THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1964 by The American Society of Biological Chemists, Inc.

Vol. 259, No. 17, Issue of September 10, pp. 10S23-10S26,1984 Prrnted in U.S.A.

Decreases in Rates of Lipid Exchange between Mycoplasma gallisepticurn Cells and Unilamellar Vesicles by Incorporation of Sphingomyelin*

(Received for publication, March 26, 1984)

Sanda Clejan and Robert BittmanS From the Department of Chemistry, Queens College of the City University of New York, Flushing, New York 11367

The kinetics of exchange of radiolabeled cholesterol and phospholipids between Mycoplasma gallisepticum cells and an excess of small unilamellar phospholipid/ cholesterol vesicles were studied using cells enriched with different phospholipid classes but the same fatty acyl content. The rates of the rapidly exchanging cho- lesterol and phospholipid pools were markedly slower in sphingomyelin-containing cells than in phosphtidyl- choline-containing cells. The decreased rates of spon- taneous cholesterol and protein-mediated phospholipid exchange from the outer leaflet of the mycoplasma membrane are attributed to stronger interactions be- tween sphingomyelin and cholesterol than between phosphatidylcholine and cholesterol. On the other hand, an increase in cholesterol exchange rate was found on incorporation of a cyclopentano-1,3-diacyl- 2-phosphatidylglycerol, which interacts less with cho- lesterol than does the analogous glycero- lY2-diacyl-sn- 3-phosphatidylglycerol.

The interbilayer transfer of membrane components repre- sents an important process by which the composition and properties of membranes may be varied. A correlation was noted between the cholesterol and SPM’ contents of mem- branes (l), which may reflect preferential SPM-cholesterol interaction. Further support for the conclusion that the extent of phospholipid-cholesterol interactions contribute to the en- dogenous levels of cholesterol that are found in a given mem- brane was gained from studies of cholesterol depletion from cells or virions using serum enriched with different phospho- lipids (2,3) and from examination of partitioning differences of cholesterol between vesicles prepared from different phos- pholipids (4). Moreover, cholesterol was found to undergo more extensive exchange in membranes having high levels of PC compared with those having high SPM content (5, 6). However, the contribution of SPM to cholesterol partitioning was not observed in a study of cholesterol transfer into C16- SPM and DPPC vesicles, suggesting that these phospholipids (which have an equal extent of fatty acid saturation) had

*This research was supported in part by National Institutes of Health Grant HL16660. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$. To whom reprint requests should be addressed. The abbreviations used are: SPM, sphingomyelin; CI6-SPM, N-

palmitoylsphingomyelin; PC, phosphatidylcholine; DPPC, dipalmi- toylphosphatidylcholine; DSPC, distearoylphosphatidylcholine; cy- clo-PG, all-cis-(1,2,3/0)-cyclo-l,3-dipalmitoyl-Z-phosphatidylglycero~ STM, 0.40 M sucrose, 50 mM Tris buffer, pH 7.4, containing 20 mM MgC1,.

equal affinities for cholesterol ( 7 ) . We found recently that the rates of cholesterol and phospholipid exchange in the Myco- plasma gallisepticum cell membrane were enhanced when the membrane cholesterol content was decreased (8). We describe here the influence of phospholipid composition of the myco- plasma cell membrane on the rates of cholesterol and phos- pholipid exchange between mycoplasma cells and small uni- lamellar vesicles. Mycoplasmas are well suited for such ex- periments because they contain only a single membrane and have no cell wall, cannot synthesize or modify sterols, and can take up exogenous lipids from the external medium; SPM is incorporated into M. gallisepticum without modification, whereas the exogenous PC of the growth medium is modified to give a disaturated PC (9). Furthermore, the distribution of cholesterol between the two leaflets of the membrane of Mycoplasma capricolum was not altered by incorporation of PC or SPM (10). We report here a dramatic decrease in the exchange rates of both cholesterol and phospholipids in SPM- containing cells compared with PC-containing cells. We also incorporated a cyclopentano-PG to further test the extent to which phospholipid-sterol interactions modulate the lipid ex- change rate. The cyclopentano-PG interacted less with cho- lesterol in vesicles than did glycero-PG with the same fatty acid composition; in this exchange system, it also affected the cholesterol exchange rate less than phospholipids showing higher “affinity” for cholesterol.

EXPERIMENTAL PROCEDURES

Materials-Egg PC, DPPC, DSPC, C16-SPM, bovine brain SPM, oleic and palmitic acids, and bovine serum albumin (Fraction V, fatty acid-poor) were purchased from Sigma. Albumin was treated with chloroform/methanol (2:1, v/v) to eliminate traces of lipids. The cyclopentano-PG analog (all-cis-(1,2,3/0)-cyclo-2-PG, referred to here as cyclo-PG) was kindly supplied by Dr. A. J. Hancock (University of Missouri, Kansas City, MO). Cholesterol (Sigma) was recrystallized twice from ethanol. The sources and specific activities of [4-“C] cholesterol, [6-methyL3H]thymidine, [2-3H]glycerol trioleate, [l-“C] palmitic acid, and [U-“Clalgae PC were as cited previously (8). Bovine [N-methyl-’‘C]SPM (60 Ci/mol) was purchased from New England Nuclear and [9,10-3H]oleic acid (2 Ci/mmol) was from Amersham.

Growth of Cells-M. gallisepticum strain A5969 was grown to mid- exponential phase in a modified Edward medium supplemented with cholesterol (2 pg/ml for the “adapted strain and 10 pg/ml for the “native” strain), palmitic and oleic acids (10 Gg/ml each), and 1% albumin. The final ethanol concentration did not exceed 1% (v/v). For incorporation of exogenous phospholipids, cells were grown with C16-SPM, bovine brain SPM, or DPPC (10 gg/ml). Cells were also grown in media containing egg PC plus DSPC (5 pg of each/ml). The latter mixture has approximately the same unsaturated index (the sum of the mole fractions multiplied by the number of unsaturated bonds in each fatty acyl chain) as bovine brain SPM; furthermore, the transition temperature of Cl,-SPM bilayers is 41 “C, as with DPPC bilayers (11). We grew the cultures, harvested, washed, and treated the cells, and analyzed the protein, phospholipid, and choles-

10823

10824 Decreases in Lipid Exchange Rates by Sphingomyelin

terol content as outlined previously (8). The growth medium was supplemented with 0.25 &i/ml of [3H]thymidine to monitor the recovery and leakiness of the cells during the exchange experiments. The membrane lipids were labeled with “C as indicated in the tables. To examine the fatty acyl composition of PG in cells grown with exogenous phospholipids, the medium was supplemented with -0.01 &i/ml of [i4C]palmitic acid and -0.008 &i/ml of [3H]oleic acid. The free cholesterol and total phospholipid contents of the adapted cells grown with PC and SPM were 25-28 pg/mg of membrane protein and 230-268 pg/mg of membrane protein, respectively. The lipid composition of the cell membrane was not altered after a 12-14-h incubation with vesicles of the same cholesterol/phospholipid molar ratio (0.24-0.27), demonstrating that adhesion or fusion of vesicles with cells does not occur to an appreciable extent. The retention of 90% of [3H]thymidine-labeled components after 8 h of incubation and 80% after 14 h indicated that most of the cells remained intact during the phospholipid and cholesterol exchange experiments.

Preparation of Lipid Exchange Protein-The nonspecific lipid ex- change protein was partially purified from 8 g of rat liver by the procedure described by Crain and Zilversmit (12) for the protein from beef liver cytosol. We omitted the heat treatment and substituted column chromatography on phenyl-agarose for the octyl-agarose step. In the final stage of purification, the exchange protein was eluted from a CM52 cellulose column, concentrated in an Amincon ultra- filtration cell with a Diaflo UM-10 membrane under nitrogen pres- sure, and applied to a column of phenyl-agarose. After elution at pH 3.0, the pH was increased to 7.4 and the exchange protein was stored at 4 “C at a concentration of 300 ag/ml in the elution buffer (25 mM sodium phosphate, 45 mM NaCl, 5 mM 2-mercaptoethanol, and 0.02% sodium azide, pH 7.4). There was little loss of activity for about 2-3 months. The phospholipid exchange activity for PC relative to SPM was estimated using small unilamellar vesicles containing “C-labeled phospholipid as the donor. Multilamellar liposomes containing 0.01 &i of [3H]glycerol trioleate and 10 mol % of dicetyl phosphoric acid were used as the acceptor species (12). The donor and acceptor dispersions contained equimolar amounts of DPPC and SPM (the donor had C&PM and the acceptor had bovine brain SPM); the phospholipid/cholesterol molar ratio was 4:1, which is similar to that in adapted mycoplasma cells. When the donor vesicles (containing 10 jd/ml of either [i’C]PC or [“C]SPM) and acceptor dispersions (total lipid concentrations of 0.5 and 10 mM, respectively) were mixed at 37 “C with 1.6 mg of exchange protein in 1 ml of buffer, a linear rate of “C-phospholipid transfer to acceptor was found over a period of 20 min. At time intervals, aliquots were withdrawn, the mixture was chilled briefly, and the multilamellar liposomes were sedimented for 10 min in a Beckman Airfuge at 20 p.s.i. The pellet was soluhilized and double-label scintillation counting was carried out as described previously (8). The per cent of the total ‘YXabeled phospholipid exchanged was calculated after correction for unsedimented liposomes and for the small amount of spontaneous ‘*C-phospholipid exchange. The ratio of the PC/SPM exchange rates which were mediated by the protein preparation in this hilayer system was 1.00:0.72. The specific activity of the protein for PC and SPM exchange was calcu- lated from the fraction of label transferred to acceptor and from the specific activities of the i4C-labeled phospholipids in the donor vesi- cles. For PC and SPM exchange, the specific activities of the protein preparation we used were about 34 and 31, respectively, in units of nanomoles of phospholipid transferred to liposomes/mg of exchange protein/min at 37 “C. For the exchange of “C-labeled phospholipids between M. gallisepticum and vesicles, we used 0.25 mg of exchange protein/mg of membrane protein. In experiments with cells labeled with [i4C]cholesterol, we found no acceleration of the cholesterol exchange rate when the exchange protein was added to the incubation medium containing cells, vesicles, and albumin.

Preparation of Vesicles and Measurement of ~‘ClChoksterol or ‘%- Phospholipid Exchange between Cells and Vesicles-Small unilamellar vesicles were prepared in STM buffer by sonciation under nitrogen in a Heat Systems Ultrasonics W375A sonicator equipped with a cup horn (8). The cholesterol/egg PC molar ratio in the vesicles was 0.25 or 0.90, which was the same as that in the cell membrane of the adapted or native strain, respectively. Cells and vesicles were incu- bated at 37 “C in STM buffer containing penicillin, deoxyribonu- clease, and albumin (2% w/v) (8). (This concentration of albumin was shown to enhance the rate of sterol exchange between M. galli- septicum cells and small unilamellar vesicles (8, 13, 14).) The cell pellet was obtained from assays of spontaneous [‘4C]cholesterol ex- change and protein-catalyzed i4C-phospholipid exchange; it was ana-

lyzed by double-label scintillation counting of ‘YXabeled lipid and [3H]thymidine-labeled components (8). The kinetic data and half- times for exchange were calculated as described before (8).

RESULTS AND DISCUSSION

Cholesterol Exchange Kinetics-Fig. 1 shows a comparison of the spontaneous exchange of [‘4C]cholesterol from cells containing C&-SPM and DPPC, or bovine brain SPM and a mixture of egg PC and DSPC. As shown previously, choles- terol exchange kinetics between intact A4. gullisepticum cells and excess lipid vesicles with the same sterol/phospholipid molar ratio are characterized by biphasic first-order processes (8, 13, 14). Each kinetic pool contains about 50% of the cholesterol molecules. The more rapidly exchangeable pool of cholesterol is thought to represent cholesterol molecules lo- calized in the outer leaflet of the cell membrane and the less accessible pool is considered to represent the cholesterol mol- ecules initially in the inner leaflet (8, 13). The rate of the more rapidly exchanging pool is markedly lower in SPM- containing cells than in PC-containing cells, as indicated by the decrease in the slope of the faster phase when SPM is present in the cell membrane. Table I summarizes the kinetic parameters for [‘4C]cholesterol exchange. There is a 37 to 40% decrease in the rate of the fast exponential phase in the donor cells containing &-SPM or bovine brain SPM com- pared with cells containing phosphatidylcholines with a sim- ilar extent of hydrocarbon chain saturation. However, the rate of the slow exponential phase and the relative sizes of the inner leaflet and outer leaflet cholesterol pools are not altered by incorporation of SPM or PC into the cell membrane. The ability of SPM to decrease the rate of the rapidly exchanging cholesterol pool does not depend on the presence of membrane proteins; we have found that incorporation of 44 mol % C&- SPM into egg PG vesicles caused a 100% increase in the half- time for cholesterol exchange in a vesicle/vesicle system, whereas the same level of DPPC incorporation caused an increase of only 36%.2 These results are consistent with the

201 ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ 0 2 4 6 8 IO 0 2 4 6 S IO

TIME (hours)

FIG. 1. Effect of incorporating exogenous phosphatidylcho- lines or sphingomyelins into M. gallisepticum on the kinetics of [‘4C]cholesterol exchange between adapted cells and cho- lesterol/egg PC vesicles. A, first-order plots in the presence of a mixture of exogenous egg PC plus DSPC (each supplied at 5 pg/ml of medium) (0) and bovine brain SPM (10 pg/ml) (m). B, first-order plots in the presence of 10 fig/ml DPPC (0) and &SPM (m). Exchange was carried out at 37 “C in STM buffer in the presence of albumin (2% w/v). The molar ratio of cholesterol/phospholipid in the cells and vesicles was -0.25. SPM constituted 45-48% of the polar lipids in the cell membrane when M. gallisepticum was grown in the presence of 10 pg/ml GIG-SPM or bovine brain SPM. When cells were grown with PC, the PC content of the polar lipids was 41%. In each case, the remainder was essentially PG.

* L. Fugler, S. Clejan, and R. Bittman, unpublished results.

Decreases in Lipid Exchange Rates by Sphingomyelin 10825

TABLE I Influence of PC and SPM incorporation into cell membranes on the kinetic parameters for [‘4C]cholesterol exchunge between adapted M.

gallisepticum cells and cholesterol/PC vesicles Cells and vesicles were incubated in STM in the presence of 2%

albumin as described under “Experimental Procedures.” The concen- trations of cholesterol and exogenous phospholipid in the medium were 2 and 10 pg/ml, respectively. Membrane cholesterol was labeled by supplementing the medium with [“C]cholesterol (0.005 pCi/ml). The cholesterol /phospholipid molar ratio in the cells and vesicles ranged from 0.23 to 0.27.

Exogenous phospholipid lo2 x 102 x

added to k; kg tL tH rf,.

growth medium h” h h

None 12.7 15.0 2.5 rtO.2 1.1 f 0.1 0.54(4) PC (egg + distearoyl) 10.9 13.6 2.9 f 0.3 2.5 f 0.2 0.55(2) SPM (bovine brain) 4.8 6.8 5.9 f 0.5 4.1 f 0.3 0.58(2) PC (dipalmitoyl) 10.6 11.5 3.1 f 0.2 2.8f 0.2 0.52(2) SPM (N-palmitoyl) 5.1 5.6 6.5 f 0.5 4.7 -I- 0.4 0.52(2)

k d and kk represent the first-order rate constants for the trans- location of [14C]cholesterol from the inner to outer and from the outer to inner monolayers, respectively. They were calculated from -2.303 x the slopes of the lines in the semilogarithmic plots of per cent [“C] cholesterol exchanged versus time.

The half-time of equilibration of [14C]cholesterol between the two kinetic pools in the membrane is given by tlh = 0.693/(kd + kh). The values represent the mean f S.E.

t60 is the time required for the [14C]cholesterol content to be reduced to 50%. It represents mostly a transfer of cholesterol. t50 was calculated from -0.693/the slope of the rapidly exchanging pool (Fig. 1).

router is given by k,/(kd + k k ) (19). The numbers in parentheses represent the number of kinetic experiments performed with separate cultures.

proposed preference of cholesterol for SPM compared with PC (for recent reviews, see Refs. 15-17).

We tested the possibility that the differences in the choles- terol exchange rates in the DPPC- and SPM-grown cells reflect changes in the palmitic acid/oleic acid ratios in the membrane phospholipids. We examined the fatty acyl com- positon of only the PG fraction since (a) mycoplasma species cannot synthesize saturated or unsaturated fatty acids, (6) the PC of M. gallisepticum grown in medium containing DPPC is disaturated, and (c) the SPM is incorporated without modification (9). Previous studies demonstrated that the po- sitional distribution of these fatty acids in M. gallisepticum PG is unusual, with the oleoyl chain preferentially at position 1 and palmitoyl at position 2 (9). In cells adapted to grow with low cholesterol, the radioactivity of M. gallisepticum PG derived from [3H]oleic acid and [‘4C]palmitic acid added to the growth medium was as follows: with no exogenous lipid added, I4C count~/min/~H counts/min = 1.88 f 0.08; with DPPC added, I4C count~/min/~H counts/min = 1.65 f 0.14; and with CI6-SPM added, 14C count~/min/~H counts/min = 1.71 f 0.09. (The error limits represent standard errors of the mean.) Thus, cells supplemented with DPPC or C16-SPM do not modify the relative amounts of oleic and palmitic acids incorporated into PG. In agreement with previous results obtained with the total polar lipids of M. gallisepticum cells supplemented with oleic and palmitic acids (18), we found an increase in the palmitic acid/oleic acid ratio in PG on adap- tation to growth on low cholesterol. The I4C count~/min/~H counts/min ratio was 1.38 in the native strain, compared with 1.88 in the adapted strain.

In contrast to the effect of SPM incorporation on the [“C] cholesterol exchange kinetics between cells and vesicles, in- corporation of cyclo-PG into the cell membrane increased the

TABLE I1 Influence of cyclo-PG incorporation into cell membranes on the

kinetic parameters for [14C]cholesterol exchange between native and adapted M. gallisepticum cells and cholesterol/PC vesicles

Cells and vesicles were incubated in STM in the presence of 2% albumin as described under “Experimental Procedures.” The concen- tration of cyclo-PG in the medium was 10 pg/ml. Adapted cells were grown in medium containing 2 pg/ml cholesterol and native cells grown with 10 pg/ml cholesterol. The cyclo-PG content of the polar lipids in the cell membrane was 22-25% as judged by lipid phosphorus content. The remainder was glycero-PG, which is the only de novo synthesized phospholipid of M. gallisepticum (9).

Exoeenous Strain phospholipid 10’ X 10’ x

- ~ ” - -~

addedto k, kb. th tw r,, growth medium

h” h h Native None Native

4.7 6.5 6.2 2 0.2 2.5 f 0.1 0.58

Adapted None 12.7 15.0 2.5 1.2 0.54 Adapted cyclo-PG 15.2 15.8 2.1 0.8 0.51

CyClo-PG 6.0 6.0 5.4 0.2 1.9 0.1 0.50

TABLE I11 Influence of DPPC and c16-sPM incorporation into adapted X

gallisepticum cells on the initial rates of “C-phospholipid exchange Cells were incubated with egg PC/cholesterol vesicles and 0.25 pg

of transfer protein per mg of mycoplasma membrane protein and 2% albumin as described under “Experimental Procedures.” The phos- pholipid (PL) composition of the extracted lipids was estimated by thin-layer chromatographs and lipid phosphorus analvsis (10).

“C-PL incorporated composition Exogenous PL Membrane PL

exchanged into cell tso rHbr memhrane PG PC SPM

h PGb None 95 3.9 2 0.2 0.44 (2) PG‘ DPPCd 49 47 PGb C16-SPMd 54

4.2 f 0.4 0.50 (2)

PC‘ 42 7.8 f 0.4 0.52 (2)

DPPCd 49 47 SPMf

4.3 f 0.3 0.50 (3) C1s-SPMd 54 42 7.3 f 0.2 0.50 (3)

”The numbers in parentheses represent the number of kinetic experiments performed with separate cultures.

Membrane lipids were labeled by growing the cells with 0.002 pCi of [l-’4C]palmitate/ml of medium.

‘ A small amount of radiolabeled PC was also formed (-2.8% of the total radiolabeled phospholipids), presumably by deacylation of the exogenous PC followed by reacylation (9).

Exogenous DPPC or C&PM was incorporated by growing the cells in the presence of 10 pg of phospholipid/ml of medium.

e PC was labeled by growing cells with 0.005 pCi of algae [“C]PC/ ml of medium.

’SPM was labeled by growing cells with 0.006 pCi of bovine [N- methyl-14C]SPM.

rate. Table I1 shows that the half-time of the rapidly exchang- ing cholesterol pool (t5,,) decreased significantly in both the native and adapted strains when the cells were grown with exogenous cyclo-PG compared with cells containing only de nouo synthesized PG. The ability of cyclo-PG to enhance the cholesterol exchange rate relative to glycerol-PG probably results from weaker interactions between cholesterol and the conformationally rigid cycEo-PG. Indeed, we have found that the rate constant for C1- efflux from cyclo-PG vesicles above the phospholipid phase transition temperature is decreased by only 32% on incorporation of cholesterol to 25 mol %; in contrast, cholesterol had a larger effect in DPPG vesicles, causing the rate constant to decrease by 42%.3

Phospholipid Exchange Kinetics-Table I11 summarizes the results of protein-mediated ‘*C-phospholipid exchange. The

S. Clejan and R. Bittman, unpublished results.

10826 Decreases in Lipid Exchange Rates by Sphingomyelin

rapidly exchanging pool which interacts with the exchange protein represents phospholipid in the outer leaflet of the mycoplasma membrane. The effects of exogenous phospho- lipids on the remaining radiolabeled phospholipid pool were not investigated since this pool underwent exchange ex- tremely slowly. The half-time for the rapidly exchanging [“C] SPM pool (tJh = 7.3 h) is 70% longer than that for the comparable [“C]PC pool (tlh = 4.3 h), but the pool sizes (as reflected by router) are the same. The much lower exchange rate of SPM cannot be attributed solely to the lower specificity of the exchange protein toward SPM relative to PC, which was estimated in a bilayer exchange system (see “Experimen- tal Procedures”). The difference in the exchange rates may reflect a significant difference in molecular packing in the membranes enriched in these phospholipids.

We also examined the effects of DPPC and C16-SPM on the rate of the rapidly exchanging [14C]PG pool. Incorporation of CIG-SPM to 42% of the total lipid phosphorus content of the mycoplasma membrane caused a 100% decrease in the rate of [14C]PG exchange. When DPPC was incorporated to a similar level as SPM, there was only a small decrease in the phospholipid exchange rate. The contrast between the effects of CI6-SPM and DPPC on the kinetics of spontaneous cho- lesterol and protein-mediated phospholipid exchange between mycoplasma cells and vesicles could be explained by the existence of stronger interactions between SPM and choles- terol compared with PC and cholesterol. Together with the cholesterol exchange data in cells containing exogenous cyclo- PG (Table 11) and our earlier results on the effect of mem- brane cholesterol content on lipid exchange rates (8), these results indicate an inverse correlation between exchange rate and membrane lipid order. This correlation is consistent with a mechanism of exchange between donor and excess acceptor

that involves the slow dissolution of the lipid from the surface of the donor particle.

Acknowledgments-We thank Dr. A. J. Hancock for kindly provid- ing cyclo-PG and Lillian Fugler for technical assistance.

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