Proc. Nati. Acad. Sci. USA Vol. 76, No. 3, pp. 1194-1198, March 1979 Biochemistry Subcellular localization of acyl carrier protein in leaf protoplasts of Spinacia oleracea (acyl carrier protein radioimmunoassay/acyl carrier protein-dependent fatty acid synthesis/chloroplast function/site of fatty acid synthesis in plants) JOHN B. OHLROGGE*, DAVID N. KUHN, AND P. K. STUMPFt Department of Biochemistry and Biophysics, University of California, Davis, California 95616 Contributed by P. K. Stumpf, December 26,1978

ABSTRACT This communication demonstrates that all de intact organelles with a concomitant decrease in leakage of novo fatty acid biosynthesis in spinach leaf cells requires acyl organelle proteins into the cytoplasmic fraction. In addition, carrier protein (ACP) and occurs specifically in the chloroplasts. Antibodies raised to purified spinach ACP inhibited at least immunological detection of proteins avoids many of the com- 98% of malonyl CoA-ependent fatty acid synthesis by spinach plications inherent in the assay of enzyme complexes such as leaf homogenates. Therefore, the presence of ACP in a com- fatty acid synthetase. In plants, ACP is an essential soluble partment of the spinach leaf cell would serve as a marker for component in fatty acid biosynthesis (1), the specific moiety de novo fatty acid biosynthesis. A radioimmunoassa capable in the initial desaturation of stearoyl-ACP (10), and a possible of detecting 10-'S mol (10-" g) of spinach ACP was deve oped acyl carrier in triglyceride (11) and phospholipid biosynthesis to measure the levels of ACP in leaf cell components isolated by sucrose gradient centrifugation of a gentle lysate of spinach (unpublished data). Because ACP can be purified to homoge- leaf protoplasts. All of the ACP of the leaf cell could be attrib- neity, antibodies raised against it have been used to determine uted to the chloroplast. Less than 1% of the ACP associated with the proportion of ACP-dependent fatty acid biosynthesis in leaf chloroplasts resulted from binding of free ACP to chloro lasts. tissue and to ascertain the subcellular sites of all ACP-dependent Of interest, ACP from Escherichia coli, soybean, and sunflower reactions in the leaf protoplast. showed only partial crossreactivity with spinach ACP by the radioimmunoassay. These results strongly suggest that, in the leaf cell, chloroplasts are the sole site for the degnovo synthesis MATERIALS AND METHODS of C16 and C18 fatty acids. These fatty acids are then transported into the cytoplasm for further modification and are either in- Purification of Spinach ACP. Spinach ACP was purified serted into extrachloroplastic membrane lipids or returned to from 20 kg of spinach leaves by a modification of the method the chloroplast for insertion into lamellar membrane lipids. of Simoni et al. (12). The initial heat treatment step was omit- ted. After ammonium sulfate fractionation and acid precipi- In plants, acyl carrier protein (ACP) plays an essential role in tation, [1,3-14C]malonyl-ACP was added to the crude prepa- both the synthesis and the subsequent metabolism of the C16 ration and served as a marker for identification of ACP in and C18 fatty acids (1). Whereas de novo fatty acid synthesis subsequent steps of purification. Column chromatography on in isolated chloroplasts has long been known (1), the site of DEAE-cellulose and DEAE-Sephadex was carried out as de- synthesis of fatty acids that are required for the formation of scribed by Majerus et al. (13). After these steps, the preparation plasma, mitochondrial, and other extrachloroplast membranes (15 mg) was judged to be homogeneous by sodium dodecyl in the leaf cell is not clear. In sharp contrast, in both animal and sulfate/disc gel electrophoresis. E. coli ACP was purified as yeast cells, synthesis occurs in the cytoplasm (2). In Escherichi described (13). colA cells, it has been shown that ACP is localized on or near the Preparation of IgG to Spinach ACP. Three New Zealand inner face of the plasma membrane and this implies that the White male rabbits (3-4 kg) were injected intradermally at nonassociated fatty acid synthetase enzymes may be organized multiple sites with 0.5 ml of an emulsion containing 1-2 mg of in the same area in vivo (3). spinach ACP mixed with an equal volume of Freund's complete Although earlier studies have provided evidence that fatty adjuvant. Three booster injections of 0.2-0.5 mg were given at acid biosynthesis occurs in chloroplasts (1) and in proplastids 2-week intervals with incomplete adjuvant. Serum was collected (4-8), until recently the methods used for the isolation of these 2 weeks after the final injection. organelles led to substantial breakage and release of enzymes For use in the ACP radioimmunoassay, a crude gamma into the cytoplasmic fraction. Consequently, it has been difficult globulin fraction was prepared from the serum by precipitation to assign a precise site for an enzyme in the leaf cell. In addition, with 10% polyethylene glycol and solution of the precipitate after cell disruption, attempts to localize the complex set of in 0.01 M potassium phosphate, pH 7.0/0.15 M NaCl (Pi/NaCI). reactions comprising fatty acid synthesis can be further com- This fraction was further purified for use in the inhibition of plicated by cofactor dilution (particularly ACP), enzyme fatty acid synthesis by dialysis for 12 hr against 0.07 M sodium dilution, and inactivation. Hence, it has been difficult to con- acetate (pH 5.0) and passage through a DEAE-Sephadex A-50 clude from these earlier studies whether de novo fatty acid column equilibrated with the same buffer. Preimmune control synthesis takes place only in the chloroplast (or plastid) or in the serum was processed in the same manner. cytoplasm or other organelles as well. Todination of Spinach ACP. Because spinach ACP lacks Damage to organelles can be greatly reduced in the isolation tyrosine (12), iodination by a modification of the method of procedure by using protoplasts as the starting material (9). Bolton and Hunter (14) was used. Spinach ACP (30,ug), dis- Gentle lysis of isolated protoplasts gives an increase in yield of Abbreviations: ACP, acyl carrier protein; Pi/NaCl, 0.01 M potassium The publication costs of this article were defrayed in part by page phosphate, pH 7.0/0.15 M NaCl. charge payment. This article must therefore be hereby marked "ad- * Present address: Plant Growth Laboratory, 1047 Wickson Hall, vertisement" in accordance with 18 U. S. C. §1734 solely to indicate University of California, Davis, CA 95616. this fact. t To whom reprint requests should be addressed. 1194 Downloaded by guest on September 29, 2021 Biochemistry: Ohlrogge et al. Proc. Natl. Acad. Sci. USA 76 (1979) 1195 solved in 10 ml of 0.1 M borate (pH 8.5), was treated for 30 leaves were vacuum infiltrated for 3 min and incubated over- min at room temperature with 2.6 Aug of 3-(4-hydroxyphenyl)- night in the dark at room temperature, without shaking. Pro- propionic acid N-hydroxysuccinimide ester. Excess reagent was toplasts were released the following morning by gently shaking removed by acid precipitation and three washes. The conju- the leaves with a forceps. The protoplasts were filtered through gated ACP was redissolved in 30 jul of 0.1 M Tris buffer (pH 8), a 73-jum nylon mesh and pelleted by centrifugation at 140 X and 1 Al of this solution was iodinated by using 200 juCi of 125I g for 5 min at room temperature. The pelleted protoplasts were (z17 Ci/mg; 1 Ci = 3.7 X 1010 becquerels) and the chlora- washed twice by resuspension in 0.85 M mannitol and cen- mine-T method of Hunter and Greenwood (15). This yielded trifugation at 140 X g for 5 min. Filtration of the resuspended 1251-labeled ACP (1251-ACP) with a specific activity of ;250 protoplasts through a 50-jum nylon mesh removed xylem ele- Ci/mmol. ments and druses. The final pellet was resuspended to a known Radioimmunoassay. Antibody prepared as described above volume with 0.85 M mannitol and the chlorophyll content was was diluted 1:500 with bovine serum albumin (1 mg/ml) in determined. P1/NaCI (albumin/Pi/NaCI). A 25-Ml aliquot of this solution Protoplasts were lysed by three passages through a 10-ml was added to standards or samples containing unlabeled ACP. syringe with a 25-gauge needle outfitted with a Swinnex filter This mixture was incubated 4-20 hr at room temperature, after (13 mm) and two layers of Miracloth and a single layer of 20-jum which l251-ACP was added and the incubation was continued nylon mesh (9). Extent of lysis was estimated by light micros- for an additional 2 hr. ACP bound to antibody was then sepa- copy. A protoplast lysate (1-2 mg of chlorophyll) containing rated from free ACP by addition of 250,gg of Immunobeads less than 10% intact protoplasts was layered onto a 30-58% (Bio-Rad). After 1-2 hr of additional incubation, the Immu- (wt/wt) linear sucrose gradient in 0.02 M potassium N-[2- nobead-rabbit antibody complex was centrifuged and washed hydroxy-1,1-bis(hydroxymethyl)ethyl]glycinate (Tricine) at twice with 1.5 ml of albumin/Pi/NaCl. The final pellet was pH 7.6. Gradients were centrifuged for 4 hr at 27,000 rpm resuspended in 0.5 ml of albumin/Pi/NaCl and transferred to (100,000 X g) and 4VC in an SW 27 rotor. Fractions (1 ml) were scintillation vials, and the radioactivity was determined. collected from the gradient for subsequent assays. With the above method of separation of bound from free Enzyme Assays. NAD-dependent isocitrate dehydrogenase, ACP, preimmune control serum yielded 1-300 cpm of non- a marker enzyme for mitochondria, was assayed by the method specific binding. of Cox (21). Ribulose-1,5-bisphosphate carboxylase was assayed Antibody Inhibition of Fatty Acid Synthesis. The fatty acid by a modification of the method of Lorimer et al. (22), ADP- synthesis reaction mixture contained 5 MM [1,3-14C]malonyl- glucose pyrophosphorylase as in ref. 23, and NADPH-triose CoA (58 ,Ci/mol), 0.32 mM NADPH, 0.38 mM NADH, 1 mM phosphate dehydrogenase (reversible) as in ref. 24. ATP, 0.3 mM MgCI2, 0.2 mM MnCl2, and 0.1 M 2-f[2-hy- droxy-1,1-bis(hydroxymethyl)ethyl]aminofethanesulfonic acid RESULTS (TES) (pH 7.5) in a final volume of 0.15 ml. ACP Requirement for Fatty Acid Synthesis. In the absence A Brinkman Polytron-PT10 was used to homogenize 4.5 g of added ACP, spinach leaf homogenates incorporated [14C]- of spinach leaves in 10 ml of 0.1 M potassium phosphate, pH malonyl-CoA into fatty acids at a rate of -20 pmol/min per mg 7.5/2 mM dithiothreitol. The homogenate was filtered through of protein. Antibodies to spinach leaf ACP inhibited essentially Miracloth before use and then preincubated for 1 hr at 300C all fatty acid synthesis from malonyl-CoA whereas preimmune with antibody or control gamma-globulins. Fatty acid synthesis control gamma globulins showed no inhibition (Fig. 1). When was assayed for 40 min at 300C, after which the reaction was 0.15 MM ACP was added to similar incubations, a 4-fold stim- stopped by the addition of 0.25 ml of 20% KOH/50% isopro- ulation in fatty acid synthesis occurred. This ACP-stimulated panol and heated at 80°C for 30 min. The mixture was acidified activity was also abolished by addition of anti-ACP immuno- and extracted with petroleum ether, and a portion of the extract globulins. Thus, both in the presence and absence of added was assayed for radioactivity. Thin-layer chromatographic ACP, de novo fatty acid synthesis was abolished by antibodies analysis of the extract indicated that at least 95% of the radio- to ACP. These results therefore document the essential re- active products comigrated with free fatty acid standards in a quirement of ACP for all de novo fatty acid biosynthesis in the petroleum ether/diethyl ether/acetic acid, 80:20:1 (vol/vol), spinach leaf cell. solvent system. Immunological Crossreactivity of Different ACPs. The Protein and chlorophyll concentrations were estimated by anti-ACP immunoglobulins were used to develop a radioim- the methods of Bradford (16) and MacKinney (17), respec- munoassay for ACP. Fig. 2 presents a standard curve for this tively. assay in which increasing amounts of unlabeled spinach ACP Protoplast Preparation. Spinacia oleracea var. Hybrid High compete with 125I-ACP for the antibody-combining sites. Under Pack (Asgrow Seed Co., Kalamazoo, MI) was germinated and these conditions the assay is capable of detecting 10-11 g or grown in vermiculite in a growth chamber with an 8-hr- 10-' mol of ACP. light/16-hr-dark regimen. Seedlings, 3-4 weeks old, were Although ACPs from different species have similar amino transplanted into aerated jars containing a modified Hoagland's acid compositions (12) and similar amino acid sequences ad- solution (18) and grown hydroponically for 3-4 weeks in the jacent to the 4'-phosphopantetheine side chain (25), their im- same light regimen. Mature leaves were allowed to wilt for 15 munologic crossreactivity was limited. Fig. 2 indicates the min; then the lower epidermis was abraded by brushing with ability of E. coli ACP and crude homogenates of soybean and carborundum (19) and the leaves were plasmolyzed by floating sunflower seeds to compete with spinach 1251-ACP. Similar on 0.85 M mannitol/0.02 M 4-morpholinepropanesulfonic acid curves were obtained with cowpea and soybean leaf homoge- (MOPS), pH 7.0, for 30 min. nates. The non-spinach preparations apparently can compete The mannitol was replaced with a cell wall-digesting solution with only a portion of the antibody-combining sites because (15 ml/g fresh weight of leaf tissue). The solution consisted of addition of greater quantities of these preparations did not 0.5% Cellulysin (Calbiochem), 0.5% Macerase (Calbiochem), decrease the spinach '251-ACP binding beyond 20-40%. 0.5% dextran sulfate (Calbiochem), 50 mM 4-morpho- Localization of ACP in Spinach Cells. Fig. 3 shows the linethanesulfonic acid (MES), pH 5.8, 0.85 M mannitol, and a distribution of ACP after sucrose density gradient centrifuga- salt solution whose composition is described in ref. 20. The tion of lysed spinach leaf protoplasts. The maximum at 1.23 Downloaded by guest on September 29, 2021 1196 Biochemistry: Ohlrogge et al. Proc. Natl. Acad. Sci. USA 76 (1979)

1.2 _ 0 E U -0

1.1 Ca 0@2

0

50 Immunoglobulin, ,ig FIG. 1. Inhibition of fatty acid synthesis by anti-ACP immuno- globulins. Spinach leaf homogenate (100 jig of protein; 6 /ig of chlo- rophyll) was preincubated for 1 hr with immune (@) or preimmune control (0) immunoglobulins. Fatty acid synthesis from [14C]mal- onyl-CoA was then assayed.

g/cm3 and the shape of the ACP peak coincided with those for chlorophyll and for chloroplast enzymes ADP-glucose pyro- phosphorylase and ribulose-bisphosphate carboxylase. The absence of other ACP peaks in the gradient indicated that no other organelles such as mitochondria (indicated by isocitrate 0_ dehydrogenase activity in Fig. 3) or peroxisomes contained @ ACP. @2c 4.0 c The small amount of ACP recovered at the top of the sucrose 0 gradient can be attributed to release from broken chloroplasts. The extent of chloroplast breakage was estimated from the ~0 recovery of chlorophyll and the activity of the chloroplast en- @2 R 2.0 (, _s

x 16 E u 10 20 30 U12- Fraction FIG. 3. Distribution of ACP (gg/ml), chlorophyll (jig/ml), ribu- 8 X lose-bisphosphate (RubP2) carboxylase (jumol/min per ml), ADP- 0 glucose pyrophosphorylase (jimol/min per ml), and isocitrate dehy- §3 4 drogenase (4mol/min per ml) in sucrose gradients of lysates of spinach leaf protoplasts. All gradient fractions were 1 ml.

0 0.4 0.8 1.2 zymes pyro- ACP, ng ribulose-bisphosphate carboxylase, ADP-glucose phosphorylase, and NADP triosephosphate dehydrogenase FIG. 2. Radioimmunoassay of spinach ACP and crossreactivity found in the intact chloroplast (22-24). by other species. Antibody was preincubated with the indicated Table 1 summarizes the recovery of ACP, chlorophyll, and amounts of unlabeled ACP after which 1251-ACP was added. After 2 these marker enzymes for three experiments. For each exper- hr of incubation the amount of 1251-ACP bound to antibody was de- iment, chloroplast intactness was estimated by averaging the termined. A, Spinach; X, soybean; O, sunflower; 0, E. coli. The units on the abscissa are arbitrary for soybean and sunflower because pu- percentages of chlorophyll and marker enzymes recovered in rified ACP from these species was not available. the chloroplast peak. This value was then used to calculate the Downloaded by guest on September 29, 2021 Biochemistry: Ohlrogge et al. Proc. Natl. Acad. Sci. USA 76(1979) 1197 Table 1. Recovery of ACPM chlorophyll, and chloroplast marker tively, and (iv) using gently lysed protoplasts as the source for enzymes in chloroplasts isolated by sucrose gradient intact organelles-in particular, the chloroplast. centrifugation of spinach protoplast lysate The results reported here clearly establish that the incorpo- ration of [14C]malonyl-CoA into fatty acid by homogenates of Recovery in chloroplast peak, % site of RubP2 Triose-P ADP-glc spinach leaf is totally ACP-dependent and the exclusive Chloro- carbox- dehydro- pyrophos- ACP in the leaf cell is the chloroplast. Exp. ACP phyll ylase genase phorylase Because ACP appears to be a specific marker for plant nonassociated fatty acid synthetase activity, it follows that, in 1 82 75 - the leaf cell, the chloroplast is the specific site for fatty acid 2 76 82 78 87 83 synthesis. Thus, in addition to its role in CO2 fixation, ATP 3 85 80 75 80 generation, and 02 formation, the chloroplast plays a key role Abbreviations: RubP2, ribulose-bisphosphate carboxylase; Triose-P in the de novo synthesis of palmitic and oleic acids (1) which dehydrogenase, NADP triosephosphate dehydrogenase. must then be transported to the cytoplasmic compartment for further modification and incorporation into the membrane proportion of ACP at the top of the gradient attributable to lipids of the leaf cell. release from broken chloroplasts. From the three experiments It has been known for some time (1) that the primary product shown in Table 1, 109, 92, and 104% of the ACP recovered on of de novo synthesis in the chloroplast is palmitoyl-ACP which the sucrose gradient was attributable to chloroplasts. The is then elongated to stearoyl-ACP. In the chloroplast, both amount of ACP recovered from the sucrose gradient was also stearoyl-ACP desaturase and oleoyl-ACP hydrolase occur and compared with the amount of ACP in the protoplasts before function jointly in the formation of the principal product of centrifugation. Approximately 110% of the ACP applied to the fatty acid synthesis in isolated chloroplasts (1)-namely, free gradient was recovered after centrifugation. To exclude non- oleic acid with an accompanying regeneration of free ACP. specific binding of ACP to organelles during sucrose gradient Free oleic acid can then move through the envelope of the centrifugation, 3 tig of spinach 125I-ACP was added to a pro- chloroplast and be converted to oleoyl-CoA by a long chain toplast lysate before centrifugation. Less than 1% of the ra- acyl-CoA synthetase that is associated with this membrane (30). dioactive ACP in this experiment was associated with chloro- Once in the cytoplasmic compartment, oleoyl-CoA can either plasts and greater than 95% was recovered at the top of the be inserted into membrane lipids or be further modified by a sucrose gradient. poorly defined extrachloroplast organelle to linoleic and a- Essentially all of the ACP in the spinach leaf cell is therefore linolenic acids (31, 32). These acids may then be transported localized in the chloroplast as evidenced by its distribution in back to the chloroplast to be inserted into the lipids of the la- the gradient, total recovery, and the absence of nonspecific mellar membranes. This mechanism would then resolve the binding. paradox that, although isolated chloroplasts readily synthesize From the data in Fig. 3, the concentration of ACP in the palmitic and oleic acids, they do not form linoleic and a-lin- chloroplast can be calculated. The volume of spinach chloro- olenic acids which make up more than 80% of the total fatty plasts has been estimated to be 24 ,um3 (26) and the number of acids of their lamellar membrane lipids. a-Linolenic acid bio- chloroplasts per mg of chlorophyll is 1.5 X 109 (27). From these synthesis via hexadecatrienoic acid, an earlier proposal (33) data the ACP concentration in the spinach chloroplast can be makes an additional but probably minor contribution to the calculated to be approximately 8 ,uM. This value is considerably specific formation of a-linolenic acid in the spinach leaf cell. lower than the ACP level in E. coli which is calculated to be In conclusion, in documenting the absolute requirement for 100-200 ,M from the data of Kubitschek (28) and Alberts and ACP in de novo fatty acid synthesis in leaf tissue and the precise Vagelos (29). location of ACP in chloroplasts, these studies demonstrate that chloroplasts, in addition to their classic role of CO2 fixation and DISCUSSION 02 evolution, also must carry out an additional role-namely, Previous studies (1, 4-8) have demonstrated that both chloro- fatty acid synthesis and the export of the products of this syn- plasts and proplastids from nonphotosynthetic tissues isolated thesis to the cytoplasmic compartment for multiple uses. by conventional techniques are major sites of fatty acid bio- We thank Ms. Barbara Clover for her assistance in the preparation synthesis. However, clear-cut data documenting the contri- of this manuscript. This research was supported by National Science bution of various organelles and cytoplasmic proteins in the Foundation Grant PCM76-01495. plant cell to de novo fatty acid biosynthesis are difficult to ob- tain for the following reasons: (i) considerable breakage occurs 1. Stumpf, P. K. (1977) in MTP International Review of Science, during the isolation procedures, causing leakage of organelle Biochemistry of Lipids II, ed. Goodwin, T. W. (Butterworth, proteins into the cytoplasmic fraction; (ii) comparison of the London), Vol. 14, pp. 215-238. 2. Vagelos, P. R. (1974) in MTP International Review of Science, activity recovered in the organelle fractions with that present Biochemistry of Lipids I, ed. Goodwin, T. W. (Butterworth, before fractionation is incomplete; (iii) plant enzymes are London), Vol. 4, pp. 100-140. subject to major losses of activity upon cell disruption, and re- 3. van den Bosch, H., Williamson, J. R. & Vagelos, P. R. (1970) ported subcellular activities may represent minimal values; (iv) Nature (London) 228,338-340. fatty acid synthesis requires the concerted activity of at least 4. Nakamura, Y. & Yamada, M. (1974) Plant Cell Physiol. 15, six enzymes, and low activity for total synthesis may relate to 37-48. the inactivation of only one of these proteins; and (v) optimal 5. Simcox, P. D., Reid, E. E., Canvin, D. T. & Dennis, D. T. (1977) substrate and cofactor additions may not have been rigorously Plant Physiol. 59, 1128-1132. established and the omission from or addition of suboptimal 6. Weaire, P. J. & Kekwick, R. G. 0. (1975) Biochem J. 146, levels of ACP to a fraction could lead to erroneous results. 425-438. 7. Vick, B. & Beevers, H. (1978) Plant Physiol. 62, 173-178. These problems were resolved by (i) selecting a stable, pure 8. Kleinig, H. & Liedvogel, B. (1978) Eur. J. Biochem. 83, 499- marker protein for fatty acid synthesis (namely, ACP), (ii) 506. preparing antibodies to this protein, (iii) developing a radio- 9. Nishimura, M., Graham, D. & Akazawa, T. (1976) Plant Physiol. immunoassay for detecting small amounts of ACP quantita- 58,309-314. Downloaded by guest on September 29, 2021 1198 Biochemistry: Ohlrogge et al. Proc. Natl. Acad. Sci. USA 76 (1979)

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