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Subcellular Localization of Acyl Carrier Protein in Leaf Protoplasts Of

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Subcellular Localization of Acyl Carrier Protein in Leaf Protoplasts Of 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the levels of ACP in leaf cell components isolated by sucrose gradient centrifugation of a gentle lysate of spinach (unpublished data).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.
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