM Mg²⁺ present, both enzymes were totally inhibited. However, two established competitive molecules of phosphate in protein-binding sites, vanadate and molybdate (see "Experimental Procedures"), both stimulated the DGlcDAG synthase at low concentrations. This was especially the case for vanadate (more than 2× stimulation at 0.5 mM; data not shown), supporting the role of phosphate-binding sites on the DGlcDAG synthase (above).

The cellular phosphorylated compounds analyzed contain different numbers of phosphate moieties (Fig. 2) and stimulated the activity of the DGlcDAG but not the MGlcDAG synthase. Comparing ATP (3 phosphates) with FBP and PP_i (2 phosphates), it seems that three or two phosphates in a row was not as effective as the two at the opposite ends in FBP (cf. Fig. 2). This implies that a simultaneous interaction with several phosphate-binding sites is more effective.

Numbers and Positions of Lipid Head Group Phosphates Are Crucial-- The potential importance of activator phosphate numbers and localization was monitored using different phosphate-substituted phosphatidylinositol (PI) lipids (Fig. 2). Inositol phosphates are important intracellular second messengers in eukaryotic cells; however, they don't exist in A. laidlawii. The endogenous DOPG was used as the activator, at a deliberately low (20 mol %) activating level (cf. Fig. 1B).

The DGlcDAG synthase activity increased with the concentration of PI derivatives, i.e. PI efficiently supplemented PG for an enhanced activation (Fig. 5). The numbers and positions of the phosphate moieties on the inositol ring were also important (Fig. 5). A stronger increase in DGlcDAG synthesis was obtained with very low amounts of PIP₂; much higher amounts of PI were required to achieve a similar activity (Fig. 5). PIP showed no evident stimulation of the enzyme activity at similarly low fractions, indicating that the phosphate on carbon 4 in the inositol ring of PIP (cf. Fig. 2) may not be able to reach the presumed phosphate-binding pocket on the DGlcDAG synthase.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Activator lipid phosphate positions are important for the DGlcDAG synthase. Enzyme activities were monitored in CHAPS mixed-micelles containing low amounts (20 mol %) of activator DOPG and different phosphatidylinositol derivatives, i.e. PI, PIP, and PIP2 (balance DOPC). Curves are normalized relative to the common point where no PI derivatives were added. The single curve for MGlcDAG synthesis is valid for the three different additives. PIP and PIP2 could only be assayed up to 2 mol % due to precipitation by Mg2+ at higher concentrations.

These findings strongly support a cooperative mechanism for activation of the DGlcDAG synthase by lipid phosphates, where accessibility is important. The MGlcDAG synthase activity was not influenced by the small concentrations of the potent PI derivatives.

Activator Phosphates Induce a Conformational Change of the DGlcDAG Synthase-- Activator phosphates may induce a close approach of the membrane-bound enzyme (or the active site) to the bilayer surface and/or a conformational change. This was assessed by sensitivity to proteolytic degradation. The resistance of the enzyme against proteinase K digestion in the different mixed micelles was visualized by SDS-polyacrylamide gel electrophoresis (Fig. 6). With CHAPS detergent only, and no lipids present, the DGlcDAG synthase could be totally digested. However, the enzyme revealed a high protease resistance in DOPG- or phosphate-containing micelles. Some enzyme activity could be obtained in micelles containing DOPS (14), but still the enzyme was not protected against proteolytic cleavage in DOPS (Fig. 6). Denaturated DGlcDAG synthase could not be protected from proteolytic digestion even when the most protective lipid conditions were used (data not shown). The presence of Mg²⁺ had no influence; the enzyme was protected in PG micelles with or without Mg²⁺ ions. However, it was found that by extended incubation times the enzyme could be digested also at the most protective conditions. Hence, the enzyme is not totally protected from digestion by being active.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   The proteolytic resistance of DGlcDAG synthase in different lipid mixed micelles. Silver-stained SDS gel with the DGlcDAG synthase after digestion by proteinase K in different lipid/CHAPS mixed micelles for 20 min at 28 °C. Small digested fragments were rarely seen. Sample composition is as follows: enzyme (2.3 µg), 10 mM (33 mol %) lipid, 20 mM CHAPS, and 20 mM MgCl2 (no MgCl2 was added with abundant phosphate). Lanes 1 and 2, enzyme in CHAPS only; lane 3, in DOPG/CHAPS; lane 4, in DOPS/CHAPS; lane 5, in DOPG/CHAPS without Mg2+; lane 6, in 100 mM phosphate/CHAPS; and lane 7, in 800 mM phosphate/CHAPS. Lane 1 without and lanes 2-7 with proteinase K. A copurified 30-kDa protein also present (14) was degraded at all conditions (data not shown).

Soluble phosphate stimulated the activity 16-fold (Fig. 3). Could phosphate protect the enzyme by itself, without PG? Since the surface concentration of the PG activator is very high, it was necessary to raise the soluble phosphate concentration substantially. The DGlcDAG synthase was indeed protected from digestion at 800 mM (but not by 100 mM) phosphate and no PG present (Fig. 6); hence, the phosphate need not be attached to a lipid.

These results show that there is a correspondence between high activity, induced by phosphates, and proteolytic resistance for the DGlcDAG synthase. Furthermore, both lipid-associated and free (soluble) phosphate could induce this conformational change.

dsDNA Stimulates the DGlcDAG Synthase Activity-- Another major source of phosphates in a cell is the nucleic acids. The structure of nucleic acids influences how their phosphate moieties are exposed. Double-stranded (ds) DNA from A. laidlawii and phage lambda, a single-stranded (ss) DNA (oligo primer), and ssRNA were analyzed. The two dsDNAs increased the activity of DGlcDAG synthase 7-fold when 0.7 µg of DNA/ml was added (20 µg enzyme/ml), corresponding to 2 µM DNA phosphates (Fig. 7). This amount of DNA phosphate is very low compared with the amount of soluble phosphate needed for a similar stimulation (Fig. 3A) or the concentration of nucleic acids in E. coli, for example, at least 0.35 M expressed as DNA phosphates (75-120 mg of DNA/ml) (32). It is also much less than the amount of DOPG activator in the samples, i.e. 7.5 mM, or the potent PI derivatives used (up to 0.6 mM; Fig. 5). Neither the ssDNA nor RNA yielded any significant influence on DGlcDAG synthase activity, and neither of the nucleic acids affected the MGlcDAG synthase (Fig. 7).

View larger version (36K): [in this window] [in a new window]   Fig. 7.   DNA stimulates glucosyltransferase activity. The MGlcDAG synthase activity (open bars) and DGlcDAG synthase activity (filled bars) were measured in mixed micelles (25 mol % DOPG). Different concentrations dsDNA were added; µM DNA phosphates was obtained from the average base composition of A. laidlawii DNA (32 mol % G + C). Bars are normalized relative to their common point where no DNA was added. Stimulation of the DGlcDAG synthase persisted up to at least 20 µM DNA phosphate.

Stimulation by dsDNA Is Additive to Phospholipid Activation-- The phospholipids PA, PG, and diphosphatidylglycerol (DPG) have different glycerophosphate constituents (Fig. 2), analogous to the soluble phosphates analyzed in Fig. 3. Both DOPA and DPG were substantially better activators for the purified DGlcDAG synthase than DOPG, see Fig. 8. However, their potencies declined at higher concentrations, perhaps due to their stronger interaction with Mg²⁺ ions compared with PG. Addition of small amounts of dsDNA to DPG (and PG) substantially increased the extent of activation for the DGlcDAG synthase, also shifting the activation curve toward higher efficiency at lower activator amounts.

View larger version (21K): [in this window] [in a new window]   Fig. 8.   DNA potentiates activator phospholipid. The influence of dsDNA on the DGlcDAG activity was analyzed as in Fig. 7, with different amounts of DOPA, DPG, and DOPG as activator phospholipids (balance DOPC). Filled boxes and circle, 2 µM DNA phosphates added to the DPG and PG samples, respectively.

Hence, the double-stranded DNA probably interacts with the DGlcDAG enzyme, presumably by its structurally organized phosphates, and consequently strongly increases the activity. The potentiation of activation, even with the best phospholipid activators, supports the presence of several phosphate-binding sites on the enzyme. The small amounts of DNA involved rule out a general change of the micellar properties.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Recent data indicated that it is not the actual "curvature stress" (spontaneous curvature) that sets the DGlcDAG synthase activity but instead access of activators (like PG) or exposure of phosphate-binding sites by the curvature that are important (14). The amounts of activator PG maintained in A. laidlawii in vivo are substantially less than amounts needed to yield average or high synthesis rates of DGlcDAG in vitro (Fig. 1, A and B), and other factors must therefore also be involved.

Activator Phospholipids-- From the data reported here it is evident that PA, DPG, and PG can all work as efficient activator lipids, where the two former were more potent (Fig. 8). PA is a precursor present at minor and not sufficient amounts in vivo, and DPG is not normally present in the A-EF22 strain used but occurs in several other strains. Indeed, more DPG (versus PG) correlates with more DGlcDAG (versus MGlcDAG) in the latter strains (33). The efficiencies of PA and DPG (but not PG) are probably enhanced by the Mg²⁺ present (Fig. 8). DOPA and E. coli DPG both become more nonbilayer-prone upon interaction with divalent cations like Mg²⁺, decreasing head group repulsion (34-36); an enhanced curvature stress in the mixed micelles (Fig. 8) is thus likely.

The G-3-P backbone of phospholipids have a perpendicular orientation at the bilayer interface, followed by a tilted head group further out (37, 38). Potent activator lipids of the DGlcDAG synthase all have this part; positively charged moieties covalently linked outside the phosphate (like in PC) prevent activation, whereas a zwitterion (like in PS) works, and no or negative charges (like in PG, DPG, and PI derivatives) (cf. Fig. 2) are very efficient (above and see Refs. 10, 11, 13, and 14). The strong inhibition of the DGlcDAG synthase by soluble G-3-P (Fig. 3) is most likely due to a competition with the lipid activator phosphate in sites close to the interphase.

A heterogeneous distribution of lipid activator may yield higher local activator concentrations and consequently higher enzyme activities. Domain formation by PG with a concomitant stimulation of the DGlcDAG synthase can be induced by a chain length mismatch in vitro (15) and may occur in vivo (39, 40). Involvement of several PG molecules for the activation event is supported by a close fit to (cooperative) Hill kinetics, where Hill coefficients ranged between 3 and 7, and PG amounts needed were dependent on the bilayer environment (10). The bilayer surface association of protein kinase C is similarly dependent upon PS, with slightly larger Hill coefficients for binding of 8 PS molecules (41).

Activation by Phosphorylated Metabolites and DNA-- Small single phosphate-containing molecules like (soluble) inorganic or G-1-P enhanced the activation potency of PG (Fig. 3A). An interference by Mg²⁺ ions was also obvious (Fig. 3B). Note that the enzyme is associated with the micelles by hydrophobic interactions (14). A very large, soluble phosphate concentration (800 mM) could replace the demand for PG (7.5 mM/25 mol %) in inducing a conformational change (Fig. 6). Mycoplasmas may contain 40 mM orthophosphate, which decrease upon glycolysis (19, 20). Mg²⁺ binds strongly to oligo- and polyphosphates (42) and adsorbs to and lowers the bilayer surface potential of phospholipids like PG (6, 43). The intracellular concentration of Mg²⁺ is 100 mM in E. coli (44), and A. laidlawii membranes (lipids) bind 25% that in the E. coli cell envelope (114 µmol/g) (44, 45). Hence, Mg²⁺ is present and important for the DGlcDAG synthase (Fig. 3).

The di- or triphosphate-containing metabolites FBP, PP_i, and ATP were strong potentiators of PG activation in the low mM (physiological) range (Fig. 4). The lower amounts needed strongly indicates an enhanced binding to the DGlcDAG synthase. Both ATP and FBP are important energy and signaling molecules in the Gram-positive bacteria including Mycoplasmas and are correlated to the cellular growth state. The very strong effects especially by FBP (Fig. 4), similar to that of PIP₂ (Fig. 5), indicate that a certain spacing between the phosphate moieties, like in FBP but not in ATP (Fig. 2), is more optimal for binding or reach more sites on the enzyme. Since these metabolites could achieve higher activities of the DGlcDAG synthase than for PG alone, the enzyme most likely contain activator sites not reachable by PG (but probably by PIP₂). The latter sites are obviously also not as specific as the G-3-P-lipid sites at the interphase (proposed above).

The multiphosphate dsDNA had an activating potency on the DGlcDAG synthase at very low (µM) concentrations, which was additive to the best phospholipid activators (Fig. 8). The distances between phosphates in dsDNA backbone (Fig. 2) and across the minor groove, but not across the substantially larger major groove, are fairly similar to the phosphate distances in PIP₂, for example. Hence, with the dsDNA (but not ssDNA or RNA) an ordered oligophosphate template is probably exposed toward the enzyme. An average aggregate size of 100 molecules, for example, in the lipid-CHAPS micelles (14) gives a micelle concentration of 0.3 mM (enzyme 15 nM). 2 µM DNA phosphate A. laidlawii dsDNA corresponds to a few pM DNA; broken into many fragments upon purification a few nM DNA is reached, not enough for one piece of DNA per enzyme. However, one DNA fragment may engage several enzymes. The diameter of a dsDNA helix (2 nm) may be too large to be accommodated between the membrane-associated DGlcDAG synthase (40 kDa) and the bilayer surface.

Hence, the stimulation brought by the DNA supports two types of binding sites on the enzyme as follows: (i) lipid-phosphate ones close to the membrane surface, and (ii) soluble or particulate phosphate sites further out on the side of the enzyme, where the latter are accessible by metabolites and DNA. Various phosphate-containing molecules obviously can act in a synergistic manner. The lack of response by the preceding MGlcDAG synthase toward the metabolites and DNA opens up for a multifaced control of the nonbilayer (MGlcDAG)/bilayer (DGlcDAG) lipid balance.

Functional Consequences for the Cell-- The phosphorylated molecules investigated are all connected with the extent of cell growth, except the phospholipids. Hence, metabolic concentrations of these may act as signals upon the DGlcDAG synthase, causing the enzyme to make more DGlcDAG (bilayer-forming) and consequently depleting the MGlcDAG pool (nonbilayer-prone), cf. Fig. 9. It was recently found in E. coli that the major membrane nonbilayer phospholipid, phosphatidylethanolamine (PE), plays an essential role at some stage in the cell division process (46), and PE-deficient mutants failed to divide properly. Mycoplasmas seem to lack the later components of this Fts cell division assembly (47). Accumulation of nonbilayer lipids in the membrane of A. laidlawii may trigger, or be beneficial to, membrane and cell division. Expansion of the membrane, needed for cell growth, would consequently be stimulated by the phosphorylated metabolites, PG, and the (DNA) chromosome, acting in a cooperative fashion.

View larger version (38K): [in this window] [in a new window]   Fig. 9.   Regulatory mechanisms for the DGlcDAG synthase. Speculative sketch indicating the activating and stimulatory molecules, which act in a cooperative fashion. Molecular sizes are presented roughly in scale. The balance between MGlcDAG and DGlcDAG may be coupled to membrane (cell) division and growth.

	ACKNOWLEDGEMENTS

We thank Drs. Dennis Pollack, University of Ohio, Columbus, and Kevin Dybvig, University of Alabama at Birmingham, for valuable comments on the manuscript.

	FOOTNOTES

^* This work was supported by the Swedish Natural Science Research Council and the K. and A. Wallenberg Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry, Umeå University, 901 87 Umeå, Sweden. Fax: +46-90-786-7661; E-mail: ake@biokemi.su.se or susanne.vikstrom@chem.umu.se.

	ABBREVIATIONS

The abbreviations used are: PA, phosphatidic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; 1, 2-DAG, 1,2-diacylglycerol; DGlcDAG, 1,2-diacyl-3-O-[-D-glucopyranosyl-(12)-O--D-glucopyranosyl]-sn-glycerol; DO, dioleoyl; 1, 3-DOG, 1,3-dioleoylglycerol; DPG, diphosphatidylglycerol; FBP, fructose 1,6-bisphosphate; Glc, glucose; G-1-P, sn-glycero-1-phosphate; G-3-P, sn-glycero-3-phosphate; MGlcDAG, 1,2-diacyl-3-O-(-D-glucopyranosyl)-sn-glycerol; P_i, inorganic phosphate; PP_i, pyrophosphate; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PEP, phosphoenolpyruvate; PG, phosphatidylglycerol; PS, phosphatidylserine; PI, phosphatidylinositol; PIP, phosphatidylinositol-4-phosphate; PIP₂, phosphatidylinositol-4,5-bisphosphate; dsDNA, double-stranded DNA; UFA, unsaturated fatty acid; DOPA, 1,2-dioleoylphosphatidic acid; DOPG, 1,2-dioleoylphosphatidylglycerol; DOPS, 1,2-dioleoylphosphatidylserine; DOPC, 1,2-dioleoyl-phosphatidylcholine; DOPI, 1,2-dioleoyl-phosphatidylinositol; ss, single-stranded.

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