Coordinate regulation of unsaturated phospholipid, RNA, and protein synthesis in Mycoplasma capricolum by cholesterol.

Abstract

The effect of cholesterol, epicoprostanol, and phosphatidylcholine on phospholipid, RNA, and protein synthesis was investigated in the sterol auxotroph Mycoplasma capricolum. Cells growing poorly on lanosterol were stimulated to grow more rapidly by supplementing the medium with either 2 jig of cholesterol or 2.2 gg of egg phosphatidylcholine per ml, In such cells cholesterol caused a sequential stimulation of phospholipid, RNA, and protein synthesis. Enhanced oleate incorporation into phospholipid occurred early; the rates of RNA and protein synthesis increased later. In cells supplemented with phosphatidylcholine only RNA and protein syntheses were enhanced. The addition of 2 pzg of epicoprostanol per ml to cells growing on lanosterol promptly inhibited the rate ofunsaturated phospholipid synthesis and subsequently the rate of growth. Inhibition of both processes was relieved by supplying 2 pig of cholesterol or 2.2 pag of phosphatidylcholine per ml along with the inhibitory sterol. The results suggest that cholesterol, in small amounts exerts a positive regulatory effect and epicoprostanol exerts a negative one on unsaturated phospholipid synthesis and, in turn, that RNA and protein synthesis are coordinately controlled with phospholipid synthesis. Recent studies in this laboratory have furnished evidence in support ofthe view that the structure ofcholesterol is optimally designed for modulating the properties of both artificial and natural membranes (1-3). In order of importance, cholesterol appears to owe its unique properties to the following structural features: planarity of the a face in the region of C-14, absence of methyl substituents at C-4, and insertion of a 5,6 double bond.Further work, notably the discovery of sterol synergism, has led to the postulate that cholesterol may serve in more than one role as a constituent of biological membranes (4). It was suggested that, apart from its well-established ability to modify the bulk physical state of the lipid bilayer (5), cholesterol may function more specifically in localized membrane domains influencing certain metabolic processes. Cholesterol availability was suggested to be coupled to the synthesis of phospholipid in Mycoplasma capricolum, a sterol-and fatty acid-requiring prokaryote. In essence, a control mechanism distinct from that affecting the bulk physical state of the lipid bilayer was proposed (6), prompted by the following observations. A low level of cholesterol (0.5 ug/ml), insufficient to support significant growth, elicits a 2-fold stimulation of the Mycoplasma growth rate when combined with lanosterol (10 tkg/ml). Alone the latter sterol supports growth only poorly. The response is synergistic, not additive (4). A significant result was that the membranes ofcells grown on lanosterol with low cholesterol show the same low microviscosity value as membranes isolated from cells cultured on lanosterol alone (4, 6). In experiments designed to elucidate the basis of the synergistic sterol effect, we have obtained evidence that chole...

Lipid acyl chain-dependent effects of sterols in Acholeplasma laidlawii membranes

“…The ability of 12 different sterols to affect (i) cell growth, (ii) lipid head group composition, (iii) the order parameter of the acyl chains, and (iv) the phase equilibria of in vivo lipid mixtures was studied. The following two effects were observed with respect to cell growth: (i) with a given acyl chain composition of the membrane lipids, growth was stimulated, unaffected, reduced, or completely inhibited (lysis), depending on the sterol structure; and (ii) the effect of a certain sterol depended on the acyl chain composition (most striking for epicoprostanol, cholest-4-en-3-one, and cholest-5-en-.3-one, which stimulated growth with saturated acyl chains but caused lysis with unsaturated chains (8,18,26), Of the membrane physical properties affected by sterols, the most commonly investigated are the gel-to-liquid crystalline phase transition temperature (Tm) of the lipids (11,17,20), the order parameter of the lipid acyl chains (17,30,32), and the permeability of the membranes or the derived lipids (1, 10, 12). Recently, studies have also been performed on the ability of some sterols to induce the transition between a lamellar phase and a nonlamellar phase, most often a reversed hexagonal (HI,) phase, in lipid-water model systems (7,15,25 …”

“…However, more specific functions of some sterols have recently been observed for two organisms auxotrophic for sterols, Mycoplasma capricolum and Saccharomyces cerevisiae. Addition of minor concentrations of a certain sterol to a growth medium already containing another sterol in a manifold higher concentration enhances the rate of important metabolic processes (8,18,26), Of the membrane physical properties affected by sterols, the most commonly investigated are the gel-to-liquid crystalline phase transition temperature (Tm) of the lipids (11,17,20), the order parameter of the lipid acyl chains (17,30,32), and the permeability of the membranes or the derived lipids (1,10,12). Recently, studies have also been performed on the ability of some sterols to induce the transition between a lamellar phase and a nonlamellar phase, most often a reversed hexagonal (HI,) phase, in lipid-water model systems (7,15,25).…”

Sterol control of the phosphatidylethanolamine-phosphatidylcholine conversion in the yeast mutant GL7.

“…Formally, the PtdEtn --PtdCho conversion proceeds by three N-methylations. Whether in any of the systems studied one or more transmethylases catalyze the overall process is not clear (15,16 Analysis of the phenomenon of sterol synergism in M. capricolum has led to the demonstration that in this prokaryotic organism cholesterol in trace amounts controls some step in the phospholipid pathway by a process independent of protein synthesis and apparently not related to modulating sterol effects on bulk membrane fluidity (4,20). For the yeast system under study corresponding information is not yet available beyond the fact that a eukaryotic cell can also be shown to exhibit sterol synergism (2, 13) and sterol-sensitive metabolic events (2).…”

Uptake of fatty acids by Mycoplasma capricolum

“…Either cell-associated radioactivity was assayed directly (7) or phospholipids and fatty acids were extracted by swirling the filters in 2 ml of chloroform-methanol (2:1). After reextraction with 1 ml of chloroform, lipids were separated by thin-layer chromatography, and radioactivity was quantified as described previously (5 After disruption by passage through a French pressure cell at 4,000 lb/in2, crude lysates were either used without further treatment or were subjected to centrifugation at 100,000 x g for 30 min. The membrane fraction was washed once and suspended to a final protein concentration of 5 mg/ml.…”

“…In M. capricolum the major phospholipids are phosphatidylglycerol and cardiolipin (9). We have shown previously that fatty acid uptake and utilization are influenced by the structure of the membrane-associated sterol (5,7). A small amount of cholesterol in lanosterol-rich cells lowers the apparent Km for oleate uptake compared with that found in cells harboring lanosterdl as their only sterol (7).…”