Plant Physiol. (1998) 116: 251-258

Fatty Acid Elongation Is Independent of Acyl-Coenzyme A Synthetase Activities in Leek and Brassica napus¹

Alenka Hlousek-Radojcic, Kimberly J. Evenson², Jan G. Jaworski, and Dusty Post-Beittenmiller^*

Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73402 (A.H.-R., K.J.E., D.P.-B.); and Chemistry Department, Miami University, Oxford, Ohio 45056 (J.G.J.)

	ABSTRACT

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In both animal and plant acyl elongation systems, it has been proposed that fatty acids are first activated to acyl-coenzyme A (CoA) before their elongation, and that the ATP dependence of fatty acid elongation is evidence of acyl-CoA synthetase involvement. However, because CoA is not supplied in standard fatty acid elongation assays, it is not clear if CoA-dependent acyl-CoA synthetase activity can provide levels of acyl-CoAs necessary to support typical rates of fatty acid elongation. Therefore, we examined the role of acyl-CoA synthetase in providing the primer for acyl elongation in leek (Allium porrum L.) epidermal microsomes and Brassica napus L. cv Reston oil bodies. As presented here, fatty acid elongation was independent of CoA and proceeded at maximum rates with CoA-free preparations of malonyl-CoA. We also showed that stearic acid ([1-¹⁴C]18:0)-CoA was synthesized from [1-¹⁴C]18:0 in the presence of CoA-free malonyl-CoA or acetyl-CoA, and that [1-¹⁴C]18:0-CoA synthesis under these conditions was ATP dependent. Furthermore, the appearance of [1-¹⁴C]18:0 in the acyl-CoA fraction was simultaneous with its appearance in phosphatidylcholine. These data, together with the results of a previous study (A. Hlousek-Radojcic, H. Imai, J.G. Jaworski [1995] Plant J 8: 803-809) showing that exogenous [¹⁴C]acyl-CoAs are diluted by a relatively large endogenous pool before they are elongated, strongly indicated that acyl-CoA synthetase did not play a direct role in fatty acid elongation, and that phosphatidylcholine or another glycerolipid was a more likely source of elongation primers than acyl-CoAs.

	INTRODUCTION

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Very-long-chain fatty acids (>18 carbons) are found in the glucocerebrosides of plant plasma membranes (Cahoon and Lynch, 1991), in the storage lipids of many oil seed species (Harwood, 1980), and as precursors of plant epicuticular waxes (Post-Beittenmiller, 1996). These fatty acids are synthesized by successive 2-carbon additions donated from malonyl-CoA. In each cycle of elongation, an acyl-primer condenses with malonyl-CoA; this step is followed by a reduction of the 3-ketoacyl-CoA, dehydration of the hydroxyacyl-CoA, and reduction of the enoyl-CoA to the saturated and fully reduced acyl-CoA. These reactions are analogous to those of de novo fatty acid biosynthesis (Ohlrogge and Browse, 1995). However, unlike the soluble, stroma-localized enzymes of fatty acid synthase, the enzymes of acyl elongation are membrane bound and generally thought to be associated with the ER and possibly the plasma membranes in vegetative cells (von Wettstein-Knowles, 1993) or with the ER (Agrawal et al., 1984) and oil bodies of developing seeds (Imai et al., 1995). Furthermore, the intermediates of acyl elongation are esterified to CoA rather than to the acyl carrier protein cofactor of fatty acid synthase (Fehling and Mukherjee, 1991).

Studies on acyl elongation in plants and animals indicate that, in general, the properties of acyl elongation in plants and animals are quite similar (for review, see Cinti et al., 1992; Cassagne et al., 1994). Both plant and animal elongases require malonyl-CoA and NAD(P)H and use a variety of primers with varied requirements for ATP. For example, relatively high rates of acyl elongation can be achieved without supplied primer, i.e. by ATP-dependent elongation of endogenous substrates. This implies that there is a sufficient endogenous primer pool, but that ATP is necessary to use it.

The extent of the ATP requirement for elongation of exogenous acyl-CoAs apparently depends on the system studied. In Brassica napus seeds both microsomes and oil body fractions have acyl-CoA elongation activity. The ATP-independent activity is found almost exclusively in the microsomal fraction (Fuhrmann et al., 1994), whereas the ATP-dependent activity is found in both microsomes and oil body fractions (Whitfield et al., 1993; Hlousek-Radojcic et al., 1995; Imai et al., 1995). Oil bodies prepared by Suc gradients showed minimal levels of the ER marker enzyme cholinephosphotransferase (Whitfield et al., 1993), whereas oil bodies prepared by differential centrifugation showed moderate levels (Imai et al., 1995). However, in leek (Allium porrum L.) microsomes, acyl-CoAs are elongated in the absence of ATP, although 1 mm ATP stimulates elongation 7- to 10-fold (Evenson and Post-Beittenmiller, 1995). This difference between leek microsomes and B. napus oil bodies may reflect the size of the primer pools in oil bodies and in microsomes. In some cases, high concentrations (100 µm) of supplied acyl-CoAs can apparently eliminate the need for ATP (Lassner et al., 1996).

The exact nature of the endogenous primer has not been carefully studied and its identity has lately been questioned in both animal (Cinti et al., 1992) and plant systems (Evenson and Post-Beittenmiller, 1995; Hlousek-Radojcic et al., 1995). Microsomal preparations from plants and animals will synthesize very-long-chain fatty acids in the presence of ATP and malonyl-CoA from endogenous primer pools, exogenous free fatty acids, or acyl-CoAs. Specifically, elongation of fatty acids has been reported in microsomes isolated from pea (Bolton and Harwood, 1977), leek (Evenson and Post-Beittenmiller, 1995), and animals (see Cinti et al., 1992, and refs. therein). This elongase activity has been attributed to the activation of the supplied fatty acids (or endogenous fatty acids in which no primer is supplied) to acyl-CoAs by an endogenous ACS.

ACS is an ATP-dependent enzyme that has activity that has been reported in microsomes prepared from leek epidermis (Lessire and Cassagne, 1979), oil seeds (Ichihara et al., 1993), and rat liver (Guchait et al., 1966). Thus, the ATP requirement for endogenous acyl elongation and elongation of supplied fatty acids has been cited as evidence for ACS involvement (Nugteren, 1965). However, ACS activity is also CoA dependent, and under standard acyl elongase assay conditions no CoA is supplied. If CoA is not supplied to safflower (Ichihara et al., 1993) or leek microsomes (Lessire and Cassagne, 1979), no ACS activity is detected. Therefore, the endogenous CoA level is apparently insufficient for ACS activity under standard elongase assay conditions. Similarly, ACS activity in rat liver microsomes does not correlate with acyl elongase activity rates in the absence of exogenously supplied CoA, fatty acid, and Mg²⁺ (Cinti et al., 1992). Nugteren (1965) suggested that small amounts of CoA may be present in malonyl-CoA preparations, which would presumably be sufficient to support ACS activity under elongase assay conditions. However, this hypothesis had not been tested before the studies reported here. To elucidate the relationship of ACS activity and fatty acid elongation, we examined both ACS and elongase activities in the same preparations of leek microsomes or B. napus oil bodies under conditions in which maximum rates of fatty acid elongation were achieved.

	MATERIALS AND METHODS

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Substrates and Reagents

Plant Material, Enzyme Assays, and Product Analyses

All enzyme assays were conducted between two and four times with essentially the same qualitative results. The results of a single experiment are shown in each figure. Because of variations among microsomal or oil body preparations, mean values were not reported. ACS activity was assayed according to Groot et al. (1974) with the following modifications: the 25-µL assay mixture contained 80 mm Hepes-KOH, pH 7.2, 1 mm ATP, 125 mm MgCl₂, 0.5 mm NADPH, and 15 µm [1-¹⁴C]oleate (NH₄⁺ salt) with B. napus oil bodies or [1-¹⁴C]stearate (NH₄⁺ salt) with leek microsomes. Oleate and stearate were used because monounsaturated and saturated C-18 primers are the preferred substrates for B. napus oil bodies and leek microsome elongases, respectively. The concentrations of CoA used were as indicated in ``Results'' or in figure legends, and whenever indicated, 100 µm malonyl-CoA or 100 µm acetyl-CoA was added. Assays (2 and 30 min) were started with the addition of B. napus oil bodies (2-3 µg of protein) or leek microsomes (24-38 µg of protein), and stopped with an equal volume of 100 mm acetic acid and 4 volumes of water. Unreacted fatty acids were removed by four successive extractions into 800 µL of diethyl ether. The diethyl ether remaining on the surface of the aqueous phase was removed under a stream of nitrogen for 3 min. Acyl-CoAs and polar lipids were then extracted into n-butanol (3 × 100 µL). The butanol phases were pooled and the volume was reduced under a stream of nitrogen. Acyl-CoA and polar lipids were separated on Silica Gel 60 A plates (Whatman) by first developing plates to the full length in chloroform:methanol:glacial acetic acid:water (85:15:10:3, v/v) and then developing to one-third of the length in n-butanol:glacial acetic acid:water (5:2:3, v/v). Quantification of radioactivity was carried out as described above.

Acyl chain lengths of acyl-CoAs, PCs, and free fatty acid fractions were analyzed by RPTLC. Briefly, acyl-CoAs and PCs were eluted from scraped silica samples with n-butanol:glacial acetic acid:water (5:2:3, v/v), and free fatty acids were eluted with chloroform:methanol (2:1, v/v). Solvent volumes were reduced under nitrogen and lipids were transmethylated according to the procedure described by Sattler et al. (1996). Fatty acid methyl esters were prepared from the lipid fractions, separated by RPTLC, and quantified as described above.

Malonyl-CoA and [1-¹⁴C]malonyl-CoA were purified (>99.8% CoA free) by HPLC (HP 1100, Hewlett-Packard) equipped with a diode array detector set to 260 nm, using a reverse-phase column (Microsorb-MV C18, Rainin, Woburn, MA) at a flow rate of 8 mL/min with an isocratic elution using 18% acetonitrile in 50 mm KPO₄ buffer, pH 5.2, for 20 min. Under these conditions, malonyl-CoA eluted at 9 min and CoASH eluted at 11 min. The column was then washed and reequilibrated by increasing the acetonitrile from 3 to 30% in 50 mm KPO₄ buffer, pH 5.2, over 1 min, followed by a 5-min wash with 30% acetonitrile, 50 mm KPO₄ buffer, pH 5.2, and finally returned to 3% acetonitrile, 50 mm KPO₄ over 1 min and held for an additional 14 min. Fractions that contained malonyl-CoA were collected, pooled, diluted 1:10 with water, and desalted on SepPak C₁₈ minicartridges. Briefly, SepPak cartridges were prepared by washing (5 mL each) with decreasing concentrations of methanol (100, 75, 50, and 25%, v/v), followed by 1 mL of water, and, finally, 5 mL of 10 mm acetic acid. Samples were loaded by gravity and the procedure was carried out at 4°C. The column was washed with 5 mL of 10 mm acetic acid followed by 1 mL of water. Malonyl-CoA was eluted with 20% acetonitrile in 1-mL fractions. Peak fractions were identified either by radioactivity or by A₂₆₀, as applicable, and were combined and dried in a Speed-Vac (Savant Instruments, Farmingdale, NY). Dried malonyl-CoA was dissolved in water (adjusted to pH 3.0 with acetic acid) to 8 to 10 mm and stored at 20°C until used. Malonyl-CoA solutions remained free of CoA for at least 1 month when prepared and stored in this manner. The purity of the malonyl-CoA was analyzed using a reverse-phase C₁₈ column at a flow rate of 1 mL/min and the following solvent system: a 5-min isocratic elution with 3% acetonitrile in 50 mm KPO₄ buffer, pH 5.2, followed by a 40-min gradient from 3 to 30% acetonitrile. Under these conditions malonyl-CoA eluted at 10.3 min and CoASH eluted at 11.9 min.

	RESULTS AND DISCUSSION

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B. napus Oil Bodies Will Effectively Elongate Fatty Acids in the Absence of CoA

View larger version (14K): [in this window] [in a new window]   Figure 1. Comparison of fatty acid and acyl-CoA primers for acyl elongation in B. napus oil bodies. Either 15 µm [1-14C]18:1 () or 15 µm [1-14C]18:1-CoA () was provided as the primer in acyl elongation assays. At the indicated times, assays were stopped and methyl esters were prepared from the saponified fatty acids. The elongated products were separated from the starting substrates by RPTLC and the radioactivity was quantified using a PhosphorImager and Image Quant (Molecular Dynamics).

ACS and Fatty Acid Elongation Activities

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To assess the CoA requirements for ACS and acyl elongation activities, a series of CoA concentrations, from 0 to 50 µm, was used with leek microsomes and B. napus oil bodies. As shown in Figure 2, in the absence of CoASH, ACS activities were very low in both the leek microsomes and the B. napus oil bodies. In leek microsomes (Fig. 2, left), ACS activity increased 38-fold with increasing concentrations of CoASH, indicating a clear dependence of ACS activity on supplied CoA. In contrast, elongase activity in the same preparations was unaffected by increasing CoA concentrations. Fatty acid elongation rates were similar (2.2-2.6 nmol h¹ mg¹ protein) from 0 to 50 µm CoA. In B. napus oil bodies, ACS activity likewise was dependent on supplied CoA, whereas elongase activity was largely unaffected by CoA (Fig. 2, right). These data together indicated that microsomal and oil body preparations did not contain sufficient levels of CoA to support ACS activity, yet they supported high rates of fatty acid elongation. Furthermore, in B. napus oil bodies, ACS activity was 3- to 11-fold higher than fatty acid elongation activity when CoA was supplied at greater than 3 µm. Therefore, if ACS provides substrate for acyl elongation, a stimulation of acyl elongation would be expected unless the level of primer was saturating. However, the concentration of [1-¹⁴C]18:0 (15 µm) was below saturating levels of the primer (Evenson and Post-Beittenmiller, 1995); therefore, we concluded that fatty acid elongation rates should have responded to increasing synthesis of acyl-CoAs if acyl-CoAs rather than fatty acids were a more direct substrate. Thus, the results clearly indicated that elongation rates were unaffected by increased ACS activities.

View larger version (24K): [in this window] [in a new window]   Figure 2. Effect of increasing CoA concentrations on fatty acid elongation and ACS activities in leek microsomes (left) and B. napus oil bodies (right). Standard ACS assays (open bars), using [1-14C]18:0 with leek microsomes and [1-14C]18:1 with B. napus oil bodies, or elongase assays (solid bars), using [1-14C]18:0 with leek microsomes and [2-14C]malonyl-CoA with B. napus oil bodies, were carried out with increasing CoA concentrations. Acyl-CoAs and fatty acid methyl esters were prepared and analyzed as described in ``Materials and Methods'' and the legend to Figure 1.

HPLC Purification of Malonyl-CoA

View larger version (12K): [in this window] [in a new window]   Figure 3. Essentially pure malonyl-CoA (>99.8%) was obtained from commercial preparations of malonyl-CoA after separation by HPLC chromatography using a C18 reverse-phase column (top). Commercial preparations of malonyl-CoA were shown to contain 1 to 3% CoASH and smaller amounts of oxidized CoA (bottom). mAU, Milliabsorbance unit.

CoA-Free Malonyl-CoA Supported Fatty Acid Elongation

View larger version (14K): [in this window] [in a new window]   Figure 4. Comparison of fatty acid elongation activities in leek microsomes using [1-14C]18:0 as the acyl primer and HPLC-purified malonyl-CoA (), commercial malonyl-CoA (), or HPLC-purified malonyl-CoA supplemented with 3 µm CoASH (). Assays were carried out and elongated products analyzed as described in Figure 1. prot, Protein.

We also considered whether hydrolysis of malonyl-CoA during the elongation reaction could provide sufficient CoA for activation of fatty acids. Such hydrolysis would presumably be independent of the purity of the malonyl-CoA. However, the data presented here (Fig. 4) appear inconsistent with malonyl-CoA hydrolysis being a source of CoA, unless this hydrolysis was very rapid. At 1 and 3 min the extent of elongation with CoA-free malonyl-CoA was equal to or greater than the extent with commercial or CoA-supplemented malonyl-CoA. Thus, if this were a CoA-dependent elongation, malonyl-CoA hydrolysis would need to occur at rates greater than 2 to 3% per min to provide adequate CoA for the earliest time points. To evaluate the possibility of rapid hydrolysis of malonyl-CoA under elongase assay conditions, we examined the fatty acid elongation reactions for the presence of CoA and loss of malonyl-CoA at different time periods. Using HPLC analysis, we were unable to detect any CoASH, oxidized CoA, or rapid loss of malonyl-CoA. We cannot rule out the possibility that any hydrolyzed CoA was rapidly reesterified to a fatty acid, although this is improbable because the levels required for significant ACS activity (Fig. 2) would be easily detected. Thus, if hydrolysis occurred during the elongase assay, it was insufficient to contribute substantially to the elongation activity (considering that HPLC-purified malonyl-CoA performed at least as well as malonyl-CoA supplemented with 3 µm CoA).

18:0-CoA Was Synthesized during Elongation Reactions

View larger version (39K): [in this window] [in a new window]   Figure 5. Effect of increasing CoA concentrations on [1-14C]18:0-CoA formation in the presence (open bars) or absence (solid bars) of HPLC-purified malonyl-CoA in leek microsomes. Fatty acid elongation assays were carried out in the presence (100 µm) or absence of malonyl-CoA; reactions were stopped after 30 min, and the acyl-CoAs were extracted into butanol and separated by TLC as in Figure 1.

The CoA-independent synthesis of 18:0-CoA observed in the presence of malonyl-CoA was not expected, and to our knowledge, it has not been previously reported. Consequently, we examined the cofactor requirements for this reaction using leek epidermal microsomes to determine if this synthesis was ATP dependent and if acetyl-CoA could substitute for malonyl-CoA (Fig. 6). Acyl-CoA synthesis occurred with both acetyl-CoA and malonyl-CoA, but only in the presence of ATP. The ATP requirement and the involvement of a free fatty acid suggest that the acyl-CoA synthesis was not a simple acyl-exchange reaction. Both the malonyl-CoA and the acetyl-CoA were analyzed by HPLC and were essentially CoA free (data not shown). Thus, the acyl-CoA synthesis could not be attributed to a large contamination of CoA, which is what would be required because this activity was 1.5 to 3.6 times greater than ACS activity with 3 µm CoA.

View larger version (44K): [in this window] [in a new window]   Figure 6. Effect of malonyl-CoA (MCoA), acetyl-CoA (AcCoA), ATP, and CoA on acyl-CoA synthesis in leek microsomes. Assays were carried out under standard elongase assay conditions using [1-14C]18:0 and indicated cofactors (100 µm acetyl-CoA, 100 µm malonyl-CoA, 1 mm ATP, 3 µm CoA). The lipids were extracted and separated by TLC and the radioactive acyl-CoA and PC bands were quantified using a PhosphorImager and Image Quant (Molecular Dynamics) as described in Figure 1.

Does 18:0-CoA Synthesis Correlate with Fatty Acid Elongation?

View larger version (20K): [in this window] [in a new window]   Figure 7. Progress curve using leek microsomes showing the appearance of 14C in acyl moieties of acyl-CoAs and PC. Fatty acid elongation assays were carried out using [1-14C]18:0 and stopped at the indicated times. The lipids were extracted and separated by TLC. The radioactive acyl-CoA and PC bands were scraped and the lipids were eluted, then saponified, methylated, separated by RPTLC, and quantified as described in ``Materials and Methods'' and in Figure 1. , [1-14C]18:0; , [1-14C]20:0; , [1-14C]22:0; , [1-14C]24:0; , [1-14C]26:0.

As has been well documented, acyl groups are rapidly exchanged from acyl-CoAs into PC (Stymne and Glad, 1981; Stymne and Stobart, 1984; Griffiths et al., 1988a, 1988b). Because of this rapid exchange, acyl-CoAs can be used as substrates for in vitro desaturation assays, even though the in vivo substrate is PC (Slack et al., 1979). Although we have not demonstrated this to be the case for acyl elongation, the data so far are consistent with this hypothesis. Similarly, the wax synthase activity, which esterifies fatty alcohols to a fatty acyl moiety, can use acyl-CoAs, but the in vivo substrate may be a phospholipid (Kolattukudy, 1967). The idea that PC or another glycerolipid is the source of acyl primer is an attractive hypothesis for the following reasons. First, acyl-CoAs are unlikely substrates for acyl elongation (Hlousek-Radojcic et al., 1995; this work). Second, PC as a major ER membrane component has accessibility to the elongase enzyme and provides a medium for solubilizing the hydrophobic growing acyl chain. Third, PC is a pool for desaturation precursors and therefore functions as a reservoir for some acyl chain modifications.

	CONCLUSIONS

Several lines of evidence have led us to question whether acyl-CoAs are the immediate substrates for acyl elongation. First, rates of acyl elongation measured by [1-¹⁴C]malonyl-CoA incorporation are 2.5-fold higher than the rates of elongation measured by either ¹⁴C-fatty acid or [¹⁴C]acyl-CoA incorporation, suggesting that a significant endogenous primer pool is present (Hlousek-Radojcic et al., 1995). Second, the specific activity of the supplied [¹⁴C]acyl-CoA substrate is considerably higher than the specific activity of the synthesized elongated product in B. napus oil bodies. This implies that the product acyl-CoA is derived from a large endogenous pool. Measurements indicate that the 18:1-CoA pool in B. napus oil body preparations is less than 0.6 µm, which is at least 20-fold less than the concentration of [¹⁴C]acyl-CoA supplied, and therefore, cannot be the pool that dilutes the supplied substrate. Information on the size of fatty acid pools in plants has not been reported to date, but free fatty acid levels in animal microsomes are reported to be 0.03 µmol mg¹ protein (Cinti et al., 1992, and refs. therein). Third, nonesterified fatty acids are elongated at higher rates and to greater levels than acyl-CoAs (Fig. 1) (Evenson and Post-Beittenmiller, 1995). Fourth, the elongation of fatty acids is accomplished in the absence of added CoA (Fig. 4) (Evenson and Post-Beittenmiller, 1995). Finally, preincubation studies with acyl-CoAs show that although the acyl-CoA pool is reduced by >95%, the subsequent acyl elongation activity is reduced only 2- to 3-fold (Evenson and Post-Beittenmiller, 1995; Hlousek-Radojcic et al., 1995).

In this study we provide evidence that ACS activity in the absence of supplied CoA cannot account for the ATP-dependent fatty acid elongation in leek microsomes and B. napus oil bodies. Furthermore, although acyl-CoAs were synthesized in vitro under conditions that support high rates of acyl elongation, the formation of acyl-CoAs was ATP and malonyl-CoA dependent, and it did not correlate with fatty acid elongation activity. Thus, although exogenous acyl-CoAs are readily elongated in B. napus and leek, we propose that ATP plays a role in the elongation process other than supporting ACS activity.

In conclusion, we have shown that fatty acid elongation in leek microsomes and B. napus oil bodies was CoA independent and that CoA-free malonyl-CoA preparations were able to support high rates of fatty acid elongation. Therefore, ACS activity did not play a direct role in providing primer for acyl elongation in plants. Furthermore, we described an activity present in plant microsomes that synthesized acyl-CoA from [1-¹⁴C]18:0 in the presence of malonyl-CoA or acetyl-CoA and that was dependent on ATP. In addition, [1-¹⁴C]18:0 appeared in the PC and acyl-CoA fractions simultaneously with the appearance of the elongated products, which suggests that PC or other glycerolipid is a likely primer for acyl elongation.

	FOOTNOTES

   Received July 25, 1997; accepted October 10, 1997.

	ABBREVIATIONS

Abbreviations: ACS, acyl-CoA synthetase. CoASH, reduced CoA. PC, phosphatidylcholine. RPTLC, reverse-phase TLC. X:Y, a fatty acyl group containing X carbon atoms and Y cis double bonds.

	ACKNOWLEDGMENTS

We thank Grattan Roughan and John Ohlrogge for helpful discussions and critical reading of the manuscript, and Kathy Schmid and Susanne Rasmussen for critical reading of the manuscript.

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