Proc. Natd Acad. Sci. USAVol. 79, pp.1.698-1702, March 1982Biochemistry

Animal cells dependent on exogenous phosphatidylcholine formembrane biogenesis

(Chinese hamster ovary cells/CDP-choline synthetase mutant/liposomes/jysophosphatidylcholine/lipid bilayer assembly)

JEFFREY D. ESKO*, MASAHIRO NISHIJIMAt, AND CHRISTIAN R. H. RAETZtDepartment of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706

Communicated by M.J. Osborn, November 5, 1981

ABSTRACT A Chinese hamster ovary cell (CHO) mutant(strain 58), defective in CDP-choline synthetase (cholinephosphatecytidylyltransferase; CTP:cholinephosphate cytidylyltransferase,EC 2.7.7.15), is temperature sensitive for growth and contains lessthan half of the normal amount ofphosphatidylcholine under non-permissive conditions [Esko, J. D. & Raetz, C. R. H. (1980) Proc.NatL Acad. Sci USA 77, 5192-5196]. We now report that the ad-dition of 40 ,uM egg phosphatidylcholine or-lysophosphatidylcho-line to the medium suppresses the temperature sensitivity of mu-tant 58 and permits the growth of colonies at the restrictivetemperature. Phospholipids with different polar headgroups, li-poprotein-bound phospholipids, sphingomyelin, and glycerophos-phocholine do not support prolonged growth at 40C, whereasphosphatidylcholine analogs, such as phosphatidyldimethyleth-anolamine, D-phosphatidylcholine, and f-phosphatidylcholineare quite effective. A broad range of saturated phosphatidylcho-lines, especially those with fatty acids 12-18 carbons in length,suppresses the phenotype. Phospholipids containing ether-linkedhydrocarbons are.ineffective, whereas polyunsaturated phospha-tidykholines are toxic. Residual endogenous synthesis of phos-phatidylcholine by.the mutant is not stimulated under conditionsof phenotypic bypass, but the uptake of exogenous lipid is en-hanced considerably compared to the wild type. Our findings dem-onstrate that exogenous phospholipid can provide at least 50% ofthe phosphatidylcholine required for membrane biogenesis in an-imal cells and that uptake of exogenous phospholipids may beregulated.

Previous reports from this laboratory (1, 2) have described theisolation ofstrain 58, a temperature-sensitive mutant ofChinesehamster ovary (CHO) cells defective. in CDP-choline synthetase(cholinephosphate cytidylyltransferase; CTP:choline phosphatecytidylyltransferase, EC 2.7.7.15). Under nonpermissive con-ditions (40°C), de novo synthesis of phosphatidylcholine is dra-matically reduced, resulting is a reduction to one-half to one-quarter in the content of phosphatidylcholine'compared to pa-rental cells (1, 2). The observation that mutant 58 is temperaturesensitive for growth in the presence of fetal bovine -serum isespecially intriguing. Serum, which is commonly used for grow-ing cells in tissue culture (3, 4) provides a considerable amountof choline-linked phospholipid bound to various serum pro-teins, especially lipoproteins (5). If CHO cells were able toutilize the intact phospholipid molecules present in 10% serum,the temperature-sensitive phenotype of the mutant would besuppressed. Because phenotypic suppression does not occur(1, 2) under typical growth conditions, CHO cells may not pos-sess adequate mechanisms for intact utilization of serumphospholipids.We now demonstrate that mutant 58 growing in tissue cul-

ture can utilize large quantities of phosphatidylcholine and ly-

sophosphatidylcholine added as dispersions to the medium, incontrast to the situation with serum phospholipids. Phospho-lipid uptake- under these conditions results in the suppressionofthe temperature-sensitive phenotype ofmutant 58 (indicatingthat the phosphodiester bond is left intact) and supplies as muchas 50% of the phosphatidylcholine required for membrane as-sembly in this setting. Mutant 58 incorporates much more ofthe added choline phosphoglycerides than the parental cells do,suggesting that phospholipid incorporation by CHO cells isregulated.

EXPERIMENTAL PROCEDURES

Materials. 32Pi (carrier-free) was obtained from New EnglandNuclear. Ham's F-12 culture medium, trypsin, and fetal bovineserum were obtained from GIBCO. Dilauroyl, dimyristoyl, anddistearoyl phosphatidylcholines and lauroyl, myristoyl, palmi-toyl, stearoyl, and oleoyl lysophosphatidylcholines were ob-tained from Sigma. Bovine brain sphingomyelin was purchasedfrom Applied Science Laboratories, and dipalmitoyl phospha-tidylmonomethylethanolamine was supplied by Calbiochem-Behring. All other phospholipids were obtained from SerdaryResearch Laboratories, Ontario, Canada. Hexanoyl, octanoyl,and decanoyl lysophosphatidylcholines were prepared fromtheir respective phosphatidylcholines by phospholipase A2treatment as described below. All phospholipids were judged>95% pure by thin-layer chromatography (6),§ except.diarachi-donoyl phosphatidylcholine, which was first purified by thin-layer chromatography. Organic solvents, including methyletha-nolamine and dimethylethanolamine, were redistilled beforeuse.

Egg phosphatidylcholine was purified by a modification ofthe procedures of Singleton et al. (8). Egg lysophosphatidyl-choline was generated from, this material by digestion withphospholipase A2 (9). Final purification was achieved by chro-matography on silicic acid.

Cell Lines and Media. CHO-K1 were obtained from theAmerican Type Culture Collection (CCL-61), Rockville, MD,and were grown in Ham's F-12 medium (GIBCO), supple-mented with 10% fetal calf serum, penicillin-G (100 units/ml),streptomycin sulfate (100 ,ug/ml), and NaHCO3 at 1.176 g/

Abbreviations: CHO, Chinese hamster ovary; PINaCl, phosphate-buf-fered saline; PtdCho, phosphatidylcholine; l-PtdCho, lysophosphati-dylcholine; PtdEtn, phosphatidylethanolamine; PtdIns, phosphatidyl-inositol; PtdSer, phosphatidylserine; SPH, sphingomyelin; PDME,dipalmitoyl phosphatidyldimethylethanolamine.* Present address: Molecular Biology Institute, University ofCalifornia,Los Angeles, CA 90024.

t Present address: The National Institute of Health, 10-35, 2-Chome,Kamiosaki, Shinagawa-Ku, Tokyo, 141 Japan.

t To whom reprint requests should be addressed.§ Silica gel 60 (E. Merck) plates were employed. These were incorrectlydesignated silica gel "G" in previous publications (6, 7).

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The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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liter. Bovine pancreatic insulin (Sigma) was also included at 20,ug/ml. The isolation and characterization of mutant 58 weredescribed in previous publications (1, 2). Cultures were main-tained at 330C or 40.0 ± 0.20C in a 5% CO2 atmosphere at 100%relative humidity. In all of the studies reported here, only di-alyzed fetal calf serum (10) was employed.When phospholipids were added to the growth medium,

concentrated lipid stocks were prepared in the following ways.Pure lipids in chloroform or 2:1 (vol/vol) chloroform/methanolwere dried in acid-washed glass centrifuge tubes under a streamof nitrogen. Phospholipid samples were resuspended at 2-6mM in phosphate-buffered saline (PjNaCl) at pH 7.2 (11) andsonically irradiated twice for 5 min at power setting 5 with aW185F ultrasonic disrupter (Heat System Ultrasonics, Plain-view, NY) equipped with a no. 419 micro-tip. Unsaturated lipidswere prepared at 0C, whereas saturated lipids were dispersedwithout cooling. Lysophosphatidylcholines were dissolved at2mM in PjNaCl without sonication. Pig liver phosphatidyleth-anolamine and phosphatidylmonomethylethanolamine (andalso in some instances phosphatidylcholine) were dissolved at4 mM in distilled ethanol. All samples were sterilized withSwinnex 13-mm-diameter, 0.22-,um-pore filters (Millipore,Bedford, MA). The recovery ofphospholipids after filtration wastypically 90% or more, and lysophospholipid dispersions orethanol solutions gave quantitative recovery.

Other Procedures. Quantitation ofcellular phospholipid wasachieved by perchloric acid digestion (7, 12) ofsamples obtainedfrom two-dimensional thin-layer chromatography (6). Proteinwas measured by the method of Lowry et al. (13).

RESULTSSerum Phospholipid Does Not Bypass Mutant 58. As de-

scribed previously (1, 2), mutant 58 is temperature sensitive forgrowth in medium supplemented with 10% (vol/vol) fetal bo-vine serum. Chloroform extraction of different lots of serumobtained from GIBCO revealed that the phospholipid contentvaried between 0.1 and 0.5 mM. Two-dimensional thin-layerchromatography (6)§ revealed that over 70% ofthe phospholipidwas phosphatidylcholine and lysophosphatidylcholine. In a typ-ical experiment, approximately 6 X 104 cells of mutant 58 in a60-mm culture dish were shifted to 40'C. After 1 day of incu-bation at 40°C the cells stopped dividing, and the density re-mained below 2 x 105 cells per dish (1, 2), whereas 2 x 106 wassaturating for CHO-K1. On the basis of our previous estimates(1, 2, 6) of the CHO-KL phosphatidylcholine content, the mu-tant would have required about 5-10 nmol of choline-linkedphospholipid for membrane assembly to divide one more time.Because 5 ml of growth medium supplemented with 10% serumwas used in these experiments, the medium provided 5- to 25-fold more choline phosphoglycerides than required for a furtherdoubling at a density of2 x 105. In other experiments, newborncalf, calf, and horse sera (GIBCO) were found to contain 1.1,2.6, and 1.9 mM phospholipid, respectively. When mutant 58was tested for growth at 40°C in Ham's F-12 medium supple-mented with 10% (vol/vol) of each of these sera, the temper-ature-sensitive phenotype was the same as in fetal calf serum,but parental cells grew normally.

Exogenous Choline Phosphoglycerides Bypass Mutant 58.Because the choline-linked phospholipids in serum apparentlywere not utilized for membrane assembly by the mutant, addedphosphatidylcholines in the form of sonicated liposomes orethanol solutions were tested for their ability to rescue mutant58 at 40°C. As shown in Fig. 1, addition of 40 uM phosphati-dylcholine liposomes largely suppressed the temperature-sen-sitive phenotype of the mutant, and the cells looked viable un-der the microscope. Under bypassing conditions, mutant 58

nio~o10~M0| Q4 ILM40OAM / 8 p

1.0 120 IAM0.5

20 40 60 80 100 120 20 40 60 80 100 120Time, hr

FIG. 1. Concentration dependence of the phosphatidylcholine by-pass phenomenon. Mutant and wild-type (parental) cells cultured at330C were harvested with trypsin (14) and added to multiple 60-mm-diameter plastic tissue culture dishes containing 5 ml of growth me-diumtoyield6 x 104 and4 x 104 cells, respectively. After 24 hr at 330C,an appropriate amount of 2 mM phosphatidylcholine liposomes pre-pared in P/NaCl (10) was added to the cultures to give the indicatedconcentration of added lipid. The dishes also received an aliquot of PiNaCl so that the final dilution of growth medium was the same in allcultures (6%, vol/vol). The cells were then shifted to 40'C. At the in-dicated times the cells from duplicate cultures were harvested withtrypsin and counted with a model B Coulter Counter. (A) Mutant 58at 400C. (B) Wild type at 400C. All cultures also contained 100 XM cho-line, a component of F-12 medium.

divided every 24 hr, whereas the parental cells doubled every14 hr independently of added phospholipid. The final cell den-sities attainable by the mutant were still one-half to one-thirdthe wild type, but they were 5-fold greater than without addedlipid. At concentrations greater than 80 AM, phosphatidylcho-line was toxic to mutant and parental cells, whereas concentra-tions less than 10 ,M had little effect on either strain (Fig. 1).Thus, a very narrow range of phospholipid concentrations(30-60 AM) was required to achieve optimal bypass.

Chemical Specificity of the Bypass Phenomenon. Variousother phospholipids were tested for their growth-promotingproperties. Phosphatidylethanolamine, phosphatidylserine, andphosphatidylinositol were ineffective (Fig. 2A), whereas 40 .Mlysophosphatidylcholine suppressed the phenotype very well(Fig. 2B). Interestingly, nonphysiological isomers of phospha-tidylcholine such as D- and 13-lecithins also supported cell di-vision at 40°C (Fig. 2B), whereas sphingomyelin and glycero-phosphocholine had no effect. Results similar to those in Fig.2 were also obtained when the lipid supplements were testedfor their ability to support colony formation from single cells orwhen the lipids were added by dilution of concentrated ethanolstocks. The parental strain was unaffected by any of these lipidsat 40 ,M (not shown).

These results showed that only choline-containing phospho-glycerides effectively fulfilled the lipid requirement of the mu-tant. To examine this further, we added phosphatidylmono-methyl- and phosphatidyldimethylethanolamines to the medium(Fig. 2C). Whereas the monomethyl derivative only partiallycorrected the mutant's phenotype, the dimethyl analog actedas well as phosphatidylcholine. When the headgroup alcohols(i.e., monomethylethanolamine and dimethylethanolamine)were tested by themselves, the monomethylethanolamine de-rivative was identical to phosphatidylmonomethylethanolamine,whereas dimethylethanolamine (like choline) had no effect onthe mutant (Fig. 2C).

Because phosphatidyldimethylethanolamine (but not di-methylethanolamine) suppresses the phenotype (Fig. 2C), di-methylethanolamine appears to be activated by the same cyti-dylyltransferase involved in choline utilization, whereas

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.a 10.0 PtdChoe 4-PtdCho PMME

D-PtdCho.M.t5.0

o,!!EsoH~c~oafll,1SPHPtdEtn GroPChoM2t

Control. A-t~h~~~~~~~~PtdSer

0.5a20 40 60 80 100 120 20 40 60 80 100 120 20 40 60 80 100 120

Time, hr

FIG. 2. Lipid specificity of the bypass phenomenon. The mutant growing at 330C was harvested with trypsin (14), and approximately 6 x 104cells were added to multiple 60-mm-diameter culture dishes containing 5 ml of complete growth medium. After 1 day at 330C, the various sup-

plements were added to 40 ,uM from 2 mM stock solutions. The cells were then shifted to 4000, and at the indicated times duplicate cultures weretreated with trypsin (14). The cells were counted on a model B Coulter Counter. (A) Growth of mutant 58 supplemented with pig brain phospha-tidylserine (PtdSer, o), pig liver phosphatidylinositol (PtdIns, *), pig liver phosphatidylethanolamine (PtdEtn, *), or egg phosphatidylcholine(PtdCho, *). (B) Growth in the presence of the sodium salt of glycerophosphocholine (GroPCho, *), bovine brain sphingomyelin (SPH, x), dipal-mitoyl-sn-glycerol-1-phosphocholine (D-PtdCho, A), dipalmitoyl-sn-glycerol-2-phosphocholine (1-PtdCho, A), egglysophosphatidylcholine (l-PtdCho,*), or the dipalmitoyl ether analog of 3-sn-phosphatidylcholine (e-PtdCho, o). (C) Growth in the presence of dipalmitoyl phosphatidyldimethyletha-nolamine (PDME, *), dipalmitoyl phosphatidylmonomethylethanolamine (PMME, *), 0.1 mM dimethylethanolamine (Me2Etn, A), or 0.1 mM mon-

omethylethanolamine (MeEtn, o).

monomethylethanolamine, which is only a partial functionalsubstitute for choline (15), may be activated by a different en-

zyme (Fig. 2C). Growth of the parental cells was not inhibitedby any of these amino alcohols at 0.1 mM (not shown).To examine the role of the fatty acid moieties, we compared

various synthetic phosphatidylcholines and lysophosphatidyl-cholines to egg lecithin (Table 1). In general, compounds withone or two fatty acyl chains in the range ofC12 to C18 were mostactive. Polyunsaturated lecithins were toxic, and short-chainlipids and 16- or 18-carbon ether-linked phosphatidylcholineswere unable to bypass the mutant's phenotype (Fig. 2B). Thedifferent efficiencies ofvarious molecular species (Table 1) mayalso be influenced by variation in the rate and extent of lipiduptake.

Phospholipid Content of Mutant 58. To determine if lipidsupplementation corrected the abnormal lipid content of mu-tant 58 (1, 2), we grew cells with and without added egg lecithinat 40°C for 2 days. The cells were harvested and analyzed forprotein and phospholipid content (6). Under these conditions,mutant 58 contained 166 nmol of phospholipid per mg of pro-

tein, or about half as much as wild type (356 nmoVmg of pro-tein). Addition of 40 ,tM egg phosphatidylcholine liposomespartially corrected the abnormal phospholipid content of themutant (257 nmoVmg ofprotein) but did not affect the wild type(352 nmoVmg of protein). Two-dimensional thin-layer chro-matography and quantitation of the cellular phospholipids (6,

7, 12) revealed that the amount of phosphatidylcholine in themutant increased from 46 nmol/mg of protein in the absenceofadded phospholipid to 99 nmoVmg ofprotein in its presence,whereas supplementation had very little effect on the phos-phatidylcholine content ofwild-type cells (218 compared to 223nmol/mg of protein).

These results did not distinguish whether exogenous phos-phatidylcholine was being incorporated by the mutant or

whether the addition of lipid somehow stimulated endogenousphosphatidylcholine synthesis. To examine this, cells were in-cubated with 32Pi at 33°C to label the cellular phospholipids toconstant specific radioactivity (Table 2). Prelabeled cells werethen shifted to 40°C in the presence of 40 AM phosphatidyl-choline and 32Pi at the same specific radioactivity. After threedays, the cellular phospholipids were extracted and separatedby two-dimensional thin-layer chromatography (6). As shownin Table 2, when the phospholipid compositions were deter-mined radiochemically, the phosphatidylcholine content in themutant (16.4%) was one-third of that in the parental cells(55.4%). If, instead, the phospholipids were quantitated chem-ically, phosphatidylcholine represented 35.1% of the phospho-lipid in the mutant compared to 59.2% in the wild type. Theseresults suggested that de novo synthesis ofphosphatidylcholinefrom choline was still defective under bypass conditions and thatat least some of the exogenous phospholipid may have beenutilized intact by the mutant. Enzymatic studies of the mutant

Table 1. Growth of parent and mutant in the presence of phosphatidyicholines and lysophosphatidylcholines with defined fatty acidsRelative cell density

Phospholipid Egg NoStrain supplement yolk 6:0 8:0 10:0 12:0 14:0 16:0 18:0 18:1 18:2 18:3 20:4 supplement

Parental PtdCho 1.00 1.00 1.00 0.17 1.05 1.06 1.07 1.07 1.02 0.02 0.02 0.01 1.13CHO-K1 l-PtdCho 1.00 0.95 1.00 0.22 1.09 1.15 1.23 1.13 1.10 0.02 0.02 0.02 1.16

Mutant 58 PtdCho 1.00 0.23 0.24 0.10 1.00 1.65 1.11 1.46 0.53 0.06 0.09 0.07 0.22l-PtdCho 1.00 0.22 0.25 0.04 0.46 1.06 1.00 1.06 0.93 0.04 0.05 0.04 0.17

Multiple 60-mm-diameter culture dishes containing 5 ml of complete growth medium and 4-6 x 104 mutant or wild-type cells were incubatedfor 1 day at 330C. At this time the different phospholipids of defined fatty acids were prepared and added to the dishes (final concentration 40 PM).The cultures were then shifted to 4000. After 3 days the cells were harvested with trypsin (14) and counted on a model B Coulter Counter. Shownare the cell densities expressed relative to the cell density of cultures supplemented with the egg yolk phosphatidylcholine or lysophosphatidyl-choline. Each value is the average of at least two independent determinations. Defined choline phosphoglycerides are: 6:0, hexanoyl; 8:0, octanoyl;10:0, decanoyl; 12:0, lauroyl; 14:0, myristoyl; 16:0, palmitoyl; 18:0, stearoyl; 18:1, oleoyl; 18:2, linoleoyl; 18:3, linolenoyl; 20:4, arachidonoyl.

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Table 2. Phospholipid composition of phosphatidylcholine-supplemented mutant and wild-type cells determinedchemically and by continuous labeling with 32Pi

% of total phospholipidParental CHO-K1 Mutant 58

Chemical ChemicalPhospholipid 32P, phosphorus 32Pi phosphorus

PtdCho 55.4 59.2 16.4 34.1SPH 9.1 7.5 6.1 11.3PtdEtn 18.3 16.4 45.2 31.4PtdIns 8.0 7.2 16.0 10.1PtdSer 5.2 5.2 8.2 5.7PtdGro 0.6 0.6 1.3 1.0Other 3.4 3.9 6.8 5.4

Mutant and parental cells were incubated at 330C in growth mediumsupplemented with 32Pi (2 /Ci/ml; 1 Ci = 3.7 x 1010 becquerels) forseveral generations. Prelabeled cells were harvested and approxi-mately 2 x 105 wild-type and 3.5 x 105 mutant cells were added tomultiple 100-mm-diameter tissue culture dishes at 330C containing32Pi at the same specific radioactivity. After 1 day 0.1 ml of 6mM phos-phatidylcholine liposomes was added to each culture, and these werethen shifted to 40'C. The cells were harvested after 3 days and ex-tracted as described (6), but without carrier lipid. The chloroform ex-tract was divided between two thin-layer chromatography plates, andthe phospholipids were separated as described (6). Autoradiographyrevealed the positions of the phospholipids, which were then scrapedoff and quantitated by liquid scintillation spectrometry or perchloricacid digestion (7, 12). Each value is expressed as a percentage of thetotal recovered material. PtdGro, phosphatidylglycerol; other, lyso-phosphatidylcholine, lysophosphatidylethanolamine, phosphatidic acid,and cardiolipin.

also revealed that the CDP-choline synthetase was defective invitro under bypass conditions (not shown). Although there was

very little net uptake of exogenous phospholipid by wild-typecells, the mutant incorporated enough of the added lipid to sat-isfy over halfof its phosphatidylcholine requirement. Completerestoration of the phosphatidylcholine content to the parentallevel was not observed, resulting in somewhat higher relativelevels of the other phospholipids, especially phosphatidyletha-nolamine (Table 2).

Addition of the other growth-promoting phospholipids alsoresulted in partial restoration of the cellular phospholipid con-

tent (Table 3). As shown, lysophosphatidylcholine was the mosteffective supplement, permitting the accumulation of 46.3%phosphatidylcholine. Very little lysophosphatidylcholine was

present in cells supplemented with this lipid, suggesting thatthis material was rapidly reacylated. The addition of phospha-tidyldimethylethanolamine to the mutant resulted in the ap-

pearance of this abnormal phospholipid (up to 23.2%). The un-

natural isomers of phosphatidylcholine also permitted the"phosphatidylcholine fraction" to accumulate in the mutant, butthe possibility ofrearrangement to the physiological isomer hasnot been excluded.

DISCUSSION

Most membrane phospholipids (16, 17) and many of the mem-brane proteins of eukaryotic cells (18, 19) originate on the en-

doplasmic reticulum and are then translocated to other sites.Phosphatidylcholine is formed from diglyceride and CDP-cho-line on the cytoplasmic surface of the endoplasmic reticulum(16, 17), followed by transmembrane equilibration and intra-cellular migration. Vesicular mechanisms (20-22) and perhapsalso exchange proteins (23, 24) participate in the latter process.

Although the net movement of phospholipids and proteins oc-

curs simultaneously in some instances, net movement of lipids

Table 3. Phospholipid composition of mutant 58 grown withvarious supplements at 40'C

Phospholipid % of total phospholipidsupplement PtdCho SPH PDME PtdEtn PtdIns PtdSer Other

None 27.0 19.9 - 29.9 10.5 7.1 5.6l-PtdCho 46.3 8.0 - 27.6 7.6 6.5 4.0f-PtdCho 35.6* 9.0 - 30.0 9.7 7.5 8.2D-PtdCho 39.1* 8.8 - 31.6 9.7 6.5 4.4PDME 20.1 10.8 23.2 24.8 10.2 6.7 4.2

Multiple 100-mm-diameter dishes containing 15 ml of growth me-dium were each inoculated with 4 x 105 cells and incubated for 1 dayat 330C. At this time 0.3-ml portions of 2mM solutions of the variousphospholipids were added to duplicate cultures, which were thenshifted to 400C. After 3 days the cells were harvested and extracted asdescribed for Table 2. After two-dimensional thin-layer chromatog-raphy (6), the phospholipids were visualized with iodine vapor andquantitated as described (7, 12). Phosphatidyldimethylethanolaminewas well resolved from the other phospholipids, migrating just abovephosphatidylserine (6). Systematic names for phosphatidylcholine iso-mers are given in the legend of Fig. 2. "Other" indicates lysophospha-tidylcholine, lysophosphatidylethanolamine, phosphatidic acid, andcardiolipin.* D-Phosphatidylcholine and ,3-phosphatidylcholine did not separateduring thin-layer chromatography and comigrated with L-phos-phatidylcholine.

independent of membrane proteins must also be possible.Our studies with mutant 58 indicate that animal cells can

utilize large amounts of exogenous phosphatidylcholine formembrane biogenesis when endogenous synthesis is impaired.The following simple scheme could account for this phenome-non. (i) Phosphatidylcholine (or related diacyl lipids) insert intothe plasma membrane by fusion, a possibility supported by theobservations of Pagano and others (25-28). Lysophosphatidyl-choline may enter as a monomer, but in any case is rapidly re-acylated by acyltransferases, some of which are located in theplasma membrane (16, 29). (ii) The excess phosphatidylcholinein the surface returns to the interior of the cell by mechanismssimilar to those that bring it out, for instance by way ofendocyticvesicles (30, 31) or via phospholipid transfer proteins (23, 24).We speculate that "phospholipid sensors" exist that activate theuptake of exogenous phosphatidylcholine when endogenoussynthesis is limited. It is unlikely that the low density lipopro-tein system (32) is specifically involved, because phosphatidyl-choline in lipoproteins is not readily available for phenotypicbypass.

Unlike earlier studies (25-28, 33), which were done withnongrowing cells or in the absence of serum, our results withmutant 58 demonstrate that exogenous phosphatidylcholine canbe taken up continuously and constitute at least one-half of thecellular pool. The incorporated lipid is functional as judged bycell growth, and a large portion of the supplement is incorpo-rated without hydrolysis of the phosphodiester linkage. How-ever, extensive remodeling of the fatty acids can occur (notshown) and may be obligatory, because ether-linked phospha-tidylcholines do not support growth. The excellent phenotypicbypass observed with monoacyl derivatives also shows that ly-solipid acyltransferases (16) can generate a large fraction of thecellular phosphatidylcholine.

Limited incorporation of exogenous phospholipids has beendescribed previously in microbial systems (34-37). Jones andOsborn demonstrated that phosphatidylserine can fuse with theouter membrane of Salmonella typhimurium deep rough mu-tants and subsequently can be translocated to the inner mem-brane, where it is decarboxylated (34, 35). Escherichia coli isable to take up some lysolipid (36, 37), but this phenomenon

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has not yet been characterized extensively. The rapid functionaluptake of phospholipids by animal cells has important impli-cations for further genetic studies, because more mutants like58 should be accessible without searching for conditional alleles.

We thank Ms. M. Wermuth for her excellent assistance. This re-

search was supported in part by Grants AM 21722 and 1KO4-AM00584from the National Institute ofArthritis, Metabolism and Digestive Dis-eases to C. R. H. R. The research described here forms part of a disser-tation by J. D.E. submitted to the University of Wisconsin at Madisonin partial fulfillment of the requirements for the Ph. D. degree.

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1702 Biochemistry: Esko et aLD

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