Pathways for the Incorporation of Exogenous Fatty Acids into

THEJOURNAL OF BIOLOGICAL. CHEMISTRY 0 1985 by The American Societyof Biological Chemists, Inc.

Vol. 260, No. 23, Issue of October 15,pp. 12720-12724.1985 Printed in U.S.A.

Pathways for the Incorporation of ExogenousFatty Acids into Phosphatidylethanolamine in Escherichia coli* (Received for publication, April 22,1985)

Charles 0. RockSfi and Suzanne Jackowskis From the Departments of Biochemistry, $St. Jude Children’sResearch Hospital and $The University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38101

Two distinct pathways for the incorporation ofex- ated intophospholipid in theabsence of acyl-CoA synthetase ogenous fatty acids into phospholipidswere identified activity (11). The demonstration of an acyl-ACP synthetase in Escherichia coli. The predominant routeoriginates (12) opened the possibility that incorporation of exogenous with the activation of fatty acids by acyl-CoAsynthe- fatty acids into phospholipid proceeds via acyl-CoA hydrolysis tase followed by the distribution of the acyl moieties by one or both of the acyl-CoA thioesterases (13,14) followed into all phospholipid classes via the sn-glycerol-3- by ligation of the fatty acids to ACP. However, reduction of phosphate acyltransferase reaction. This pathway was the intracellular ACP content using an [ACP] synthase mublocked in mutants (fadD)lacking acyl-CoA synthe- tant does not inhibit the incorporation of exogenous fatty tase activity. In fadD strains, exogenous fatty acids acids into phospholipid (15), suggesting that acyl-ACPs may were introduced exclusively intothe1-positionof phosphatidylethanolamine.This secondary routeis re- not be the sole intermediates. In addition to the two possible acyl donors, there are also lated to 1-position fatty acid turnover in phosphatitwo inner membrane acyltransferase systems that could be dylethanolamine and proceeds via the acyl-acyl carrierproteinsynthetase/2-acylglycerophosphoethanola- used for phospholipid biosynthesis from exogenous fatty acids. The glycerol-P acyltransferase system is responsible for the mine acyltransferase system. The turnover pathway exhibited apreferenceforsaturated fatty acids, sequential acylation of glycerol-P to phosphatidic acid (for whereas the acyl-CoA synthetase-dependent pathway review see Refs. 1and 2) whichin turnserves as theprecursor was less discriminating. Both pathways were inhibited of all the phospholipid classes in E. coli (for review see Ref. 16). The acyl-ACP end productsof fatty acid biosynthesis are inmutants (fadL)lacking the fatty acidpermease, fadL gene product translocates directly esterified by this reaction sequence i n vivo (17), but demonstrating that the exogenousfatty acids to an intracellular pool accessible both acyl-ACPs and acyl-CoAs function equally well as acyl to both synthetases. These data demonstrate that acyl- donors in vitro (18-22). Recently, the acyl-ACP synthetase CoA synthetase is not required forfatty acid transport (12) and 2-acyl-GPE acyltransferase (23, 24) reactions have in E. coli and that the metabolism of exogenousfatty been shown to represent a second pathway for the incorpoacids is segregated from the metabolism of acyl-acyl ration of acyl moieties into phospholipid (25). The flux of carrier proteins derived from fatty acid biosynthesis. fatty acids through this pathway is only a few per centof the flux through the glycerol-P acyltransferase (25), since this The metabolic pathway for the incorporation of exogenous system functions to esterify 2-acyl-GPE formed from the fatty acids into phospholipid in Escherichia coli is not estab- turnover of fatty acids in the 1-position of PE (25). Either lished (for reviews see Refs. 1 and 2), and this uncertainty acyl-ACP or fatty acids plus ATP:Mg+ are utilized by this complicates the interpretation of labeling experiments em- system, but acyl-CoAs are not substrates(23,25). The marked ploying extracellular fatty acids. The current “vectorial acyl- preference of the acyl-ACP synthetase reaction for saturated fatty acids (12,26) is consistent with the 1-position specificity ation” model for fatty acid uptake (3-5) considers the inner membrane fatty acid permease (6-8) to be coupled to the of this acyltransferase (23,25). The biochemical properties of acyl-ACP synthetase (27) and the channeling of fatty acids soluble acyl-CoA synthetase (9) and CoA thioesters to be the activated by this enzyme into the reacylation of 2-acyl-GPE intracellularproducts of thetransport system. Thus,the (25) led us to suspect that acyl-ACP synthetase may not inhibition of phospholipid biosynthesis from extracellular provide acyl groups to the glycerol-P acyltransferase system fatty acids in mutants (fadD) defective in acyl-CoA synthein uivo. Therefore, we have structurally characterized the tase activity (10) is interpreted to reflect a transport defect in these strains rather than a lack of acyl-CoA substrates for the phospholipids synthesized from exogenous fatty acids in the glycerol-P1acyltransferase. This idea is strengthened by the absence of acyl-CoA synthetase activity to determine if the finding that fatty acids generated intracellularly are incopor- residual synthesis found in these mutants is attributable to the ACP-dependent 1-position turnoverpathway. *This research was supported by National Institutes of Health Grant GM 28035, Cancer Center (CORE) Support Grant CA 21765 from the National Cancer Society, and American Lebanese Syrian Associated Charities. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. The abbreviations used are: glycerol-P, sn-glycerol-3-phosphate; 2-acyl-GPE, 2-acylglycerophosphoethanolamine;ACP, acyl carrier protein; PE, phosphatidylethanolamine; ppGpp, guanosine 5”diphosphate 3’-diphosphate.

EXPERIMENTAL PROCEDURES

Materials-Sources for supplies were: Amersham Corp.,[1-14C] palmitic acid (specific activity, 56 Ci/mol), [l-’4C]oleic acid (specific activity, 57 Ci/mol), and ACS scintillation mixture; Analtech, Inc., thin-layer chromatography plates; Sigma, Brij 58, oleic acid, Ophiophngus hannah venom, Bacillus cereus phospholipase C, and buffers; Serdary Research Laboratories Inc., phospholipid standards. All other biochemicals and solvents were reagent grade or better. Bacterial Strains-All bacterial strains used in this workwere derivatives of E. coli K12.Strain Ymel (supF58, supE44, mel-1, F+)is

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Fatty Acid Incorporation into PE a prototrophic wild-type strain (10). Strain K27 (fadD) lacks acylCoA synthetase activity (lo), strain K19 (fadE) is defective in @oxidation due to the lack of an electron transport flavoprotein (4), and both strains3008 (fadL) (6,28) and LS5283 (fadL:TnlO, fadR) (8) lack a functional fatty acid permease. All strains hadcomparable levels of acyl-ACP synthetase (27) and 2-acyl-GPE acyl-transferase (0.1 nmol/min/mg of protein) assayed using endogenous 2-acyl-GPE as theacyl acceptor (25). Strains were grownon glycerol/M9 medium consisting of M9 minimal salts (29) containing glycerol (0.4%), vitamin-free casein hydrolysate (0.1%), thiamine (O.OOl%), and Brij 58 (5 mg/ml). When added, the oleate concentration was 0.2%. All growth experiments were conducted at 37 “C, and the cell number was determined using a Klett-Summerson colorimeter calibrated by determining the number of colony-forming units/ml as a function of colorimeter (Klett) readings. The doubling time under these conditions was 1h for all strains in the presence or absence of oleate. Thin-layer Chromatographic Systems-Bacterial phospholipids were separatedon Silica Gel H layers developed with Solvent I (ch1oroform:methanol:acetic acid, 55:205, v/v), and neutral lipid classes were separated on Silica Gel G layers developed in Solvent I1 (hexane:diethyl ether:acetic acid, 50:501, v/v). The products of the phospholipase A2 digestions were resolved on Silica Gel H layers developed with Solvent I11 (chloroform:methanol:water,70303, v/ v). The distribution of radioactivity on the plates was determined by scraping 0.5-cm sections of the silica gel (28 fractions) into scintillation vials and adding 0.1 ml of water to deactivate the silica gel followedby 3 ml of scintillation counting solution. Recoveries of radioactivity were greater than 90%. Fatty Acid Uptake Experiments-Overnight cultures were inoculated into glycerol/M9 medium containing oleate and grown to a density of 5 X lo8 cells/ml. At this point, the cells were collected on a 0.45-pm filter, washed twice with warm growth medium without oleate, and resuspended in warm medium by shaking for 10 min. At the endof this time, 25 pCi of [l-14C]oleatewas added to theculture (88 p ~ )and , 0.5-ml samples were then removed at theindicated time intervals and placed into microcentrifuge tubes in an ice bath. The cells were pelleted using a Beckman Microfuge and washed twice with cold growth medium to remove exogenous [l-14C]oleate. The cells were then extracted by the method of Bligh and Dyer (30), and the total radioactive content of the lipid extract was determined. The amount of incorporation into phospholipids was determined by quantitating the distribution of radioactivity among the phospholipid classes by thin-layer chromatography (Solvent I). Positional Analysis-Labeled P E was isolated by preparative thinlayer chromatography using Solvent I. The silica gel containing P E was removed from the plate, transferred to a vial containing 0.5 ml of 50 mM Tris-HC1, pH 7.5, 1 mM calcium chloride, and 0.4 mg/ml 0. hannah venom (31), and then slowly stirred for 3 h at 23 “C (32). Digestion with the venom phospholipase A2 was complete. The silica gel was removed from the aqueous solution with a fritted funnel and washed with several portions of methanol to completely remove the lipids. The combined filtrates were evaporated to dryness and resuspended in a small volume of ch1oroform:methanol (l:l,v/v), and the fatty acid was resolved from the 1-acyl-GPE by thin-layer chromatography using Solvent 111. The acyl group compositions of the fatty acid and 1-acyl-GPE thin-layer fractions were determined by gasliquid Chromatography (5% DEGS-PS column operated isothermally a t 165 “C), and these results were consistent with the known asymmetric distribution of fatty acids between the 1-and 2-positions of E. coli P E (33).

RESULTS

Effect of fad Mutations on the Incorporation of [1-14C]Oleate into Phospholipid-The effects of the fadD, fadE, and fadL mutations onthe amount of exogenous [l-14C]oleateincoporated into thephospholipid fraction are summarized in Table I. Strain K19 (fadE) is unable to degrade fatty acids via the /+oxidation pathway (4), and blocking this pathway significantly increased the transferof oleate into phospholipid (Table I). Strain K27 (fadD) lacks acyl-CoA synthetase activity (lo), and the amountof oleate assimilated intophospholipid was drastically decreased in this mutant compared to both the fadE mutant and thewild-type strain (Table I). Although smallerin magnitude than the incorporation observed in

12721

TABLEI Effect of fatty acid degradation (fad) mutations on the incorporation of exogenous [l -14C]oleic acid into E. coli phospholipid [l-l4C]0leic acid incorporation into the phospholipid fraction of the indicated strains was measured as described under “Experimental Procedures.” nmol/l5 min/l08 cells

Genotype Strain

Ymel K19 K27 LS5283 3008

fad+ fadE fadD fadL:TnlO fadL

1.776 5.303 0.042 0.004 0.006

strains Ymel and K19, the labeling of the phospholipid pool in strain K27 was easily measurable. The fadL gene encodes an inner membrane component of the fatty acid transport system ( 6 4 , and incorporation of extracellular oleate into phospholipid in the two fadL mutants tested was even more severely depressed than in strain K27 (fadD) (Table I ) . The important point to glean from these data is that blocking fatty acid uptake into the cell (fadL lesion) was much more effective in decreasing the incorporation of exogenous oleic acid into phospholipid than blocking fatty acid activation (fadD lesion) (Table I).

Phospholipid Classes Labeled by Exogenous [l-14C]Oleatein fad Mutants-In order to determine if the fad mutations all affect the same process, the lipid products of oleate uptake were quantitated by thin-layer chromatography (Figs. 1 and 2). Strain Ymel distributed oleate into all three major phospholipid classes in the same ratio as normally present in unlabeled cells (Fig. 1B). This pattern of incorporation was the same in strain K19 (fadE) (not shown), and the small amount of label taken up by strain LS5283 (fadL) was also distributed among PE, phosphatidylglycerol, and cardiolipin (Fig. IC). Incontrast, [l-14C]oleatespecifically labeled PE in strain K27 (fadD) (Fig. 1A). The labeling pattern in the neutral lipid fraction also fell into two groups (Fig. 2). In addition to labeled fatty acid, strains Ymel (Fig.2B), LS5283 (fadL),and K19 (fadE) (not shown) all contained detectable levels of labeled 1,2-diacylglycerol. 1,2-Diacylglycerols were not found in the fadD mutant (Fig. 2A). These data indicate that there were two pathways for oleate incorporation into phospholipid. The predominant pathway requires the fadD gene product and results in the labeling of all phospholipid classes and 1,2-diacylglycerols. The second pathway is independent of acyl-CoA synthetase activity, and the exogenous oleate is specifically transferred to the major phospholipid class, PE. Thus, the residual labeling observed in fadD mutants is not due to the incomplete inhibition of the incorporation pathway present in the wild-type strain, but rather reveals the presence of a separate, less active process. Positional Specificity of Oleate Incorporation i n fad Mutants-The positional distribution of [ l-14C]oleate incorporated into P E was determined using the wild-type strain and all three f a d mutants. [1-14C]Oleatewas localized in the 2position of PE in strain Ymel (Fig. 3B).This 2-position localization was also found for PE formed in strainK19 (fadE) (83% 2-position). In contrast, extracellular oleate was exclusively introduced into the 1-position of PE in strain K27 (fadD) (Fig. 3A). Thus, thefadD-independent route for phospholipid synthesis is fundamentally different from the fadDdependent pathway in that it isspecific for the 1-position of P E (Fig. 3). Acyl Group Specificity of the fadD-independent Pathway[l-14C]Oleatewas used in allthe previous experiments because it had a high selectivity for the 2-position of phospholipids

12722

Fatty Acid Incorporation into PE 6.4 10

h

4.8

X

5

3.2

0

I .6

PE (78 X)

4.8 0

”

6.4 4.8

3.6 X

5

5

3.2

2.4

0

1.6

I .2

0.25

0.75

0.5

Relative Mobility

3.2

FIG.2. Neutral lipids labeledby [l-’4C]oleatein strain K27 ( A ) and strain Ymel (23). Cells were labeled with [1-“Cloleate for 1 h, and the lipids were extracted and analyzed by thin-layer chromatography (Solvent 11) as describedunder “Experimental Procedures.” DAG, 1,2-diacylglycerol;FA, fatty acid.

Ir)

h

2.4

X

5

1.6

V

0.8

-

RelativeMobility

FIG.1. Incorporation of exogenous [l-14C]oleateinto the of strain K27 ( A ) ,strain Ymel ( B ) , three phospholipid classes and strain LS5283 (C). Cells were labeled with [1-“C]oleate for 1 h, and the phospholipids were extracted and analyzed by thin-layer chromatography (Solvent I) as described under “Experimental Procedures.” Lyso-PE, acylglycerophosphoethanolamine; PG, phosphatidylglycerol; CL, cardiolipin; FA, fatty acid.

and was the fatty acid of choice in previous studies (4-8, 10). Since all of the oleate was incorporated into the 1-position of PE in the fudD mutant (Fig. 3) and this position of the glycerol backbone is primarily occupied by palmitate in vivo (33),we compared the rateof [1-’*C]palmitateto [l-14C]oleate incorporation into P E in both the wild-type and acyl-CoA synthetase negative strains (Fig. 4). In strain Ymel, oleate was transferred to phospholipid at a higher rate than palmitate (Fig. 4) in agreement with earlier observations on the acyl chain specificity of fatty acid uptake (7, 10). In contrast, palmitate was incorporated into PE at approximately five times the rate of oleate in strain K27 (fudD). This specificity for the saturated fatty acid is consistent with the 1-position destination of the acyl moieties in strain K27.

0.6

FA 14x1

P X

n

FAIB8Xl

3.6 2.7 1.8

n

09

0

0.25

05

0.75

0

Relolive Mobilily

FIG.3. Positional specificityof exogenous [l-’4C]oleateincorporation into PE of strain K27 ( A ) and strain Ymel ( B ) . Cells were labeled with [l-14C]oleatefor 1h, and the PE fraction was purified and digested with phospholipase Az as described under “Experimental Procedures.”The distribution of label between the 1-and 2-positions was determined by thin-layer chromatography (Solvent 111). FA, fatty acid.

ent routes are blocked in fudD mutants which lack acyl-CoA synthetase activity (4,10) (Table I and Figs. 1 and 3). The second pathway for fatty acid utilization is related to phosDISCUSSION pholipid turnover (25), and the acyl groups are transferred Our results support the scheme for exogenous fatty acid exclusively into the 1-position of pre-existing PE (25) (Fig. metabolism presented in Fig. 5. Extracellular fatty acids are 3). Incorporation by this route is catalyzed by 8-acyl-GPE first translocated to the intracellular compartment by the acyltransferase (23-25) that ligates the fatty acid to a bound fudL gene product localized in the innermembrane (8).Acyl- ACP subunit priorto transfer to the1-position of 2-acyl-GPE CoA synthetase catalyzes the first step in the CoA-dependent (25). Although ACP thioesters are the intermediates in this pathways for utilization of exogenous fatty acids. The CoA pathway, the acyl moieties are specifically channeled intothe thioesters can be either degraded by the P-oxidation pathway 1-position of PE and are notavailable to theenzymes of fatty (4, 5 , 10) or incorporated into phospholipids (Figs. 1 and 4) acid biosynthesis or the glycerol-P acyltransferase. Thus, the by the glycerol-P acyltransferase system. Both CoA-depend- metabolism of extracellular fatty acidsis segregated from the

Incorporation into Acid Fatty

15 30 45 60

15 30 45 60

Minutes FIG. 4. A comparison of [l-"C]palmitate and [l-14C]oleate incorporation into the PE fraction of strain K 2 7 ( A )and strain Ymel ( B ) .Uptake of labeled fatty acid into P E was determined as described under"Experimental Procedures." 16.9, [l-'4C]palmitic acid; 18:1, [l-14C]oleicacid. 2-Acvl-GPE

Pid Etn

4 4 4

in I lout

I

Fatty Acid

I

-

-Fatty Acid

Acyl-CoA I

Glycerol-P

I/

B -oxidation FIG. 5. Pathwaysfortheincorporation of extracellular fatty acids into PE in E. coli. Exogenous fatty acids are first translocated across the inner membrane by the fudL gene product. The intracellular fatty acids can then be activated by either acyl-CoA or acyl-ACP synthetase. Acyl-CoAs serve as substrates for the glycerol-P acyltransferase system or the enzymes of P-oxidation. AcylACPs are intermediates in the incorporation of fatty acids into the 1-position of P E (PtdEtn).The box around the acyl-ACP pool indicates that these acyl-ACPs are segregated from the acyl-ACP pool derived from fatty acid biosynthesis and cannot be elongated or utilized by the glycerol-P acyltransferase. The fate of the fatty acids released from the 1-position of P E is unknown (?). PtdOH, phosphatidic acid.

metabolism of acyl-ACPs derived from fatty acid biosynthesis as was first proposed by Silbert et ul. (34). The segregation of the de novo and exogenous pathways for the incorporation of fatty acids into phospholipids clarifies the role of the glycerol-P acyltransferase in the regulation of phospholipid synthesis following nutritional stress. Several laboratories report that phospholipid synthesis decreases following the starvation of rel' but not relA strains, and considerable evidence has accumulated that ppGpp is the effector of lipid synthesis in vivo (for review see Refs. 1 and 2). A regulatory role for the glycerol-P acyltransferase was suggested by Merlie and Pizer (35) who reported that the acyltransferase is inhibited by ppGpp in vitro. However, the need for control at the glycerol-P acyltransferasestep was not supported by either in vivo experiments that demonstrated a direct effect of the relA gene on fatty acid biosynthesis (36, 37) or in vitro studies that showed ppGpp does not inhibit the glycerol-P acyltransferase when ACP thioesters are the acyl donors (19,ZO). A passive role for the glycerol-P acyltransferase in the stringentresponse is not consistent with the demonstration of relA-mediated control of phospholipid synthesis in strains requiring exogenous fatty acids for growth (38),but

PE

12723

at thetime the interpretation of this experiment was unclear because the acyl donor question had not been resolved (2). Evaluation of these experiments in light of the metabolic scheme in Fig. 5 suggests that utilization of acyl-CoAs bythe glycerol-P acyltransferase isrestricted during nutritional stress causing a reduction in phospholipid synthesis from exogenous fatty acids. Thus, there aretwo sites for the stringent control of phospholipid synthesis in E. coli: regulation at an early step in fatty acid biosynthesis controls phospholipid synthesis via the de novo pathway, and regulation at the glycerol-P acyltransferase step modulates phospholipid synthesis from exogenous fatty acids. Our results provide the first clear demonstration that acylCoA synthetase is not required for the transportof exogenous fatty acids. In the absence of acyl-CoA synthetase activity, incorporation of exogenous fatty acids into the 1-position of PE is easily measurable (Table I and Figs. 1 and 3), and accumulation of fatty acids by this route is abolished in mutants (fadL)lacking the fattyacid permease (Table I). We observed the same marked inhibition of fatty acid incorporation into phospholipid in acyl-CoA synthetase (fudD) (Table I) mutants as reported by previous investigators (4,5, 7, 10). This low rate of uptake in fudD mutants (4, 5, 7, 10) is exacerbated by the use of [l-14C]oleate, which yields a low estimate of the acyl-CoA synthetase-independent incorporation due to thesubstrate specificity of the alternate pathway for saturated fatty acids (Fig. 4). Therefore, the defect in phospholipid biosynthesis from extracellular fatty acids in fadD mutants reflects the lack of acyl-CoA substrates for the glycerol-P acyltransferase system ratherthan atransport defect. The metabolic scheme in Fig. 5 provides a rationale for all of the known enzyme activities involved in fatty acid activation and metabolism except for the two thioesterases. There are two of these enzymes in E. coli that are very active on acyl-CoA substrates (13, 14),butnot acyl-ACP thioesters (39). One of the interesting aspects of exogenous fatty acid metabolism is thatuptake is tightly coupled to utilization (5, 10) (Table I),and neither free fatty acids nor acyl-CoAs accumulate in the cell when utilization of these species is blocked (4,5). Itis possible that thethioesterases may participate in thisphenomenon by converting unutilized acyl-CoAs back to fatty acids to prevent the accumulation of acyl-CoA and inhibit further uptakeof exogenous fatty acids. Acknowledgments-We thank SusanPickren for her excellent technical assistance and William D. Nunn for strain LS5283. REFERENCES 1. Cronan, J. E., Jr. (1978) Annu. Reu. Biochem. 47, 163-189 2. Rock, C. O., and Cronan, J. E., Jr. (1982) Curr. Top. Membr. Trunsp. 17,207-233 3. Black, P. N., Kianian, S. F., DiRusso, C. C., and Nunn, W. D. (1985) J. Biol. Chem. 260, 1780-1789 4. Klein, K., Steinberg, R., Fiethen, B., and Overath, P. (1971) Eur. J. Biochem. 19,442-450 5. Frerman, F. E., and Bennett, W. (1973) Arch. Biochem. Biophys. 159,434-443 6. Nunn, W. D., and Simons, R. W. (1978) Proc. Natl. Acud. Sci. U. S. A. 75,3377-3381 7. Maloy, S. R., Ginsburg, C. L., Simons, R. W., and Nunn, W. D. (1981) J. Bwl. Chem. 256, 3735-3742 8. Ginsburg, C. L., Black, P. N., and Nunn, W. D. (1984) J. Biol. Chem. 259,8437-8443 9. Kameda, K., and Nunn, W. D. (1981) J. Biol. Chem. 256,57025707 10. Overath, P., Pauli, G., and Schairer, H.U. (1969)Eur. J.Bbchem. 7,559-574

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