Pl nm

Planta (1988) 175:145-152

9 Springer-Verlag 1988

Immunogold localization of acyl carrier protein in plants and Escherichia colt': Evidence for membrane association in plants A.R. Slabas* and C.G. Smith Biosciences Division, Unilever Research, Colworth House, Sharnbrook Bedford, MK44 1LQ, U K

Abstract. Immunogold labelling was used to study the distribution of acyl carrier protein (ACP) in Escherichia coli and a variety of plant tissues. In E. coli, ACP is distributed throughout the cytoplasm, confirming the observation of S. Jackowski et al. (1985, J. Bacteriol., 162, 5-8). In the mesocarp of Avocado (Persea americana) and maturing seeds of oil-seed rape (Brassica napus cv. Jet Neuf), over 95% of the ACP is localised to plastids. The protein is almost exclusively located in the chloroplasts of leaf material from oil-seed rape. Approximately 80% of the gold particles associated with the ACP were further localized to the thylakoid membrane of the chloroplast. Since acetyl-CoA carboxylase has been reported to be localized to the thylakoid membrane (C.G. Kannangara and C.J. Jensen, 1975, Eur. J. Biochem., 54, 25-30), these results are consistent with the view that the two sequential enzymes in fatty-acid synthesis are in close spacial proximity.

Key words: Acyl carrier protein - Brassica (acyl carrier protein) - Escherichia - Lipid synthesis Persea - Plastid (acyl carrier protein) - Thylakoid membrane.

Introduction

The de-novo biosynthesis of fatty acids, from malonyl CoA and acetyl CoA, is catalysed by the enzyme fatty-acid synthetase (FAS). In plants and most bacteria this enzyme complex has been demonstrated to be composed of seven discrete separable polypeptides (Stumpf and Shimakata 1983). Six of the polypeptides have a catalytic function, and the seventh, acyl carrier protein (ACP), plays a * To whom correspondence should be addressed Abbreviations: ACC = acetyl CoA carboxylase; ACP = acyl carrier protein; FAS = fatty-acid synthetase

central role as a cofactor/carrier of the growing acyl chain (Stumpf and Shimakata 1983). Acyl carrier protein thus serves an equivalent role, in the biosynthesis of fatty acids, as coenzyme A does in their breakdown by/?-oxidation. Acyl carrier protein has been the most extensively studied protein of plant FAS. It has been purified from several plant sources including leaves (Simoni et al. 1967; Hoj and Svendson 1983) seeds (Slabas et al. 1987), and oil-forming tissues such as avocado mesocarp (Simoni et al. 1967). Antibodies against spinach ACP have been shown to be immuno-cross-reactive with ACP from other plant species (Ohlrogge and Kuo 1984). The basis of this cross-reactivity is probably centred around a group of 13 amino acids Leu-Gly-Ala-Asp-SerLeu-Asp-Thr-Val-Glu-Ile-Val-Met which exhibit absolute sequence conservation in all plant ACPs sequenced to date (Slabas et al. 1987). The serine residue underlined above is the residue which is post-translationally pantethenylated, and is at the active site of the protein. Localization of ACP was first reported by Van den Bosch et al. (1970), following autoradiography and electron microscopy, of an Escherichia coli/?alanine mutant fed /?-[3H]alanine which should have been specifically incorporated into ACP. In their study they concluded that ACP was localized to the inner membrane. It has subsequently been shown using immunoelectron microscopy that ACP is distributed throughout the cytoplasm of E. coli (Jackowski et al. 1985). This latter observation is more consistent with the soluble dissociated nature of E. coli FAS and the soluble nature of the enzyme involved in the ACP-dependent biosynthesis of lipid-A precursors (Anderson and Raetz 1987). In our initial studies aimed at the immunoelectron-microscopic localization of ACP in plant tissue we independently sought to localize ACP, in E. coli, to establish the prerequisite methodolgy. Our results confirm those of Jackowski et al.

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A.R. Slabas and C.G. Smith: Immunogold localization of acyl carrier protein

(1985) i n t h a t A C P is p r e s e n t t h r o u g h o u t t h e c y t o p l a s m o f E, coli. In plants, fatty-acid biosynthesis occurs in two t y p e s o f s u b c e l l u l a r o r g a n e l l e s . T h e c h l o r o p l a s t is the site o f s y n t h e s i s i n t h e leaf, w h i l s t t h e p l a s t i d is the site i n o i l - f o r m i n g tissues ( S t u m p f a n d Shim a k a t a t a 1983). O h l r o g g e et al. (1979), u s i n g a r a d i o i m m u n o a s s a y , have localised A C P to chloroplasts derived f r o m leaf protoplasts following subc e l l u l a r f r a c t i o n a t i o n . N o o t h e r tissues h a v e b e e n e x a m i n e d t o d a t e a n d t h e r e is n o i n f o r m a t i o n o n the s u b o r g a n e l l a r d i s t r i b u t i o n o f A C P i n p l a n t tissue. I n t h e p r e s e n t s t u d y , use h a s b e e n m a d e o f t h e p o s t - e m b e d d i n g i m m u n o g o l d t e c h n i q u e to v i s u a l i s e d i r e c t l y the s u b c e l l u l a r l o c a l i s a t i o n o f ACP in a variety of plant materials, including rape l e a f a n d seed, a n d a v o c a d o m e s o c a r p .

Materials and methods Material Rape (Brassica napus L. cv. Jet NeuD seed was obtained from Quenby Price, Bedford, UK. Plants were fieldgrown and leaf material was collected from young plants. Seed material was collected from plants 30 50d post anthesis as judged by the size of the embryo. Avocado (Persea americana L) fruit was purchased from a local supplier. Escherichia coli was grown on nutrient broth (Oxoid, Basingstoke, UK)) overnight at 37~ C. Rape seed ACP was prepared as its 3H-palmityl derivative as described previously (Slabas et al. 1987). Spinach leafACP was prepared by the method of Simoni et al. 1967. Escherichia coli ACP was prepared by the method of Majerus et al. (1969). Antibodies against spinach ACP were raised in rabbits The ACP was purified to electrophoretic homogeneity and 100 pg linked to 1 mg of keyhole-limpet haemocyanin (Sigma Chemical Co., Poole, Dorset, UK) using glutaraldehyde, Three injection each of 33 Ixg of ACP were given. The first in Freund's complete adjuvant and the two subsequent booster injections in incomplete adjuvant at two-week intervals. Serum was collected two weeks after the final injection. When rape-ACP-absorbed rabbit anti-spinach ACP was used experimentally it was prepared as follows: the serum was diluted 1:250 (v/v) in 200 mM 2-amino2-(hydroxymethyl)-l,3-propanediol(Tris)-HC1 pH 7.4 containing 450 mM NaC1 and rape-seed [3Hlpalmityl ACP, 30 gg (3 nmol) in 50 gl of 0.05 M Tris-HC1 pH 7.5, was added. The complex was allowed to stand at room temperature for 4 h, after which it was used directly for immunolabelling. Initial experiments were performed with an antibody against spinach 9leaf ACP which was kindly provided by Dr. John Ohlrogge (Department of Botany and Plant Pathology, Michigan State University, East Lancing, USA). Antibodies against E. coli ACP were raised in sheep. Electrophoretically homogeneous E. coli ACP (15 rag) was cross-linked with glutaraldehyde and 5 mg was injected intramuscularly into a sheep in Freund's complete adjuvant. The animal was reinjected with 5 mg of cross-linked ACP in phosphate-buffered

saline (PBS) at two sites subcutaneously on days 40 and 44 after the initial incubation. On day 50, a final bleed was taken from ~he animal. The antibody was affinity-purified by passing it down a column of immobilised E. coli ACP. Tissue preparation for immunoetectron microscopy a) Plant material. Developing rape embryo, rape leaf and avocado mesocarp tissue (mesocarp tissue adjacent to epicarp, and tissue adjacent to endocarp) were fixed in either 1% freshly prepared paraformaldehyde +0.05% glutaraldehyde or 3"/o paraformaldehyde in 0.05 M sodium-phosphate buffer pH 6.8 at 4~ for 2 h. Following an overnight wash in phosphate buffer, the tissues were dehydrated through alcohol, and placed in several changes of hydrophilic resin (3 parts LR Gold [London Resin Co., Woking, Surrey, UK], 2 parts GMA-glycol methacrylate [Polysciences Inc., Warrington, Pa., USA] - low acid, 0.1% benzoin ethyl ether LRG/GMA). The tissue was finally embedded in the above resin, in gelatin capsules, and polymerised for 24 h at room temperature by illumination with ultraviolet light at 360 nm. b) Escherichia coli. A suspension of E. coli on growth medium was gently pelleted on a bench centrifuge, and the growth medium removed. The cells were then fixed for 30 min at ice temperature in either 1% paraformaldehyde+005% glutaraldehyde or 3% paraformaldehyde in PBS pH 7_4. The fixative was removed and the pelleted celIs embedded in 1% agar and subsequently given a further fixation for 30 min. Following an overnight wash in PBS at 4~ C the cells were dehydrated and embedded as described above. Immunostaining Ultrathin sections were collected on formvar (2% in amyl acetate)-coated nickel grids and placed on 10-1~1aliquots of 1% (w/w) ovalbumin (Sigma) in Tris-buffered saline (TBS) containing 0.2 M Tris-HCl pH 7.4, 450 mM NaC1 for 20 rain at 37~ C. a) Plant material The grids were then floated on 10-~tl aliquots of rabbit anti-ACP antibody 1 : 250 (v/v) dilution, in TBS+0.1% Tween 20 (Sigma) for 60 min at 37~ C. They were washed thoroughly with TBS, and then incubated with goat anti-rabbit/colloidalgold (Janssen, Wantage, Oxfordshire, UK) 15 nm diameter, diluted 1:50 (v/v) for 30 rain at room temperature. Grids were then washed thoroughly with TBS, and then distilled water. The sections were stained with 2% aqueous uranyl acetate, and lead citrate before examination in the transmission electron microscope. Control experiments, of the following type were performed, (1) without the primary antibody, (2) using pre-immune rabbit serum and (3) using primary antibody adsorbed with ACP. b) Eseherichia coli. Sections were floated on t0-pI aliquots of sheep-anti E. coli ACP, 1:200 (v/v) dilution in TBS-ovalbumin+0A% Tween 20 for 60 rain at 37~ C, washed throughly using TBS, and subsequently incubated in rabbit anti-sheep IgG (Miles Scientific, Slough, Bucks., UK) l:100 (v/v) dilution in TBS-ovalbumin+0.1% Tween 20 (polyethylene sorbitan monolaurate) for 60 min at 37~ C. Following washing in TBS the grids were incubated with goat anti-rabbit IgG-colloidal gold as described above.

Results S t u d i e s with E. coli W e initially investigated the localization of A C P i n E. coli u s i n g s h e e p a n t i - E , coli A C P a n t i b o d i e s .

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Fig. 1 a, b. Longitudinal (a) and transverse (b) sections of E. coli showing ACP localized throughout the cytoplasm of the cell. x 70000; b a r = 1 p-m

The results from this experiment are shown in Fig. 1 where it can be clearly seen that ACP is distributed throughout the cytoplasm and not localised to the membranes. Rabbit anti-spinach ACP antibodies and pre-immune serum failed to give any appreciable staining, indicating the specificity of the antiserum.

Studies with plant material (a) Preservation of antigenieity and ultrastruetural quality. Both fixatives allowed retention of anti-

genicity; however, 1% paraformaldehyde + 0.05% glutaraldehyde gave better ultrastructural preservation so the latter was used routinely in this study. (Fig. 2). (b) Localisation of ACP in leaf material. The localization of ACP in rape leaf material was investigated. Sheep anti-E, coli ACP antibodies failed to label the plant material investigated, indicating that the primary epitopes in E. coli ACP are different from those of plant ACP. Plant material was therefore investigated using rabbit anti-spinach ACP antibodies. Figure 3 a shows an electron mi-

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A.R. Slabas and C.G. Smith: Immunogold localization of acyl carrier protein

Fig. 2a, b. Rape leaf material fixed in 3% parafolnnaldehyde (a), and 1% paraformaldehyde+0.5% gtutaraldehyde (b). a x 24500, b x 17500; b a r s = / p m

crograph typical of such an experiment. The chloroplasts are heavily labelled. However, occasional labelling of mitochondria has been observed (data not shown). Labelling of the cell matrix is virtually absent. The chloroplasts have retained a reasonable degree of ultrastructural preservation, and the thylakoid membranes are clearly visible. The majority of gold particles are in association with the thylakoid membranes, with a low level of labelling in the stroma, the membrane-free area of the chloroplast. Simple analysis of the results allowed quantification of these observations. The data presented in Table 1, show that approx. 80% of the ACP was localized to the thylakoid membrane whilst the remaining approx. 20% was localized to the stroma of the plastid. The distribution of colloidal gold particles could be a reflection of the volumes occupied by the stroma and thylakoids in such preparations. If this were so, there would be no apparent suborganellar localization. To further investigate this possibility we have analysed the percentage area of the chloroplast, in these experiments, occupied by thylakoids and stroma. This was done using a Quantimet image-analysis system. The results, from five electron-microscope grids, gave values

for the percentage area occupied by the thylakoids of 39.45, 34.15, 50.02, 47.00 and 36.83%, which have a mean value of 41.49%. These data clearly indicate that the bias of the distribution, of gold particles to the thylakoid cannot be simply explained by the percentage area occupied by the thylakoids, and that there is indeed a true subcellular concentration. Furthermore, if one considers that there is no reason to believe that ACP is localized internally in the thylakoids, since it has to interact with other FAS components in the stroma, then the data on the percentage area occupied by the thylakoids will be a gross over-estimate of the actual area occupied by its external membrane surface. These considerations taken together, lead us to our conclusion, that ACP is concentrated around the thylakoids. Similar experimental results (data not shown) were obtained with leaf material from courgette (Cucurbita pepo ouifera) with 76% of the ACP thylakoid-associated. The use of rape-ACP-adsorbed serum resulted in total loss of gold labelling (Fig. 3 b). This demonstrates the specificity of the antiserum. (c) Localisation of ACP in rape-seed material. To our knowledge, no one has previously attempt-

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Fig. 3. a Localization of ACP in rape leaf showing ACP localized to the chloroplast (ch). nuc, nucleus; cw, cell wall. b Control experiment in which ACP antibody was adsorbed with ACP. a x 18425, b x 20000; b a r = 1 gm Fig. 4. Localization of ACP in rape embryo. The chloroplasts (ch), have weakly developed thylakoids and contain starch grains (sg). Localization of A C P is restricted to the plastid, nuc, nucleus; cw, cell wall; oh, oil body. x ~6250; b a r = 1 gm

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Table 1. Immunogold localization of ACP in rape leaf chloroplasts Sample (Micrograph)

Chloroplast

Total gold particies per chloroplast

Thylakoid-membraneassociated gold particles

%Thylakoidassociated gold particles

1

a b c a b c a b c a b c a b c d a b a b

37 55 24 26 36 24 38 60 38 56 48 29 33 52 18 49 36 43 46 39

30 40 17 21 32 19 31 49 32 39 34 25 26 43 15 34 30 31 39 31

81.09 72.72 70,83 80.76 88.88 79.16 81.57 81.66 84.21 69.64 70.83 86.20 78.78 82.69 83.33 69.38 83,33 72.09 84.78 79.48

2

3

4

5

6 7

79.07%

ed to localize ACP in any material other than leaf (Ohlrogge et al. 1979). It is known that lipid biosynthesis occurs predominantly in plastids in such material. Figure 4 shows an electron micrograph of immunogold labelling of an oil-seed-rape embryo. The label is almost exclusively localized to the plastids, but again some mitochondrial labelling is occasionally seen (not shown). The plastids resemble the chloroplast of the leaf in ultrastructure, and the ACP seems to be associated with the thylakoid membranes. N o appreciable labelling is seen in or around the oil bodies. The plastid localization of ACP is consistent with their role as a major site of lipid biosynthesis. (d) Localization of ACP in avocado mesocarp. Examination ofmesocarp tissue from the inner and outer regions of the mesocarp showed that, as suspected, plastid ultrastructure differed in these two regions. The plastids from the outer layer of mesocarp, adjacent to the epicarp showed a chloroplastlike ultrastructure with thylakoid membranes (Fig. 5 a), whilst the plastids in the mesoderm adjacent to the endocarp (Fig. 5b) were typically etioplast-like, containing prolamellar bodies. Despite the difference in plastid ultrastructure, ACP localization was again confined to the plastid in both inner and outer layers of mesocarp tissue. No firm conclusion can, however, be drawn on a sub-plastid localization of ACP in such tissue.

Discussion

The use of immunogold labelling has allowed us to observe the distribution of ACP in a wide variety of experimental tissues. In E. coli we have observed that ACP is distributed throughout the cytoplasm, so confirming the observation of Jackowski et al. (1985). In all the plant tissues investigated, ACP was localized almost exclusively to plastids - consistent with the plastid localization of lipid biosynthesis. Although the plastids investigated differed considerably in ultrastructure, ACP has been demonstrated in these studies to be a universal immunocytochemical marker for these organelles. This indicates that it could potentially be used to demonstrate the developmental relatedness of various plastids. Such a procedure would overcome any bias resulting from morphological similarity or artifacts brought about by homogenization procedures which can damage cellular components, releasing soluble proteins. As an example of a homogenization artifact, Hanks et al. (1981) could find only about 15% of the total uricase activity in the peroxisomal fraction following differential centrifugation, presumably because of the fragile nature of the organelles - despite the unique localization of this enzyme to peroxisomes. The immunogold-localization technique has proved particularly valuable in demonstrating directly the suborganellar distribution of ACP in

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151

Fig. 5 a, b. Localization of ACP in Avocado mesocarp. Plastids from the outer mesocarp (a) show typical chloroplast (ch) ultrastructure, whilst ptastids from the inner mesocarp (b) are etioplast-like with prolamellar bodies (pb). In both examples the ACP is localized to the plastid, a • 25000; bar = 1 gm. b x 50000; bar=0.1 p.m

chloroplasts. Biochemical isolations of ACP from leaf material have not indicated any association of ACP with organeUes. The protein is soluble, and does not require detergent to solubilise it (Simoni et al. 1967); this is in contrast to the situation with seed ACP which does requires detergent (Slabas et al. 1987). The membrane association of ACP is particularly interesting when one considers that early studies by Kannangara and Jensen (1975) had led them to the conclusion that acetyl-CoA carboxylase is thylakoid-bound, a view substantiated by the requirement of detergent to solubilise ACC from rape-seed material (Slabas and Hellyer 1985). Since ACP provides the substrate for plant FAS, it is possible that the two enzymes are in close proximity in vivo. This indicates that there is potentially a localized high concentration of malonyl CoA around FAS. If this were so, then measurements of interorganellar concentrations of malonyl CoA, and their correlation to Kms of the enzyme, and its activity, would be relatively meaningless. It has not been possible to demonstrate an association of components of plant FAS. Shimakata and Stumpf (1982), in a series of elegant

experiments, purified several of the individual components of plant FAS. Using the purified preparations it did not, however, prove possible for them to demonstrate any re-association of an enzyme complex. If such a complex does exist in vivo, our study indicates that immunoelectron microscopy would be an ideal method of resolving the issue, causing minimal perturbation to the system. We are currently purifying several of the components of plant FAS such as enoyl ACP reductase (Slabas et al. 1986) in order to examine this hypothesis. In view of the occasional detection of very low levels of ACP localization in mitochondria and the low concentration of mitochondria in plants we have endeavoured to ascertain the significance of this. Using the spadix of Arum maculatum, which is highly enriched with mitochondria, we have been unable to detect any ACP using our colloidal-gold method. This indicates to us that the occasional detection in mitochondria is probably a background-staining effect. We would like to thank Dr. John Ohlrogge (Michigan State University, East Lansing, USA) for kindly providing us with rabbit anti-spinach ACP antibodies which were used in initial studies.

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References Anderson, M.S., Raetz, C.R.H. (1987) Biosynthesis of lipid A precursors in Escherichia eoli. J. Biol. Chem 261, 5159-5169 Hoj, P.B., Svendsen, I. (1983) Barley acyl carrier protein: its amino acid sequence and assay using purified malonyl CoA: ACP transacylase. Carlsberg Res. Commun. 48, 285-305 Hanks, J.F., Tolbert, N.E., Schubert, K.R. (1981) Localization of enzymes of ureide biosynthesis in peroxisomes and microsomes of nodules. Plant Physiol. 68, 65-69 Jackowski, S., Edwards, H.H., Davis, D., Rock, C.O. (1985) Localization of acyl carrier protein in Eseherichia eoli. J. Baeteriol. 162, 5-8 Kannangara, C.G., Jensen, C.J. (1975) Biotin carboxyl protein in barley chloroplast membranes. Eur. J. Biochem. 54, 25-30 Majerus, P.W., Alberts, A.W., Vagelos, P.R. (1969) Acyl carrier protein from Eseheriehia eoli. Methods Enzymol. 14, 43-50 Ohlrogge, J.B., Kuhn, D.N., Stumpf, P.K, (1979) Subcellular localisation of acyl carrier protein in leaf protoplasts of Spinaeia oleraeea. Proc. Natl. Acad. Sci. USA 76, 11941198 Ohlrogge, J.B., Kuo, T. (1984) Control of lipid synthesis during soybean seed development: Enzymic and immunochemical assay of acyl carrier protein. Plant Physiol. "/4, 622-625 SimoN, R.D., Criddle, R.S., Stumpf, P.K. (1967) Fat metabolism in higher plants. XXXI. Purification and properties of plant and bacterial acyl carrier proteins. J. Biol Chem. 242, 573-581

Shimakata, T., Stumpf, P.K. (1982) Purification and characterisation of fl-ketoacyl-[acyl-carrier-protein] reductase, flhydroxyacyl-[acyl-carrier-protein] dehydrase, and enoyl[acyl-carrier-protein] reductase from Spinacia oleracea leaves. Biochem. Biophys. 218, 77-91 Slabas, A.R., Hellyer, A. (1985) Rapid purification of a high molecular weight sub-unit polypeptide form of rape seed acetylCoA carboxylase. Plant Sci. Lett. 39, 177-182 Slabas, A.R., Sidebottom, C.M., Hellyer, A., Kessell, R.M.J., Tombs, M.P. (1986) Induction, purification and characterisation of NADH-specific enoyl acyl carrier protein reductase from developing seeds of oil seed rape (Brassica napus). Biochim. Biophys. Acta 877, 271-280 Slabas, A.R., Harding, J., Hellyer, A., Roberts, P., Bambridge, H.E. (1987) Induction, purification and characterisation of acyl carrier protein from developing seeds of oil seed rape (Brassiea napus). Biochim. Biophys. Acta 921, 50-59 Stumpf, P.K., Shimakata, T. (1983) Molecular structures and functions of fatty acid synthetase enzymes. In: Biosynthesis and function of plant lipids, pp. 1-15, Thomson, W.W., Mudd, J.B., Gibbs, M., eds., Waverley Press, Baltimore, Md., USA Van den Bosch, H., Williamson, J.R., Vagelos, P.R. (1970) Localization of acyl carrier protein in Eseherichia eoli. Nature 228, 338-341

Received 27 August; accepted 15 December 1987