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Proc. Natd Acad. Sci. USA Vol. 79, pp.1.698-1702, March 1982

Animal cells dependent on exogenous for membrane biogenesis (Chinese hamster ovary cells/CDP- synthetase mutant//jysophosphatidylcholine/ bilayer assembly) JEFFREY D. ESKO*, MASAHIRO NISHIJIMAt, AND CHRISTIAN R. H. RAETZt Department of Biochemistry, College ofAgricultural and Sciences, University of Wisconsin, Madison, Wisconsin 53706 Communicated by M.J. Osborn, November 5, 1981 ABSTRACT A Chinese hamster ovary (CHO) mutant sophosphatidylcholine added as dispersions to the medium, in (strain 58), defective in CDP-choline synthetase (cholinephosphate contrast to the situation with serum phospholipids. Phospho- cytidylyltransferase; CTP:cholinephosphate cytidylyltransferase, lipid uptake- under these conditions results in the suppression EC 2.7.7.15), is temperature sensitive for growth and contains less ofthe temperature-sensitive phenotype ofmutant 58 (indicating than halfofthe normal amount ofphosphatidylcholine under non- that the phosphodiester bond is left intact) and supplies as much permissive conditions [Esko, J. D. & Raetz, C. R. H. (1980) Proc. as 50% of the phosphatidylcholine required for membrane as- NatL Acad. Sci USA 77, 5192-5196]. We now report that the ad- sembly in this setting. Mutant 58 incorporates much more of dition of 40 ,uM egg phosphatidylcholine or-lysophosphatidylcho- the added choline phosphoglycerides than the parental cells do, line to the medium suppresses the temperature sensitivity of mu- suggesting that phospholipid incorporation by CHO cells is tant 58 and permits the growth of colonies at the restrictive temperature. Phospholipids with different polar headgroups, li- regulated. poprotein-bound phospholipids, , and glycerophos- phocholine do not support prolonged growth at 40C, whereas EXPERIMENTAL PROCEDURES phosphatidylcholine analogs, such as phosphatidyldimethyleth- anolamine, D-phosphatidylcholine, and f-phosphatidylcholine Materials. 32Pi (carrier-free) was obtained from New England are quite effective. A broad range of saturated phosphatidylcho- Nuclear. Ham's F-12 culture medium, trypsin, and fetal bovine lines, especially those with fatty 12-18 carbons in length, serum were obtained from GIBCO. Dilauroyl, dimyristoyl, and suppresses the phenotype. Phospholipids containing ether-linked distearoyl and lauroyl, myristoyl, palmi- hydrocarbons are.ineffective, whereas polyunsaturated phospha- toyl, stearoyl, and oleoyl were ob- tidykholines are toxic. Residual endogenous synthesis of phos- tained from Sigma. Bovine brain sphingomyelin was purchased phatidylcholine by.the mutant is not stimulated under conditions from Applied Science Laboratories, and dipalmitoyl phospha- of phenotypic bypass, but the uptake of exogenous lipid is en- tidylmonomethylethanolamine was supplied by Calbiochem- hanced considerablycompared to thewildtype. Our findings dem- Behring. All other phospholipids were obtained from Serdary onstrate that exogenous phospholipid can provide at least 50% of Research Laboratories, Ontario, Canada. Hexanoyl, octanoyl, the phosphatidylcholine required for membrane biogenesis in an- and decanoyl lysophosphatidylcholines were prepared from imal cells and that uptake of exogenous phospholipids may be their respective phosphatidylcholines by A2 regulated. treatment as described below. All phospholipids were judged >95% pure by thin-layer chromatography (6),§ except.diarachi- Previous reports from this laboratory (1, 2) have described the donoyl phosphatidylcholine, which was first purified by thin- isolation ofstrain 58, atemperature-sensitive mutant ofChinese layer chromatography. Organic solvents, including methyletha- hamster ovary (CHO) cells defective.in CDP-choline synthetase nolamine and dimethylethanolamine, were redistilled before (cholinephosphate cytidylyltransferase; CTP:choline phosphate use. cytidylyltransferase, EC 2.7.7.15). Under nonpermissive con- Egg phosphatidylcholine was purified by a modification of ditions (40°C), de novo synthesis ofphosphatidylcholine is dra- the procedures of Singleton et al. (8). Egg lysophosphatidyl- matically reduced, resulting is a reduction to one-half to one- choline was generated from, this material by digestion with quarter in the content ofphosphatidylcholine'compared to pa- (9). Final purification was achieved by chro- rental cells (1, 2). The observation that mutant 58 is temperature matography on silicic . sensitive for growth in the presence of fetal bovine -serum is Cell Lines and Media. CHO-K1 were obtained from the especially intriguing. Serum, which is commonly used for grow- American Type Culture Collection (CCL-61), Rockville, MD, ing cells in tissue culture (3, 4) provides a considerable amount and were grown in Ham's F-12 medium (GIBCO), supple- of choline-linked phospholipid bound to various serum pro- mented with 10% fetal calf serum, penicillin-G (100 units/ml), teins, especially (5). If CHO cells were able to streptomycin sulfate (100 ,ug/ml), and NaHCO3 at 1.176 g/ utilize the intact phospholipid molecules present in 10% serum, the temperature-sensitive phenotype of the mutant would be Abbreviations: CHO, Chinesehamster ovary; PINaCl, phosphate-buf- suppressed. Because phenotypic suppression does not occur fered saline; PtdCho, phosphatidylcholine; l-PtdCho, lysophosphati- (1, 2) under typical growth conditions, CHO cells may not pos- dylcholine; PtdEtn, ; PtdIns, phosphatidyl- sess adequate mechanisms for intact utilization of serum ; PtdSer, ; SPH, sphingomyelin; PDME, phospholipids. dipalmitoyl phosphatidyldimethylethanolamine. We now that mutant 58 in tissue cul- * Present address: Molecular Institute, University ofCalifornia, demonstrate growing Los Angeles, CA 90024. ture can utilize large quantities of phosphatidylcholine and ly- t Present address: The National Institute of Health, 10-35, 2-Chome, Kamiosaki, Shinagawa-Ku, Tokyo, 141 Japan. The publication costs ofthis article were defrayed in part by page charge t To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertise- § Silica gel 60 (E. Merck) plates were employed. These were incorrectly ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. designated silica gel "G" in previous publications (6, 7). 1698 Downloaded by guest on October 3, 2021 Biochemistry: Esko et aL Proc. Natl. Acad. Sci. USA 79 (1982) 1699 liter. Bovine pancreatic (Sigma) was also included at 20 ,ug/ml. The isolation and characterization of mutant 58 were described in previous publications (1, 2). Cultures were main- nio~o10~M0| Q4 ILM tained at 330C or 40.0 ± 0.20C in a 5% CO2 atmosphere at 100% 40OAM / 8 p 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. 1.0 120 IAM Pure in chloroform or 2:1 (vol/vol) chloroform/methanol 0.5 were dried in acid-washed glass centrifuge tubes under a stream of nitrogen. Phospholipid samples were resuspended at 2-6 20 40 60 80 100 120 20 40 60 80 100 120 mM in phosphate-buffered saline (PjNaCl) at pH 7.2 (11) and sonically irradiated twice for 5 min at power setting 5 with a Time, hr W185F ultrasonic disrupter (Heat System Ultrasonics, Plain- FIG. 1. Concentration dependence of the phosphatidylcholine by- view, NY) equipped with a no. 419 micro-tip. Unsaturated lipids pass phenomenon. Mutant and wild-type (parental) cells cultured at were prepared at 0C, whereas saturated lipids were dispersed 330C were harvested with trypsin (14) and added to multiple 60-mm- without cooling. Lysophosphatidylcholines were dissolved at diameter plastic tissue culture dishes containing 5 ml of growth me- liver diumtoyield6 x 104 and4 x 104cells, respectively. After 24 hr at 330C, 2mM in PjNaCl without sonication. Pig phosphatidyleth- an appropriate amount of 2 mM phosphatidylcholine liposomes pre- anolamine and phosphatidylmonomethylethanolamine (and pared in P/NaCl (10) was added to the cultures to give the indicated also in some instances phosphatidylcholine) were dissolved at concentration of added lipid. The dishes also received an aliquot of Pi 4 mM in distilled ethanol. All samples were sterilized with NaCl so that the final dilution of growth medium was the same in all Swinnex 13-mm-diameter, 0.22-,um-pore filters (Millipore, cultures (6%, vol/vol). The cells were then shifted to 40'C. At the in- Bedford, MA). The recovery ofphospholipids after filtration was dicated times the cells from duplicate cultures were harvested with dispersions or trypsin and counted with a model B Coulter Counter. (A) Mutant 58 typically 90% or more, and lysophospholipid at 400C. (B) Wild type at 400C. All cultures also contained 100 XM cho- ethanol solutions gave quantitative recovery. line, a component of F-12 medium. Other Procedures. Quantitation ofcellular phospholipid was achieved by perchloric acid digestion (7, 12) ofsamples obtained from two-dimensional thin-layer chromatography (6). divided every 24 hr, whereas the parental cells doubled every was measured by the method of Lowry et al. (13). 14 hr independently of added phospholipid. The final cell den- sities attainable by the mutant were still one-half to one-third RESULTS the wild type, but they were 5-fold greater than without added Serum Phospholipid Does Not Bypass Mutant 58. As de- lipid. At concentrations greater than 80 AM, phosphatidylcho- scribed previously (1, 2), mutant 58 is temperature sensitive for line was toxic to mutant and parental cells, whereas concentra- growth in medium supplemented with 10% (vol/vol) fetal bo- tions less than 10 ,M had little effect on either strain (Fig. 1). vine serum. Chloroform extraction of different lots of serum Thus, a very narrow range of phospholipid concentrations obtained from GIBCO revealed that the phospholipid content (30-60 AM) was required to achieve optimal bypass. varied between 0.1 and 0.5 mM. Two-dimensional thin-layer Chemical Specificity of the Bypass Phenomenon. Various chromatography (6)§ revealed that over 70% ofthe phospholipid other phospholipids were tested for their growth-promoting was phosphatidylcholine and . In a typ- properties. Phosphatidylethanolamine, phosphatidylserine, and ical experiment, approximately 6 X 104 cells of mutant 58 in a were ineffective (Fig. 2A), whereas 40 .M 60-mm culture dish were shifted to 40'C. After 1 day of incu- lysophosphatidylcholine suppressed the phenotype very well bation at 40°C the cells stopped dividing, and the density re- (Fig. 2B). Interestingly, nonphysiological isomers of phospha- mained below 2 x 105 cells per dish (1, 2), whereas 2 x 106 was tidylcholine such as D- and 13- also supported cell di- saturating for CHO-K1. On the basis of our previous estimates vision at 40°C (Fig. 2B), whereas sphingomyelin and glycero- (1, 2, 6) of the CHO-KL phosphatidylcholine content, the mu- had no effect. Results similar to those in Fig. tant would have required about 5-10 nmol of choline-linked 2 were also obtained when the lipid supplements were tested phospholipid for membrane assembly to divide one more time. for their ability to support colony formation from single cells or Because 5 ml ofgrowth medium supplemented with 10% serum when the lipids were added by dilution ofconcentrated ethanol was used in these experiments, the medium provided 5- to 25- stocks. The parental strain was unaffected by any ofthese lipids fold more choline phosphoglycerides than required for a further at 40 ,M (not shown). doubling at a density of2 x 105. In other experiments, newborn These results showed that only choline-containing phospho- calf, calf, and horse sera (GIBCO) were found to contain 1.1, effectively fulfilled the lipid requirement ofthe mu- 2.6, and 1.9 mM phospholipid, respectively. When mutant 58 tant. To examine this further, we added phosphatidylmono- was tested for growth at 40°C in Ham's F-12 medium supple- methyl- and phosphatidyldimethylethanolamines to the medium mented with 10% (vol/vol) of each of these sera, the temper- (Fig. 2C). Whereas the monomethyl derivative only partially ature-sensitive phenotype was the same as in fetal calf serum, corrected the mutant's phenotype, the dimethyl analog acted but parental cells grew normally. as well as phosphatidylcholine. When the headgroup alcohols Exogenous Choline Phosphoglycerides Bypass Mutant 58. (i.e., monomethylethanolamine and dimethylethanolamine) Because the choline-linked phospholipids in serum apparently were tested by themselves, the monomethylethanolamine de- were not utilized for membrane assembly by the mutant, added rivative was identical to phosphatidylmonomethylethanolamine, phosphatidylcholines in the form of sonicated liposomes or whereas dimethylethanolamine (like choline) had no effect on ethanol solutions were tested for their ability to rescue mutant the mutant (Fig. 2C). 58 at 40°C. As shown in Fig. 1, addition of 40 uM phosphati- Because phosphatidyldimethylethanolamine (but not di- dylcholine liposomes largely suppressed the temperature-sen- methylethanolamine) suppresses the phenotype (Fig. 2C), di- sitive phenotype ofthe mutant, and the cells looked viable un- methylethanolamine appears to be activated by the same cyti- der the microscope. Under bypassing conditions, mutant 58 dylyltransferase involved in choline utilization, whereas Downloaded by guest on October 3, 2021 1700 Biochemistry: Esko et aL Proc. Natl. Acad. Sci. USA 79 (1982)

.a 10.0 PtdCho e 4-PtdCho PMME D-PtdCho.M.t 5.0 o,!!Eso H~c~oafll,1SPH PtdEtn GroPChoM2t

Control. A-t~h ~~~~~~~~PtdSer 0.5a 20 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 104 cells 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 were treated 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--1-phosphocholine (D-PtdCho, A), dipalmitoyl-sn-glycerol-2-phosphocholine (1-PtdCho, A), egg lysophosphatidylcholine (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 functional 7, 12) revealed that the amount of phosphatidylcholine in the substitute for choline (15), may be activated by a different en- mutant increased from 46 nmol/mg of protein in the absence zyme (Fig. 2C). Growth of the parental cells was not inhibited ofadded phospholipid to 99 nmoVmg ofprotein in its presence, by any of these amino alcohols at 0.1 mM (not shown). whereas supplementation had very little effect on the phos- To examine the role ofthe moieties, we compared phatidylcholine content ofwild-type cells (218 compared to 223 various synthetic phosphatidylcholines and lysophosphatidyl- nmol/mg of protein). cholines to egg (Table 1). In general, compounds with These results did not distinguish whether exogenous phos- one or two fatty acyl chains in the range ofC12 to C18 were most phatidylcholine was being incorporated by the mutant or active. Polyunsaturated lecithins were toxic, and short-chain whether the addition of lipid somehow stimulated endogenous lipids and 16- or 18-carbon ether-linked phosphatidylcholines phosphatidylcholine synthesis. To examine this, cells were in- were unable to bypass the mutant's phenotype (Fig. 2B). The cubated with 32Pi at 33°C to label the cellular phospholipids to different efficiencies ofvarious molecular species (Table 1) may constant specific radioactivity (Table 2). Prelabeled cells were also be influenced by variation in the rate and extent of lipid then shifted to 40°C in the presence of 40 AM phosphatidyl- uptake. choline and 32Pi at the same specific radioactivity. After three Phospholipid Content of Mutant 58. To determine if lipid days, the cellular phospholipids were extracted and separated supplementation corrected the abnormal lipid content of mu- by two-dimensional thin-layer chromatography (6). As shown tant 58 (1, 2), we grew cells with and without added in Table 2, when the phospholipid compositions were deter- at 40°C for 2 days. The cells were harvested and analyzed for mined radiochemically, the phosphatidylcholine content in the protein and phospholipid content (6). Under these conditions, mutant (16.4%) was one-third of that in the parental cells mutant 58 contained 166 nmol of phospholipid per mg of pro- (55.4%). If, instead, the phospholipids were quantitated chem- tein, or about half as much as wild type (356 nmoVmg of pro- ically, phosphatidylcholine represented 35.1% of the phospho- tein). Addition of 40 ,tM egg phosphatidylcholine liposomes lipid in the mutant compared to 59.2% in the wild type. These partially corrected the abnormal phospholipid content of the results suggested that de novo synthesis ofphosphatidylcholine mutant (257 nmoVmg ofprotein) but did not affect the wild type from choline was still defective under bypass conditions and that (352 nmoVmg of protein). Two-dimensional thin-layer chro- at least some of the exogenous phospholipid may have been matography and quantitation of the cellular phospholipids (6, utilized 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 acids Relative cell density Phospholipid Egg No Strain 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 1.13 CHO-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 0.22 l-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 incubated for 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. Shown are 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. Downloaded by guest on October 3, 2021 Biochemistry: Esko et aL Proc. Natl. Acad. Sci. USA 79 (1982) 1701

Table 2. Phospholipid composition of phosphatidylcholine- Table 3. Phospholipid composition of mutant 58 grown with supplemented mutant and wild-type cells determined various supplements at 40'C chemically and by continuous labeling with 32Pi Phospholipid % of total phospholipid % of total phospholipid supplement PtdCho SPH PDME PtdEtn PtdIns PtdSer Other Parental CHO-K1 Mutant 58 None 27.0 19.9 - 29.9 10.5 7.1 5.6 Chemical Chemical l-PtdCho 46.3 8.0 - 27.6 7.6 6.5 4.0 Phospholipid 32P, 32Pi phosphorus f-PtdCho 35.6* 9.0 - 30.0 9.7 7.5 8.2 PtdCho 55.4 59.2 16.4 34.1 D-PtdCho 39.1* 8.8 - 31.6 9.7 6.5 4.4 SPH 9.1 7.5 6.1 11.3 PDME 20.1 10.8 23.2 24.8 10.2 6.7 4.2 PtdEtn 18.3 16.4 45.2 31.4 Multiple 100-mm-diameter dishes containing 15 ml of growth me- PtdIns 8.0 7.2 16.0 10.1 dium were each inoculated with 4 x 105 cells and incubated for 1 day PtdSer 5.2 5.2 8.2 5.7 at 330C. At this time 0.3-ml portions of 2 mM solutions of the various PtdGro 0.6 0.6 1.3 1.0 phospholipids were added to duplicate cultures, which were then Other 3.4 3.9 6.8 5.4 shifted to 400C. After 3 days the cells were harvested and extracted as described for Table 2. After two-dimensional thin-layer chromatog- Mutant and parental cells were incubated at 330C in growth medium raphy (6), the phospholipids were visualized with iodine vapor and supplemented with 32Pi (2 /Ci/ml; 1 Ci = 3.7 x 1010 becquerels) for quantitated as described (7, 12). Phosphatidyldimethylethanolamine several generations. Prelabeled cells were harvested and approxi- was well resolved from the other phospholipids, migratingjust above mately 2 x 105 wild-type and 3.5 x 105 mutant cells were added to phosphatidylserine (6). Systematic names for phosphatidylcholine iso- multiple 100-mm-diameter tissue culture dishes at 330C containing mers are given in the legend of Fig. 2. "Other" indicates lysophospha- 32Pi at the same specific radioactivity. After 1 day 0.1 ml of 6mM phos- tidylcholine, lysophosphatidylethanolamine, , and phatidylcholine liposomes was added to each culture, and these were . then shifted to 40'C. The cells were harvested after 3 days and ex- * D-Phosphatidylcholine and ,3-phosphatidylcholine did not separate tracted as described (6), but without carrier lipid. The chloroform ex- during thin-layer chromatography and comigrated with L-phos- tract was divided between two thin-layer chromatography plates, and phatidylcholine. the phospholipids were separated as described (6). Autoradiography revealed the positions of the phospholipids, which were then scraped off and quantitated by liquid scintillation spectrometry or perchloric independent of membrane must also be possible. acid digestion (7, 12). Each value is expressed as a percentage of the with mutant 58 indicate animal cells can total recovered material. PtdGro, ; other, lyso- Our studies that phosphatidylcholine, lysophosphatidylethanolamine, phosphatidic acid, utilize large amounts of exogenous phosphatidylcholine for and cardiolipin. membrane biogenesis when endogenous synthesis is impaired. The following simple scheme could account for this phenome- non. (i) Phosphatidylcholine (or related diacyl lipids) insert into also revealed that the CDP-choline synthetase was defective in the vitro under bypass conditions (not shown). Although there was the plasma membrane by fusion, a possibility supported by very little net uptake of exogenous phospholipid by wild-type observations of Pagano and others (25-28). Lysophosphatidyl- cells, the mutant incorporated enough ofthe added lipid to sat- choline may enter as a monomer, but in any case is rapidly re- some which are located in the isfy over halfofits phosphatidylcholine requirement. Complete acylated by acyltransferases, of restoration of the phosphatidylcholine content to the parental plasma membrane (16, 29). (ii) The excess phosphatidylcholine by mechanisms level was not observed, resulting in somewhat higher relative in the surface returns to the interior of the cell levels of the other phospholipids, especially phosphatidyletha- similar to those that bring it out, for instance by way ofendocytic nolamine (Table 2). vesicles (30, 31) or via phospholipid transfer proteins (23, 24). Addition of the other growth-promoting phospholipids also We speculate that "phospholipid sensors" exist that activate the resulted in partial restoration of the cellular phospholipid con- uptake of exogenous phosphatidylcholine when endogenous tent (Table 3). As shown, lysophosphatidylcholine was the most synthesis is limited. It is unlikely that the low density lipopro- effective supplement, permitting the accumulation of 46.3% tein system (32) is specifically involved, because phosphatidyl- phosphatidylcholine. Very little lysophosphatidylcholine was choline in lipoproteins is not readily available for phenotypic present in cells supplemented with this lipid, suggesting that bypass. this material was reacylated. The addition of phospha- Unlike earlier studies (25-28, 33), which were done with rapidly with tidyldimethylethanolamine to the mutant resulted in the ap- nongrowing cells or in the absence of serum, our results can pearance of this abnormal phospholipid (up to 23.2%). The un- mutant 58 demonstrate that exogenous phosphatidylcholine natural isomers of phosphatidylcholine also permitted the be taken up continuously and constitute at least one-half of the "phosphatidylcholine fraction" to accumulate in the mutant, but cellular pool. The incorporated lipid is functional as judged by the possibility ofrearrangement to the physiological isomer has cell growth, and a large portion of the supplement is incorpo- not been excluded. rated without hydrolysis of the phosphodiester linkage. How- ever, extensive remodeling of the fatty acids can occur (not shown) and may be obligatory, because ether-linked phospha- DISCUSSION tidylcholines do not support growth. The excellent phenotypic Most membrane phospholipids (16, 17) and many of the mem- bypass observed with monoacyl derivatives also shows that ly- brane proteins of eukaryotic cells (18, 19) originate on the en- solipid acyltransferases (16) can generate a large fraction of the doplasmic reticulum and are then translocated to other sites. cellular phosphatidylcholine. Phosphatidylcholine is formed from and CDP-cho- Limited incorporation of exogenous phospholipids has been line on the cytoplasmic surface of the described previously in microbial systems (34-37). Jones and (16, 17), followed by transmembrane equilibration and intra- Osborn demonstrated that phosphatidylserine can fuse with the cellular migration. Vesicular mechanisms (20-22) and perhaps outer membrane of Salmonella typhimurium deep rough mu- also exchange proteins (23, 24) participate in the latter process. tants and subsequently can be translocated to the inner mem- Although the net movement ofphospholipids and proteins oc- brane, where it is decarboxylated (34, 35). Escherichia coli is curs simultaneously in some instances, net movement oflipids able to take up some lysolipid (36, 37), but this phenomenon Downloaded by guest on October 3, 2021 1702 Biochemistry: Esko et aL Proc. Natl. Acad. Sci. USA 79 (1982)

has not yet been characterized extensively. The rapid functional 15. Glaser, M., Ferguson, K. A. & Vagelos, P. R. (1974) Proc. Nati uptake of phospholipids by animal cells has important impli- Acad. Sci. USA 71, 4072-4076. more mutants 16. Bell, R. M. & Coleman, R. A. (1980) Annu. Rev. Biochem. 49, cations for further genetic studies, because like 459-487. 58 shouldbe accessible without searching for conditional alleles. 17. Bell, R. M., Ballas, L. M. & Coleman, R. A. (1981)J. Lipid Res. 22, 391-403. 18. Blobel, G. (1980) Proc. NatL Acad. Sci. USA 77, 1496-1500. We thank Ms. M. Wermuth for her excellent assistance. This re- 19. De Pierre, J. W. & Ernster, L. (1977) Annu. Rev. Biochem. 46, search was supported in part by Grants AM 21722 and 1KO4-AM00584 201-262. from the National Institute ofArthritis, and Digestive Dis- 20. Palade, G. (1975) Science 189, 347-358. eases to C.R. H. R. The research described here forms part of a disser- 21. Rothman, J. E. & Fine, R. E. (1980) Proc. Nati Acad. Sci. USA tation by J.D.E. submitted to the University of Wisconsin at Madison 77, 780-784. in partial fulfillment of the requirements for the Ph. D. degree. 22. Novick, P., Field, C. & Schekman, R. (1980) Cell 21, 205-215. 23. Wirtz, K. W. A. (1974) Biochim. Biophys. Acta 344, 95-117. 24. Crain, R. C. & Zilversmit, D. B. (1980) Biochemistry 19, 1. Esko, J. D. & Raetz, C. R. H. (1980) Proc. Natl Acad. Sci. USA 1433-1439. 77, 5192-5196. 25. Pagano, R. E. & Weinstein, J. N. (1978) Annu. Rev. Biophys. 2. Esko, J. D., Wermuth, M. & Raetz, C. R. H. (1981) J. Biol Bioeng. 7, 435-468. Chem. 256, 7388-7393. 26. Struck, D. K. & Pagano, R. E. (1980) J. BioL Chem. 255, 3. Barnes, D. & Sato, G. (1980) Cell 22, 649-655. 5404-5410. 4. Ham, R. G. & McKeehan, W. L. (1979) Methods Enzymol 58, 27. Poste, G. & Papahadjopoulos, D. (1976) Proc. NatL Acad. Sci. 44-93. USA 73, 1603-1607. 5. Albott, E. C. (1966) J. Med. Lab. Tech. 23, 61-82. 28. Gregoriadis, G. (1978) Nature (London) 271, 112-113. 6. Esko, J. D. & Raetz, C. R. H. (1980) J. Biol. Chem. 255, 29. Colard, O., Bard, D., Bereziat, G. & Polonovski, J. (1980) 4474-4480. Biochim. Biophys. Acta 618, 88-97. 7. Nishijima, M. & Raetz, C. R. H. (1979) J. Biol. Chem. 254, 30. Goldstein, J. L., Anderson, R. W. G. & Brown, M. S. (1979) Na- 7838-7844. ture (London) 279, 679-685. 8. Singleton, W. S., Gray, M. S., Brown, M. L. & White, J. L. 31. Pearse, B. M. F. & Bretscher, M. S. (1981) Annu. Rev. Biochem. (1965) J. Am. Chemists Soc. 42, 53-61. 50, 85-101. 9. Wells, M. A. & Hanahan, D. J. (1969) Methods Enzymol. 14, 32. Goldstein, J. L. & Brown, M. S. (1977) Annu. Rev. Biochem. 46, 178-184. 897-930. 10. Esko, J. D. & Raetz, C. R. H. (1978) Proc. Natl. Acad. Sci. USA 33. Stein, Y. & Stein, 0. (1966) Biochim. Biophys. Acta 116, 95-107. 75, 1190-1193. 34. Jones, N. C. & Osborn, M. J. (1977) J. BioL Chem. 252, 11. Dulbecco, R. & Vogt, M. (1954)1. Exp. Med. 99, 167-182. 7398-7404. 12. Gerlach, E. & Deuticke, B. (1963) Biochem. Z. 337, 377-379. 35. Jones, N. C. & Osborn, M. J. (1977) J. BioL Chem. 252, 13. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. 7405-7412. (1951)J. Biol Chem. 193, 265-275. 36. Homma, H., Nishijima, M., Kobayashi, T., Okuyama, H. & No- 14. Litwin, J. (1973) in Tissue Culture, eds. Kruse, P. F. & Patter- jima, S. (1981) Biochim. Biophys. Acta 663, 1-13. son, M. K. (Academic, New York), pp. 188-192. 37. McIntyre, T. M. & Bell, R. M. (1978)J. Bacteriot 135, 215-226. Downloaded by guest on October 3, 2021