Plant Physiol. (1983) 72, 735-740 0032-0889/83/72/0735/06/$00.50/0

Acyl-CoA Synthetase Is Located in the Outer Membrane and Acyl-CoA Thioesterase in the Inner Membrane of Pea Chloroplast Envelopes1 Received for publication December 28, 1982 and in revised form March 16, 1983

JAEN ANDREWS' AND KENNETH KEEGSTRA Department of Botany, University of Wisconsin, Madison, Wisconsin 53706

ABSTRACT acyl-CoA + glycerol 3-P [LPA3 + CoA Both acyl-CoA synthetase and acyl-CoA thioesterase activities are acyl transferase 1 present in chloroplast envelope membranes. The functions ofthese enzymes in lipid metabolism remains unresolved, although the synthetase has been acyl-CoA + LPA )PA + CoA proposed to be involved in either plastid galactolipid synthesis or the export acyl transferase 2 of plastid-synthesized fatty acids to the cytoplasm. We have examined the locations of both enzymes within the two envelope membranes of pea PA - DG + Pi (Pisum sativum var Laxton's Progress No. 9) chloroplasts. Inner and outer phosphatidate envelope membranes were purified from unfractionated envelope prepara- phosphatase tions by linear density sucrose gradient centrifugation. Acyl-CoA synthe- DG + tase was located in the outer envelope membrane while acyl-CoA thioes- UDP-gal. MGDG + UDP terase was located in the inner envelope membrane. Thus, it seems unlikely galactosyl transferase that the synthetase is directly involved in galactolipid assembly. Instead, This scheme was deduced from the observations that all but one its localization supports the hypothesis that it functions in the transport of of the enzyme activities are localized to the envelope membranes plastid-synthesized fatty acids to the endoplasmic reticulum. (13); the first acyl transferase appears to be a soluble plastid activity (3). Thus, incubation ofisolated spinach or pea chloroplast envelopes with CoA, ATP, glycerol 3-P, and stromal components results in the synthesis of DG, which can be converted to galac- tolipids upon the addition of UDP-gal (14; Andrews and Keegstra, unpublished observations). However, recent reports indicate that the first acyl transferase in pea chloroplasts may use either acyl- The plastid is a major site of lipid metabolism in the plant cell. CoA or acyl-ACP as acyl donors (12). Fatty acid synthesis occurs within the stromal space (19, 20, 27- An alternative role for acyl-CoA synthetase has been proposed, 30), and it has been suggested that this is the only site of fatty acid in which it functions in the export of fatty acids from the plastid synthesis in plants (21). In addition, the enzymes involved in to the cytoplasm (22). The acyl-CoA synthesized in the envelope galactolipid synthesis are located primarily in the chloroplast by this enzyme is the water-soluble form in which stromal synthe- envelope membranes (14). Inasmuch as galactolipids constitute 60 sized fatty acids are transported to the ER for use in phospholipid to 90%o of chloroplast membrane polar lipids (18), and the chlo- assembly. In this scheme, the acyl donor for galactolipid synthesis roplast membranes constitute about 70%o of total leaf cell mem- is thought to be stromal acyl-ACP, rather than the acyl-CoA (24). branes (17), it follows that galactolipids are major lipids in green The role of the plastid envelope acyl-CoA thioesterase, which tissues and that plastid galactolipid synthesis represents a consid- is a separate enzyme from the synthetase (16), is still unknown. erable portion of leaf cell lipid metabolism. Because of its preference for shorter-chain acyl groups, it has been Thus, the role of the plastid in lipid metabolism has become the suggested to prevent either the export out of the chloroplast or the subject of increasing investigation. However, many details con- improper insertion into plastid-synthesized lipids of fatty acids cerning the nature and interrelationships of various metabolic with less than 16 carbons (15). pathways are currently poorly understood. For example, both Localizing these biosynthetic enzymes within the chloroplast acyl-CoA synthetase and acyl-CoA thioesterase activities are pre- envelope, to either the inner or outer envelope membrane, may sent in chloroplast envelope membranes (14, 15, 23), but the aid in answering these questions of function. Until recently, such functions of these enzymes are unresolved or unknown. localizations were not feasible because the two membranes of the Acyl-CoA synthetase has been suggested to function in plastid envelope had not been separated. However, we have developed a galactolipid assembly (9, 10, 13), as indicated in the scheme below: method which separates the envelope into two membrane fractions CoA + fatty acid + ATP + identified as the inner and outer membranes (7; Cline et al., -) acyl-CoA AMP + PPi manuscript in preparation). In this report, we present evidence acyl-CoA that the acyl-CoA synthetase is localized to the outer envelope synthetase membrane of pea chloroplasts. In addition, we present evidence that the acyl-CoA thioesterase is localized to the inner envelope 'Supported in part by a grant from the United States Department of Agriculture Competitive Research Grants Office. 3Abbreviations: LPA, lysophosphatidic acid; PA, phosphatidic acid; 2 Supported by National Institutes of Health Grant 5 T32 GM07215. DG, diacylglycerol; MGDG, monogalactosyldiacylglycerol. 735 736 ANDREWS AND KEEGSTRA Plant Physiol. Vol. 72, 1983 membrane. Portions of this work have been reported previously RF, by reactivity with specific stains, and by comparison with in abstract form (1). standards. Radioactive lipids were first located by autoradiogra- phy, and then quantitated by scraping and counting the silica MATERIALS AND METHODS which contained them. Protein was determined by the Bradford procedure (6) using Materials. [1l-4C]Oleic acid was purchased from either New BSA as the standard. Chl was determined by the method ofArnon England Nuclear or Amersham; [l-14C]palmitoyl-coenzyme A was (2). purchased from Amersham. Tricine, oleic acid, CoA, ATP, DTT, P-enolpyruvate and pyruvate kinase were obtained from Sigma. All other chemicals were reagent grade. RESULTS Purification and Fractionation of Chloroplasts. Intact chloro- Characterization ofAcyl-CoA Synthetase and Acyl-CoA Thioes- plasts were obtained from homogenates of 12- to 16-d-old pea terase Activities. Neither acyl-CoA synthetase nor acyl-CoA seedlings (Pisum sativum var Laxton's Progress No. 9) by differ- thioesterase have been characterized in pea chloroplasts, although ential centrifugation followed by Percoll density gradient centrif- both enzymes have been extensively studied in spinach chloro- ugation as described (7). plasts (e.g. 15, 16). Thus, our initial experiments were designed to Chloroplast envelope membranes were then isolated and characterize each enzyme in pea chloroplast envelopes. The results subfractionated as previously described (7). Briefly, intact chlo- (Table I) indicate that the characteristics of the pea enzymes are roplasts were lysed by suspension in hypertonic (0.6 M sucrose) nearly identical to those observed for the same enzymes in spinach buffer followed by slow freezing (-20°C) and thawing (+20°C). chloroplast envelopes (15, 16). These results were also used to The broken chloroplasts were adjusted to 1.3 M sucrose, and establish optimal assay conditions for subsequent localization unfractionated envelope membranes isolated by flotation centrif- ugation to a 1.2/0.3 M sucrose interface; thylakoid membranes and soluble 'stromal' proteins remained in the 1.3 M layer. The enve- Table I. Characterization ofAcyl-CoA Synthetase and Acyl-CoA lope membranes were then diluted to 0.45 M sucrose and subfrac- Thioesterase Activities in Pea Chloroplast Envelopes tionated by linear density gradient centrifugation (0.6-1.2 M su- Unfractionated envelope membrane preparations, prepared as described crose) into a heavy (p = 1.13 g/cm3) and a light (p = 1.08 g/cm3) in "Materials and Methods," were used for enzyme characterizations. membrane fraction, identified as the inner and outer envelope Protein and enzyme assays are described in "Materials and Methods." membranes, respectively. Purified envelope membranes were re- Information contained within parentheses indicates the conditions used covered by dilution and centrifugation at 90,000gmax for 1.5 to 2 for subsequent enzyme assays. ND = not determined. h. The buffer for isolating, fractionating, and resuspending enve- Acyl-CoA Synthetase Acyl-CoA Thioesterase lope membranes was 10 mm Tricine/NaOH (pH 7.5). Thylakoid membranes were separated from soluble stromal pH Optimum Broad, 7-9 (8) Sharp, 9 (9) at 63% control proteins by dilution of the 1.3 M sucrose layer to 0.26 M followed MgC12 effect Required: optimum Inhibitory: by centrifugation at 90,000ggma,, for 1.5 to 2 h. The thylakoid 5 mM (5 mM) at 2 mm (0 mM) membranes were pelleted, while the soluble stromal proteins Protein curve Linear at 2.5-50 ig/as- Linear at 0.5-20 ,ug/as- remained in the supernatant solution. say (20,ug/assay) say (5 ug/assay) Assays. Acyl-CoA synthetase was assayed by a modification of Substrate concn. CoA: saturated at 0.05 Palmitoyl-CoA: ND (2- the procedure described by Joyard and Stumpf (16). Unless other- mM (0.5 mM) 3 uM, the critical mi- at 0.5 wise stated, the reaction mixture contained: 0.1 M Tricine/NaOH Oleate: saturated celle concn.) (pH 8), 1 mM [14CJoleic acid (0.47-3.1 mCi/mmol), 0.5 mm CoA, mM (1.0 mM) 5 mm ATP, 5 mM MgC12, 5 mm DTT, 5 mm P-enolpyruvate, and Storage effects 334 ,ug/ml pyruvate kinase. Reactions were initiated by addition 40C Activity dec by -85%/ Activity slowly dec to of 20 ,ug membrane protein and incubated for 0 to 5 min at 22 to day 72% by 6 d 25°C. Reactions were terminated by adding 2 ml isopropyl alco- -200C Variable; activity dec Activity slowly dec to hol:H20:acetic acid (1:1:0.03, v/v/.v) to either a 200-,il total reac- by -10-40%o/day 80%1o by 6 d tion volume or 60 ,ul withdrawn from a 360- to 600-pl total reaction volume. Newly synthesized [14C]oleoyl-CoA was separated from Table II. Localization ofAcyl-CoA Synthetase and Acyl-CoA free ['4CJoleic acid by extraction of the latter 3 times into 2 ml Thioesterase Activities within Pea Chloroplasts petroleum ether (ligroine, bp 35-60°C). The aqueous layer was Isolated intact chloroplasts were fractionated, and both enzymes as- counted in 8 ml scintillation cocktail in a Packard Tri-Carb liquid as described in "Materials and Methods." The results scintillation counter, model 3385. Initial reaction rates determined sayed, presented here are from a single experiment; similar results for each enzyme were from plots of product appearance versus time were used to deter- observed in at least one additional experiment. mine enzyme activities. Acyl-CoA thioesterase was assayed essentially as described by Enzyme Activity Joyard and Stumpf (15). Unless otherwise stated, the reaction mix Enzyme Chloroplast Protein contained 100 mm Tricine/NaOH (pH 9) and 3 pLM [14Cjpalmitoyl- Fraction Distribution Specific Activity CoA (57 mCi/mmol). Reactions were initiated by addition of 5 Activity Distribution ,ug membrane protein, and incubated for 0 to 3 min at 22 to 25°C. p.mol % total Reactions were terminated by withdrawing 60-,uI aliquots at var- % total mg-h ious times (from a total reaction volume of 360-600 ,ul) and adding Acyl-CoA to 2 ml isopropyl alcohol:H20:acetic acid (1:1:0.03, v/v/v). Newly Synthetase Envelopes 1.1 37.85 89.7 liberated [ 4C]palmitate was separated from [t4Cjpalmitoyl-CoA 31.7 0.153 10.3 ether as described above. The Thylakoid by extraction into petroleum petro- 'Stroma' 67.2 <0.2 leum ether extracts were pooled, dried under air, and counted in cocktail. Acyl-CoA 10 ml of scintillation Thioesterase 1.1 0.249 21.9 extracted into ether as described Envelopes Membrane lipids petroleum Thylakoid 31.7 0.029 72.9 were two-dimensional TLC were above analyzed by (11). Lipids 'Stroma' 67.2 0.001 5.2 visualized by reversible staining with 12 vapor, and identified by LOCATION OF PLASTID Acyl-CoA SYNTHETASE AND THIOESTERASE 737 studies. The variation in both the stability of acyl-CoA synthetase synthetase and acyl-CoA thioesterase activities (Table III). when stored frozen (-20°C) (Table I) and the length of time The specific activity of acyl-CoA synthetase was greatest in required to prepare membrane samples led to significant differ- pools 3 and 4, indicating that it was located in the outer membrane. ences in the observed levels of activity between experiments. The specific activity in pool 2 was similar to that observed in However, within a single experiment, all membrane fractions were unfractionated envelope membranes, in agreement with the iden- subjected to the same storage conditions, allowing comparisons to tification of this fraction as a mixture of inner and outer mem- be made within each experiment. branes. However, a significant amount ofactivity occurred in pool The possibility that the '4C-acyl group of ['4C]acyl-CoA could 1, the purest inner membrane fraction. This activity was probably be incorporated into membrane lipids was examined by analyzing due to contamination by outer membrane, as protein analysis by the lipids after both enzyme assays. These analyses demonstrated SDS-polyacrylamide gel electrophoresis indicates that the total that >98% of the petroleum ether soluble radioactivity migrated protein of this fraction contains 5 to 20%1o outer membrane protein with free fatty acids, indicating that membrane lipids were not (8; Andrews and Keegstra, unpublished observations). labeled from ['4C]acyl-CoA during the course of either enzyme The specific activity of acyl-CoA thioesterase was greatest in assay. The same results were observed when either gently lysed pool 1, indicating that it was located in the inner membrane. This whole chloroplasts or isolated unfractionated envelopes were used determination of specific activity was probably low, due to the as the source for either enzyme. presence in this fraction ofcontaminating outer membrane protein Localization of Acyl-CoA Synthetase and Acyl-CoA Thioester- (see above). The very low amounts ofthioesterase activity in pools ase Activities within Pea Chloroplasts. Acyl-CoA synthetase and 3 and 4 further support the conclusion that it was an inner acyl-CoA thioesterase activities were first localized within pea envelope membrane enzyme. chloroplasts. Isolated intact chloroplasts were ruptured by slow Assays of both enzyme activities across linear density sucrose freezing and thawing, and then fractionated into envelope, thyla- gradients confirmed the localizations determined from the pooled koid, and stromal fractions. Each fraction was then assayed for fractions described above. To obtain sufficient material from one the presence of both enzymes. The specific activities of both acyl- gradient for the assays, isolated envelope membranes were first CoA synthetase and acyl-CoA thioesterase were greatest in the concentrated by sedimentation before subsequent fractionation. envelope membrane fraction (Table II). From this, it appeared The total activity of acyl-CoA synthetase was greatest in the outer that both enzymes were located in the envelope membranes ofpea envelope membrane peak (Fig. 2A). It also exhibited a smaller chloroplasts. Acyl-CoA thioesterase activity has been previously peak of activity centered on the trailing edge of the heavy mem- reported to be localized in pea chloroplast envelopes, although brane peak, which is the region most extensively contaminated by supporting data were not included (4). In addition, these two outer envelope membranes. The highest specific activities also enzymes are also considered to be located in the envelope mem- occurred in the outer membrane peak. For acyl-CoA thioesterase, branes of spinach chloroplasts (15, 16). the total activity was greatest in the heavy membrane peak, which However, we consistently found a small but significant propor- is greatly enriched in inner envelope membranes (Fig. 2B). The tion of the total activity of acyl-CoA synthetase associated with increasing specific activity over the inner envelope membrane the thylakoid fraction (Table II). This distribution of acyl-CoA fraction suggested that the purity of the inner membranes in- synthetase activity among pea chloroplast fractions is similar to creased as the density increased. A shoulder in the specific activity that observed in spinach chloroplasts by Roughan and Slack (23). profile was seen over the trailing edge of the heavy membrane A very different distribution of total activity in spinach chloro- peak. plasts was noted by Joyard and Stumpf (16), who reported that the largest proportion oftotal acyl-CoA synthetase activity (-60%1o) DISCUSSION is found in the thylakoid fraction, while about 30%o is found in the The results presented here demonstrate that acyl-CoA synthe- envelope fraction. The distribution of total acyl-CoA thioesterase tase is located in the outer membrane ofpea chloroplast envelopes. activity within pea chloroplasts was quite different from that of This location suggests that acyl-CoA synthetase is not involved in acyl-CoA synthetase, as the majority of the total thioesterase galactolipid synthesis as proposed by the scheme outlined in the activity was associated with the thylakoids. This is similar to the introduction, as this scheme would necessitate an extensive trans- distribution in spinach chloroplasts as determined by Joyard and fer of synthetic intermediates between compartments, based upon Stumpf (15). what is now known about the localizations ofthe enzymes believed Because the chloroplast fractionation schemes cited above in- to be involved. Specifically, fatty acids, which are synthesized in volved hypotonic lysis for breaking chloroplasts, we compared the stroma, would have to be transported to the outer envelope this method with ours, which involves lysis caused by slow freezing membrane for incorporation into acyl-CoA. Of the next several and thawing under hypertonic conditions. The protein distribu- enzymes involved in galactolipid assembly, the first transferase is tion, and the specific activities and total activity distribution of a soluble plastid enzyme and thus probably located in the stroma each enzyme, were nearly identical for both methods (data not (3). The second acyl transferase appears to be located in the inner shown). Thus, the two methods of lysis yield equivalent enzyme envelope membrane, as demonstrated by preliminary in vitro segregation during subsequent fractionation of chloroplasts into labeling studies with inner and outer envelope membrane fractions envelopes, thylakoids, and soluble components. from pea chloroplasts (Andrews and Keegstra, unpublished ob- Envelope-bound acyl-CoA synthetase and acyl-CoA thioester- servations). Thus, the acyl-CoA synthesized at the outer envelope ase activities were then localized to either the inner or outer membrane would then have to be shuttled inward for the acyl membrane of pea chloroplast envelopes. Isolated envelope mem- transfer reactions in the stroma and inner envelope membrane. It branes were subfractionated by centrifugation through a linear seems more reasonable to assume that stromal acyl-ACP, rather density sucrose gradient (0.6-1.2 M sucrose) (Fig. 1). The light than outer envelope membrane synthesized acyl-CoA, serves as membrane peak (p = 1.08 g/cm3) has been identified as the outer the acyl donor for LPA and PA synthesis. In fact, it has been envelope membrane, and the heavy membrane peak (p = 1.13 g/ recently demonstrated that acyl-ACP is the preferred substrate in cm3) as the inner envelope membrane, although it is significantly vitro for both acyl transferases from either spinach or pea chloro- contaminated with outer membrane (7, 8, and Cline et al., man- plasts (12). uscript in preparation). Fractions of the gradients were pooled as Further support for the conclusion that the acyl-CoA synthetase indicated (Fig. 1) and the membranes collected by dilution and is not involved in galactolipid assembly comes from two sets of centrifugation. These membranes were then assayed for acyl-CoA experiments. Bertrams et al. (4, 5) noted that for isolated intact 738 ANDREWS AND KEEGSTRA Plant Physiol. Vol. 72, 1983

0 0 0 I 1.16 n E E U 0 00 1.12 E

0 1.08 C 0 0

1.04

5 10 15 20 25 30 BOTTOM TOP Fraction Number FIG. 1. Fractionation of chloroplast envelope membranes by linear density gradient centrifugation. Unfractionated envelope membranes isolated from 20 mg Chi of intact chloroplasts were sedimented through a 0.6 to 1.2 M sucrose linear density gradient. Gradient fractions were 1.25 ml. For analysis of the membranes, pools I to 4 were made as shown, and the membranes recovered by dilution and centrifugation.

Table III. Localization ofAcyl-CoA Synthetase and Acyl-CoA Thioesterase Activities within Pea Chloroplast Envelope Membranes Envelope membranes were fractionated and pooled as indicated in Figure I and "Materials and Methods." Acyl-CoA synthetase activity was assayed in the experiment reported in Figure 1; acyl-CoA thioesterase activity was assayed in a separate experiment. Similar results were observed for each enzyme in at least two additional experiments. Membrane Preparation

Pool 1: Pool 2: Pool 3: Pool4: Unfractionated inner mixed inner outer outer envelopes membrane membranes membrane membrane Acyl-CoA Specific activity Synthetase (uLmol/mg.h) 3.23 1.49 3.73 12.6 13.8 Activity distribution (% total) 3.9 17.4 40.5 38.2 Protein distribution (% total) 42.9 25.0 17.2 14.9 Acyl-CoA Specific activity Thioesterase (,umol/mg.h) 0.238 0.357 0.306 0.044 0.005 Activity distribution (% total) 60.4 37.8 1.7 0.13 Protein distribution (% total) 47.2 34.4 10.8 7.6 pea chloroplasts, exogenous acyl-CoA is not used for de novo An alternative role for acyl-CoA synthetase has been proposed synthesis of either LPA or PA, even in the presence of glycerol 3- in which this enzyme functions in the transport of plastid-synthe- P. Thus, if the acyl-CoA synthesized by the outer envelope mem- sized fatty acids to the ER (22). In this scheme, newly synthesized brane is released to the cytoplasm, then their results would imply fatty acids are released from ACP and subsequently incorporated that this acyl-CoA is not subsequently used by the plastid during into acyl-CoA in the chloroplast envelope. These fatty acids are synthesis of the galactolipid precursors LPA and PA. In addition, then transported as water-soluble acyl-CoA to the ER for use in Roughan et al. (22) observed that, when isolated intact spinach phospholipid synthesis. The location of acyl-CoA synthetase in chloroplasts are actively synthesizing lipids from labeled acetate, the outer envelope membrane supports this hypothesis. This lo- the addition of CoA and ATP does not result in an increased cation is also consistent with the possibility that both the ATP synthesis of PA or DG. The only change observed is an increase and CoA required for this reaction may be provided from outside in acyl-thioester synthesis with a parallel decrease in nonesterified the chloroplast. Several lines of evidence support this possibility. fatty acid synthesis. Subsequent incubation of chloroplasts in the First, Roughan et al. (25) reported that, when isolated intact dark reveals that the acyl-CoA synthesized from nonesterified spinach chloroplasts synthesize fatty acids from I'4Clacetate, an fatty acids is not used for polar lipid synthesis, even in the presence activity which requires active photosynthesis, the addition of of glycerol 3-P. Their results suggest that the newly synthesized exogenous CoA and ATP results in a 26-fold increase in the acyl-CoA is located outside the chloroplasts. amount oflabeled acyl-CoA synthesis. The addition of CoA alone LOCATION OF PLASTID Acyl-CoA SYNTHETASE AND THIOESTERASE 739

0 0 .0go C-E E

.t c a

a) E-c

E n a) E

5 15 BOTTOM TOP BOTTOM TOP Fraction Number Fraction Number FIG. 2. Enzyme acivities ofsubfractionated chloroplast envelope membranes. A, Acyl-CoA synthetase. Unfractionated envelope membranes obtained from 42 mg Chl of intact chloroplasts were first concentrated by centrifuging at 43,500g for 60 min; the membrane pellet was then resuspended in 9 ml buffer and the membranes sedimented through a 29 ml, 0.6 to 1.2 M sucrose linear density gradient. Each fraction (1.25 ml) was assayed for protein and acyl-CoA synthetase activity. B, Acyl-CoA thioesterase. Unfractionated envelope membranes obtained from 31 mg Chl of intact chloroplasts were first concentrated by centrifuging at 43,500g for 60 min; the membrane pellet was then resuspended in 5 ml buffer, and the membranes sedimented through a 12 ml, 0.5 to 1.25 M linear density sucrose gradient. Each fraction (0.75 ml) was assayed for protein and acyl-CoA thioesterase activity. increases labeled acyl-CoA synthesis only 2-fold. In addition, will assist in evaluating various hypotheses concerning its function. Roughan and Slack observed that oleoyl-CoA formation from Further work is necessary to define its role in plant lipid metab- oleate and CoA by illuminated photosynthetically active intact olism. spinach chloroplasts is stimulated 10-fold by the presence of ATP The segregation of envelope enzymes among chloroplast frac- in the surrounding media (23). Finally, similar results with spinach tions allows us to evaluate the contamination of these fractions by chloroplasts were reported by Sanchez and Mancha (26). Only the envelope membranes. When pea chloroplasts were fractionated addition of both ATP and CoA results in significant stimulation into envelope, thylakoid, and soluble stromal components, the (90-fold) of long-chain acyl-CoA during fatty acid synthesis from highest specific activities for both acyl-CoA thioesterase and acyl- acetate. ATP alone did not stimulate acyl-CoA synthesis, while CoA synthetase were observed in the envelope fraction. These CoA alone results in a very small stimulation (2-fold). results led us to conclude that both enzymes were located in the The acyl-CoA thioesterase activity in pea chloroplast envelopes envelope membranes of pea chloroplasts; similar envelope locali- is due to a separate enzyme from the acyl-CoA synthetase, and is zations for these enzymes were determined in spinach chloroplasts not simply a reversal of the reaction catalyzed by the synthetase. (15, 16). However, in pea chloroplasts, the majority of the total Several pieces of data support this conclusion. First, the thioester- thioesterase activity was consistently associated with the thylakoid ase activity was quite different from the synthetase activity in membranes. This is in contrast to the much lower proportion of several respects, including pH optima, effects of MgCl2, and total acyl-CoA synthetase activity associated with the thylakoid stability during storage. This confirms the work of Joyard and membranes. In addition, the distribution of the total activity of Stumpf, who observed similar and additional differences between another outer envelope membrane enzyme, galactosyl transferase, the acyl-CoA thioesterase and synthetase activities in spinach among pea chloroplast fractions (8) is quite similar to that ob- chloroplast envelopes, and therefore concluded that these activities served for acyl-CoA synthetase. The thylakoid-associated galac- also represent separate enzymes (15, 16). In addition, our dem- tosyl transferase activity is thought to be due to contamination by onstration that the acyl-CoA thioesterase was located in the inner envelope membranes (8). Thus, if the activity of both the thioes- envelope membrane of pea chloroplasts, while the synthetase was terase and the synthetase in the thylakoid fraction is also due to located in the outer membrane, provides definitive support for the contamination of the thylakoid membranes by envelopes, then the conclusion that the two activities are due to separate enzymes. higher proportion of thioesterase activity found in the thylakoids The function of acyl-CoA thioesterase remains unclear. Al- suggests that the inner envelope membranes preferentially con- though the localization of this enzyme to the inner envelope taminate the thylakoids. Alternatively, a separate thioesterase membrane did not suggest a role for this enzyme, such information activity may exist in thylakoid membranes, resulting in the higher 740 ANDREWS AND KEEGSTRA Plant Physiol. Vol. 72, 1983 proportion of total activity seen in these membranes. Currently, galactolipid synthesis? Yes! In L-A Appelquist, C Liljenberg, eds, Advances in the Biochemistry and Physiology of Plant Lipids. Elsevier/North-Holland we are unable to distinguish between these two possibilities. Biomedical Press, New York, pp 181-186 14. JOYARD J, R DoUCE 1977 Site ofsynthesis ofphosphatidic acid and diacylglycerol Acknowledgments-We thank Ken Cline and Maggie Werner-Washburne for in spinach chloroplasts. Biochim Biophys Acta 486: 273-285 vigorous, stimulating and encouraging discussions, and Joan Wilber for adroit 15. JOYARD J, PK STUMPF 1980 Characterization ofan acyl-coenzyme A thioesterase technical assistance. associated with the envelope of spinach chloroplasts. Plant Physiol 65: 1039- 1043 LITERATURE CITED 16. JOYARD J, PK STUMPF 1981 Synthesis of long-chain acyl-CoA in chloroplast envelope membranes. Plant Physiol 67: 250-256 1. ANDREWS J, K KEEGSTRA 1982 Localization of lipid synthesizing enzymes in pea 17. FORDE J, MW STEER 1976 The use of quantitative electron microscopy in the chloroplast envelopes. Plant Physiol 69: S-133 study of lipid composition of membranes. J Exp Bot 27: 1137-1141 2. ARNON DI 1949 Copper enzymes in isolated chloroplasts. Polyphenoloxidase in 18. MAZLIAK P 1977 Glyco- and phospholipids of biomembranes in higher plants. Beta vulgaris. Plant Physiol 24: 1-15 In M Tevini, HK Lichtenthaler, eds, Lipids and Lipid Polymers in Higher 3. BERTRAMS M, E HEINZ 1979 Soluble, isomeric forms of glycerophosphate acyl- Plants. Springer-Verlag, Berlin, pp 48-72 transferase in chloroplasts. In L-A Appelquist, C Liljenberg, eds, Advances in 19. NAKAMURA Y, M YAMADA 1975 Fatty acid synthesis by spinach chloroplasts. I. the Biochemistry and Physiology of Plant Lipids. Elsevier/North-Holland Property of fatty acid synthesis from acetate. Plant Cell Physiol 16: 139-149 Biomedical Press, New York, pp 139-144 20. NOTHELFER HG, RH BARCKHAUS, F SPENER 1977 Localization and characteri- 4. BERTRAMS M, E HEINZ 1980 Long chain acyl-coenzyme A thioesters as substrates zation of the fatty acid synthesizing system in cells of Glycine max (soybean) in glycerolipid biosynthesis of chloroplasts. Dev Plant Biol 6: 67-71 suspension cultures. Biochim Biophys Acta 489: 370-380 5. BERTRAMS M, K WRAGE, E HEINZ 1981 Lipid labeling in intact chloroplasts from 21. OHLROGGE JB, DN KUHN, PK STUMPF 1979 Subcellular localization of acyl exogenous nucleotide precursors. Z Naturforsch 36: 62-70 carrier protein in leaf protoplasts of Spinacia oleracea. Proc Natl Acad Sci 6. BRADFORD M 1976 A rapid and sensitive method for the quantitation of micro- USA 76: 1194-1198 gram quantities of protein utilizing the principles of protein-dye binding. Anal 22. ROUGHAN PG, R HOLLAND, CR SLACK 1980 The role of chloroplasts and Biochem 72: 248-254 microsomal fractions in polar-lipid synthesis from [1-14C] acetate by cell-free 7. CLINE K, J ANDREWS, B MERSEY, EH NEWCOMB, K KEEGSTRA 1981 Separation preparations from spinach (Spinacia oleracea) leaves. Biochem J 188: 17-24 and characterization of inner and outer envelope membranes of pea chloro- 23. ROUGHAN PG, CR SLACK 1977 Long-chain acyl-coenzyme A synthetase activity plasts. Proc Natl Acad Sci USA 78: 3595-3599 of spinach chloroplasts is concentrated in the envelope. Biochem J 162: 457- 8. CLINE K, K KEEGSTRA 1983 Galactosyl transferases involved in galactolipid 459 biosynthesis are located in the outer membrane of pea chloroplast envelopes. 24. ROUGHAN PG, CR SLACK 1982 Cellular organization ofglycerolipid metabolism. Plant Physiol 71: 366-372 Annu Rev Plant Physiol 33: 97-132 9. DOUCE R, J JOYARD 1979 The chloroplast envelope: an unusual cell membrane 25. ROUGHAN PG, CR SLACK, R HOLLAND 1976 High rates of [1-'C1 acetate system. In L-A Appelquist, C Liljenberg, eds, Advances in the Biochemistry incorporation into the lipid of isolated spinach chloroplasts. Biochem J 158: and Physiology of Plant Lipids. Elsevier/North-Holland Biomedical Press, 593-601 New York, pp 79-96 26. SANCHEZ J, M MANCHA 1981 Synthesis of acyl-CoAs by isolated spinach 10. DOUCE R, J JOYARD 1979 Structure and function of the plastid envelope. Adv chloroplasts in relation to added CoA and ATP. Planta 153: 519-513 BotRes7: 1-116 27. STUMPF PK, AT JAMES 1963 The biosynthesis of long-chain fatty acids by lettuce 11. DOUCE R, J JOYARD 1980 Chloroplast envelope lipids: detection and biosynthesis. chloroplast preparations. Biochim Biophys Acta 70: 20-32 Methods Enzymol 69: 290-301 28. VICK B, H BEEVERS 1978 Fatty acid synthesis in endosperm ofyoung castor bean 12. FRENTZEN M, E HEINZ, T MCKEON, PK STUMPF 1982 De novo-biosynthesis of seedlings. Plant Physiol 62: 173-178 galactolipid molecular species by reconstituted enzyme systems from chloro- 29. WEAIRE PJ, RGO KEKWICK 1975 The synthesis of fatty acids in avocado plasts. In JFGM Wintermans, PJC Kuiper, eds, Biochemistry and Metabolism mesocarp and cauliflower bud tissue. Biochem J 146: 425-437 of Plant Lipids. Elsevier Biomedical Press, New York, pp 141-152 30. ZILKEY BF, DT CANVIN 1972 Localization of oleic acid biosynthesis enzymes in 13. JOYARD J, M CHUZEL, R DOUCE 1979 Is the chloroplast envelope a site of the proplastids of developing castor endosperm. Can J Bot 50: 323-326