The Plant Cell, Vol. 2, 195-206, March 1990 O 1990 American Society of Plant Physiologists

Acyl Carrier Protein (ACP) lmport into Chloroplasts Does not Require the Phosphopantetheine: Evidence for a Chloroplast Holo-ACP Synthase

Michael D. Fernandez’ and Gayle K. Lamppab3’ a Department of Biochemistry and Molecular Biology, University of Chicago, 920 East 58th Street, Chicago, lllinois 60637

lllinois 60637 Department of Molecular Genetics and Cell Biology, University of Chicago, 920 East 58th Street, Chicago,

lmport of the acyl carrier protein (ACP) precursor into the chloroplast resulted in two products of about 14 kilodalton (kD) and 18 kD when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Time, course experiments indicate that the latter is a modification derivative of the 14-kD peptide after the removal of the transit peptide. Substitution of serine 38 by alanine, eliminating the phosphopantetheine prosthetic group attachment site of ACP, produced a precursor mutant that gave rise to only the 14-kD peptide during import, showing that the modified form depends on the presence of serine 38. Furthermore, these results demonstrate that the prosthetic group is not essential for ACP translocation across the envelope or proteolytic processing. Analysis of the products of import by nondenaturing, conformationally sensitive gels showed reversal of the relative mobility of the 14-kD peptide and the modified form, raising the possibility that the modification is the addition of the phosphopantetheine. Proteolytic processing and the modification reaction were reconstituted in an organelle-free assay. The addition of coenzyme A to the organelle-free assay completely converted the 14-kD peptide to the modified form at 10 micromolar, and this only occurred with the wild-type substrate. Reciprocally, treatment of the products of a modification reaction with Escherichia coli phosphodiesterase converted the modified ACP form back to the 14-kD peptide. These results strongly support the conclusion that there is a holo-ACP synthase in the soluble compartment of the chloroplast capable of transferring the phosphopantetheine of coenzyme A to ACP.

INTRODUCTION

Acyl carrier protein (ACP) is a key component of the fatty acid synthetase complex in all organisms studied. ACP functions as a carrier of growing fatty acid chains, which are attached to the protein through a 4‘-phosphopante- theine prosthetic group. ACP is an acidic protein but, in contrast to ACP of fungi and animals, which exist as part of a large multi-functional fatty acid synthetase polypep- tide, the ACPs of plants and bacteria are small (9 kD to 1 O kD), nonassociated soluble proteins. The amino acid se- quences of Escherichia co// (Vanaman, Wakil, and Hill, 1968), spinach leaf (Kuo and Ohlrogge, 1984), and barley leaf [H0j and Svendsen, 1983) ACPs have been determined and exhibit extensive homology, particularly in the se- quence surrounding the phosphopantetheine binding site, at a “special” serine residue that is 38 amino acids from the N terminus in spinach ACP. In higher plants there are at least two isoforms of the protein that are expressed in a tissue-specific manner (H0j and Svendsen, 1984; Ohl- rogge and Kuo, 1985) and may have different functions (Guerra, Ohlrogge, and Frentzen, 1986).

’ To whom correspondence should be addressed

In higher plant leaves, ACP is localized in the chloroplast, where it appears that de novo fatty acid biosynthesis occurs (Ohlrogge, Kuhn, and Stumpf, 1979; Stumpf, 1980). lsolation and analysis of cDNAs generated from poly A’ selected RNA for a spinach ACP indicates that the genes for ACP are nuclear-encoded (Scherer and Knauf, 1987). Similar results have been presented for Brassica ACP (Safford et al., 1988). Nuclear-encoded chloroplast proteins are generally synthesized in the cytoplasm as precursor polypeptides with N-terminal transit peptides and post-translationally imported into the organelle, where they are proteolytically processed to their mature forms (Schmidt and Mishkind, 1986). The transit peptide of spin- ach ACP was tentatively identified as a 56-amino acid sequence (Scherer and Knauf, 1987) that is typically rich in serine, threonine, and the basic amino acids arginine and lysine.

The study of the import of proteins into the chloroplasts of higher plants has focused primarily on proteins involved in the reactions of photosynthesis (for review, see Keegs- tra, 1988). The analysis of the import and processing of an essential protein not involved in photosynthesis, such

196 The Plant Cell

as ACP, might, therefore, add new insights to our under- standing of these events, as well as confirm those more general features of the import pathway. Furthermore, post- translational modification of ACP provides an opportunity to study another aspect of this complex process. A recent report (Elhussein, Miernyk, and Ohlrogge, 1988) indicates that the enzyme holo-ACP synthase, responsible for the transfer of the phosphopantetheine prosthetic group from COA to ACP, is found in the cytoplasm of spinach leaf cells. This observation raised the possibility that the addi- tion of the phosphopantetheine is an essential step for ACP import, conferring an import competent conformation on the ACP precursor (pre-ACP). Only a minor amount of holo-ACP synthase activity was found associated with the chloroplast. On the other hand, apo-ACP has been found in the chloroplast upon overexpression of spinach ACP in transformed tobacco plants (Post-Beittenmiller, Schmid, and Ohlrogge, 1989).

We have undertaken an analysis of the import and proteolytic processing of spinach ACP. Mutational analysis provides direct evidence that the addition of the prosthetic group is not required for the import of the precursor protein. Furthermore, our results show that, upon import, the processed protein is rapidly modified to a slower migrating form. The modification is dependent on the spe- cial serine at position 38. In the presence of a soluble chloroplast extract that is capable of processing pre-ACP, the modification is stimulated by the addition of COA and Mg*+. Treatment of the modified form with E. coli ACP phosphodiesterase regenerates the 14-kD peptide. These results strongly support the hypothesis that the chloroplast itself indeed contains a holo-ACP synthase activity respon- sible for the addition of the phosphopantetheine prosthetic group. The implications of this are discussed in terms of the function of ACP.

RESULTS

lmport of Pre-ACP into Chloroplasts Yields Two Processed Forms

A 500-bp Hindlll-EcoRI fragment of the plasmid pCGNl SOL (Scherer and Knauf, 1987) containing a cDNA sequence coding for the spinach ACP I protein as well as a putative transit peptide sequence of 56 amino acids was subcloned into the plasmid vector ~16131. Figure 1 shows the resulting construct, which was linearized with EcoRl and used as a template to synthesize pre-ACP RNA in vitro. As shown by SDS-PAGE in Figure 2A, lane 1, trans- lation of this RNA by a reticulocyte lysate in the presence of "S-methionine produced a single band with an apparent molecular mass of about 21 kD, which is about 6 kD larger than expected for the ACP precursor based on amino acid composition, but consistent with previous SDS-PAGE es-

Figure 1. Structure of plBI-ACP500 Plasmid Used for in Vitro RNA Synthesis and Strategy To Produce the Mutant ACPASer.

The spinach ACP cDNA sequence was placed 3' to the T7 promoter as diagrammed. The plasmid was linearized with EcoRl for RNA synthesis with T7 RNA polymerase. The DNA sequence flanking the serine 38 codon (see asterisk) of the wild-type gene is shown, along with the corresponding amino acids for which it codes. The oligonucleotide sequence used to produce the serine- to-alanine substitution is presented immediately below, and the nucleotides changed are indicated in bold face. The bottom line gives the sequence of the mutant precursor, pre-ACPASer. The smaller arrow indicates the position of the diagnostic Mlul restric- tion site created immediately 5' to the new alanine codon.

timates of ACP precursor protein translated from spinach mRNA (Ohlrogge and Kuo, 1984). AI1 acyl carrier proteins migrate with an anomalously high apparent molecular weight, presumably because of their small size and relative lack of SDS binding sites (Rock and Cronan, 1979).

To investigate the ability of the putative transit peptide sequence to direct ACP into chloroplasts, radiolabeled precursor protein was incubated with intact chloroplasts isolated from young spinach leaves. After incubation, half of the organelles were treated with the protease thermo- lysin to remove any proteins on the exterior of the chloro- plasts, and then both the protease-treated and nontreated chloroplasts were washed, lysed, and separated into their membrane and soluble components. As shown in Figure 2A, lane 5 , the putative transit peptide was functional and sufficient to direct ACP transport into the chloroplast. From the single precursor, there arose two products in the

Chloroplast Import of ACP 197

A Spinach

I 2 3

B pea

1 2 3 4 5

T.P. M MT

T.P. M M S ST T

Figure 2. in Vitro Import of Pre-ACP into Chloroplasts Yields TwoProducts.

(A) The reticulocyte lysate translation products of pre-ACP (T.P.,lane 1) were incubated with spinach Chloroplasts, and the totalmembrane (M, lane 2), membrane after thermolysin treatment (M+ T, lane 3), total soluble (S, lane 4), and soluble proteins afterthermolysin treatment (S + T, lane 5) were analyzed. The openarrow indicates the position of the 14-kD product and the filledarrow indicates the position of the 18-kD product.(B) In vitro import of pre-ACP into pea Chloroplasts. The lanes arenumbered the same as (A).

soluble fraction, both lower in molecular weight and resist-ant to thermolysin treatment, indicating that the precursorwas proteolytically processed and sequestered to thestromal compartment of the chloroplast during the importreaction.

The size of the lower band, which we estimated asabout 14 kD, was consistent with the removal upon importof the 56-amino acid putative transit peptide by the chlo-roplast processing activity, i.e., "transit peptidase." Theappearance of the upper band (about 18 kD), typically themore abundant of the two products, was unexpected.Significantly, the relative ratio of these two bands wasvariable. Although not yet systematically analyzed in detail,our results suggest that the ratio of these two products issensitive to seasonal changes affecting the physiological/developmental state of the plant. In contrast to spinach,Chloroplasts isolated from young pea leaves consistentlygenerated more of the lower molecular weight product(see Figure 2B). The nature of these two products of pre-ACP import is investigated in the experiments describedbelow.

The Larger Import Product Is not a ProteolyticProcessing Intermediate

To explore the possibility that the upper band was aprecursor processing intermediate, a time course of import

was carried out in which spinach Chloroplasts were incu-bated with radiolabeled precursor protein for 1 min, 5 min,and 15 min, as illustrated in Figure 3. We argued that, ifthe upper band represented an intermediate form, it shouldappear first and then gradually give rise to the lower, fullyprocessed approximately 14-kD form over time. We sawthe opposite effect where the lower band appeared ingreater abundance at the 1 min time point and then theratio rapidly shifted so that by 15 min the upper band wasthe most prominent. This result indicated that the highermolecular weight product was a derivative of the lowermolecular weight form, suggesting a covalent modifica-tion of the 14-kD product upon import of ACP into theChloroplasts.

Conversion of the "Special" Serine to Alanine Doesnot Prevent Import but Eliminates Production of theLarger Import Product

Spinach ACP is known to be modified by the addition of a4'-phosphopantetheine prosthetic group attached, via aphosphodiester bond, exclusively at a serine 38 aminoacids from the start of the mature protein. Evidence hasbeen presented that the major site of this addition is thecytoplasm (Elhussein et al., 1988), but it has not beenexcluded that there is also addition of the prosthetic groupin the chloroplast itself. To determine whether the slowermigrating, apparently higher molecular weight product thatwe observed upon import resulted from a modification ofACP through the special serine, site-directed mutagenesis

1 2 3 4 5 2 3 4 5 2 3 4 5

T.P. M MT

S ST

M MT

S S M M+ +T T

S+

1 min 5 min 15 min

Figure 3. Time Course of Import Indicates That the Larger Prod-uct Is a Derivative of the 14-kD Peptide.

Reticulocyte lysate translation products are shown in lane 1, andlanes 2 to 5 are the same as those described in Figure 2 and asindicated below each lane. Translation products of pre-ACP wereincubated with Chloroplasts for 1 min, 5 min, and 15 min asindicated below each reaction. The positions of the 14-kD andmodified form are indicated by open and filled arrows, respectively.

198 The Plant Cell

was used to eliminate the phosphopantetheine binding siteby converting serine 38 to alanine (see Figure 1 andMethods). In addition to the serine-to-alanine conversion,the mutation also included a silent single-nucleotidechange immediately 5' to the new alanine codon thatcreated a unique Mlul restriction site, providing a rapidmethod of screening for mutated plasmids.

A restructured plasmid containing the desired substitu-tion was transcribed and translated as previously de-scribed for wild-type pre-ACP. The radiolabeled mutantprecursor, referred to as pre-ACPASer, was used in an invitro import reaction. If imported and processed, it should,by definition, give rise to apo-ACP with a single residuechange because the phosphopantetheine cannot beadded. The results of this experiment are shown in Figure4. The radiolabeled ACPASer precursor protein co-mi-grated with wild-type pre-ACP during SDS-PAGE (Figure4, lanes 1). The products of the import reaction, however,were strikingly different. As before, the wild-type precursorgave rise to two thermolysin-resistant products in thesoluble fraction. On the other hand, the pre-ACPASermutant produced only one band, migrating slightly fasterthan the approximately 14-kD product of wild-type import.Thus, the serine-to-alanine conversion resulted in a loss ofthe higher molecular weight derivative, indicating that the

smaller form of pre-ACP import is apo-ACP. Two importantpoints emerged from this mutational analysis. First, thelarger import product arises from a modification that de-pends on the presence of serine 38. Second, the phospho-pantetheine prosthetic group, which cannot be added topre-ACPASer, is not required for ACP translocation acrossthe chloroplast envelope or for proteolytic processing uponimport.

The minor difference in mobility of the lower molecularweight product of wild-type ACP import and the productof pre-ACPASer import is most likely due to an alteredconformation caused by the single serine-to-alanine mu-tation. A similar change in electrophoretic mobility has beenidentified for a mutant ACP that has Ser 38 converted tocysteine (Jaworski, Post-Beittenmiller, and Ohlrogge,1989b). Predictions of E. coli ACP secondary and tertiarystructure (Rock and Cronan, 1979; Mayo, Tyrell, and Pres-tegard, 1983) suggest that the special serine resides atthe base of a central hydrophobic corridor formed by foura-helices. Predictions of spinach ACP secondary structureare consistent with this model (M. D. Fernandez, unpub-lished data; Kuo and Ohlrogge, 1984). The substitution ofthe nonpolar alanine residue for the polar serine in thiscritical region might, therefore, change the overall confor-mation and migration of the protein.

ACP wild type ACP ASer

1 2 3 4 5 1 2 3 4 5

T.P. M M ST

T.P. M MT

Figure 4. In Vitro Import of the Pre-ACPASer Mutant ProducesOnly the 14-kD Form, Defined as Apo-ACP.Parallel in vitro import reactions were carried out using radiola-beled pre-ACP and pre-ACPASer proteins from reticulocyte ly-sates. Translation products are shown in lanes 1, and lanes 2 to5 are as described in Figure 2 and as indicated below each lane.The positions of apo-ACP and the modified form are indicated byopen and filled arrows, respectively.

Organelle-Free Processing Yields Two Products ThatCo-Migrate with Those from Import Reactions

We have recently developed an assay for organelle-freeprocessing of the precursor of light-harvesting chloro-phyll a/b-binding protein in which the transit peptide isremoved (Lamppa and Abad, 1987; Abad, Clark, andLamppa, 1989). In addition, the crude soluble extractprocesses the small subunit of ribulose-1,5-bisphosphatecarboxylase/oxygenase precursor, as has been observedin analogous experiments by Robinson and Ellis (1984).Using a similar protocol, we have prepared chloroplastsoluble extracts from spinach that contain an active proc-essing enzyme (see Methods). Our goal was to establishwhether direct removal of the transit peptide of pre-ACP,independent of translocation across the chloroplast enve-lope, generated the same products as the in vitro importreactions. Figures 5A and 5B show the products of orga-nelle-free processing reactions, using the wild-type andACPASer precursors synthesized in a wheat germ lysate,compared with the soluble products of import reactions,analyzed on a denaturing SDS-polyacrylamide gel. Theorganelle-free assay (lanes 2) generated products of thesame size as the import reactions (lanes 1) for both thewild-type and mutant substrates. That is, pre-ACP wascleaved and again gave two products, whereas pre-ACPASer yielded only one peptide, the smaller form. Incontrast to the import reactions, however, the solubleextracts always produced more of the 14-kD peptide thanof the higher molecular weight form from the wild-type

Chloroplast Import of ACP 199

A B1 2 3 4 1 2 3 4

D

Figure 5. An Organelle-Free Assay Reconstitutes Removal of theTransit Peptide and the Modification Reaction.

Analysis by denaturing and conformationally sensitive gels.(A) Analysis of the wild-type precursor products on denaturinggels. The soluble products of an import reaction using reticulocytelysate translation products from thermolysin-treated chloroplasts(lane 1), and the products of organelle-free processing reactionsusing translation products from a wheat germ lysate and solublechloroplast extracts prepared from pea (lane 2) and spinach (lane3) leaves were analyzed on a 15% SDS-polyacrylamide gel. Lane4 shows an organelle-free reaction to which CoA was added at afinal concentration of 10 ^M. The positions of apo-ACP and themodified form are indicated by open and filled arrows, respectively.(B) Analysis of the pre-ACPASer products on denaturing gels.Reactions identical to those described for the wild-type precursorwere carried out and the lanes are numbered the same.(C) Analysis of the wild-type precursor products on nondenatu-ring, conformationally sensitive gels. Samples from parallel reac-tions to (A) were analyzed on 20% conformationally sensitivegels. The lanes are numbered as above, and the positions of apo-ACP and the modified form are indicated by open and filled arrows,respectively.(D) Analysis of the pre-ACPASer products on conformationallysensitive gels. Reactions identical to those described for the wild-type precursor were carried out and the lanes are numbered thesame.

substrate. These results support the conclusion that the14-kD form is the primary product of the simple removalof the transit peptide. In addition, the organelle-free assayexhibits a modifying activity, yielding the 18-kD peptide,but it is less active than in intact organelles. Parallel reac-tions were carried out in the presence of a soluble extractfrom pea chloroplasts (Figures 5A and 5B, lanes 3), andwe usually found that, with a pea extract, less of the highermolecular weight form was produced than upon additionof the spinach extract. This difference in the ability of thespinach and pea extracts to produce the modified form isconsistent with the results obtained using intact organelles(see Figure 2B).

Conformationally Sensitive Gel Analysis of Import andProcessing Reaction Products

It has been well documented that modification of ACPthrough serine 38 leads to bands of altered mobility duringelectrophoresis. This phenomenon has been best charac-terized using E. coli ACP derivatives on nondenaturing or"conformationally sensitive" gels (Rock and Cronan, 1981;Rock, Cronan, and Armitage, 1981), and has also beenapplied to the analysis of spinach ACP derivatives (Jawor-ski, Clough, and Barnum, 1989a). ACP undergoes a pH-induced conformational change upon transition from thestacking gel (pH 6.8) to the separating gel (pH 9.0) that ischaracterized by the loss of «-helical content (Schulz,1975) and an expansion of the Stokes radius (Rock andCronan, 1979). Therefore, ACP derivatives that destabilizethe protein moiety migrate more slowly because of thispartial denaturation, and, conversely, ligands that stabilizethe protein migrate faster. These studies have shown thatthe conformational changes induced by addition of thephosphopantetheine prosthetic group to the unmodifiedprotein, apo-ACP, to produce holo-ACP and further addi-tion of various acyl chains yielding acyl-ACP cause pre-dictable shifts in mobility on nondenaturing gels. The orderof relative mobility of these conformers in this system isacyl-ACP > holo-ACP > apo-ACP.

To explore the nature of the ACP modification, theproducts of both the import and processing reactions wereanalyzed on conformationally sensitive gels. Figures 5Cand 5D show an analysis of the same samples as in Figures5A and 5B. As on the denaturing gels, we saw a singleband of identical mobility in both organelle-free processingand import reactions using pre-ACPASer as the substrate(see Figure 5D, lanes 1 to 3). Two products were againdetected from the wild-type precursor, but their relativemobility was reversed on the conformationally sensitivegel. The modified form of ACP, the slower migrating bandon denaturing gels, which is the major product of importreactions (Figure 5C, lane 1) and the minor product ofprocessing reactions (Figure 5C, lanes 2 and 3), was nowseen to migrate ahead of apo-ACP, as represented by the

200 The Plant Cell

pre-ACPASer products (Figure 5D, lanes 1 to 3). That is,the modification that produces the upper band on dena-turing gels causes a conformational change that enhancesthe mobility of the protein on nondenaturing gels. Impor-tantly, the 14-kD peptide and apo-ACP (pre-ACPASerproduct) have not changed their relative positions, aswould have been expected if the 14-kD peptide carried thephosphopantetheinegroup, i.e., was holo-ACP, supportingthe conclusion that their different mobilities are due to theserine-to-alanine substitution. Hence, the enhancement ofthe mobility of the modified protein in relation to apo-ACPon conformationally sensitive gels, its slower migrationduring SDS-PAGE, and the observation that its presenceis dependent on serine 38 strongly suggested that themodification of the 14-kD peptide is due to the addition ofthe phosphopantetheine prosthetic group.

CoA-Dependent Modification of the 14-kD Peptide

The enzyme holo-ACP synthase catalyzes the transfer ofthe phosphopantetheine group from CoA to apo-ACP in £.co// (Alberts and Vagelos, 1966; Elovson and Vagelos,1968) and higher plants (Elhussein et al., 1988), althoughthe properties of the latter reaction are not well under-stood. In addition to CoA as a substrate, the reaction alsorequires divalent cations such as Mg2+. We examined theeffects of the addition of MgCI2 and CoA to the organelle-free assays. MgCI2 stimulated production of the modifiedform at as little as 1 mM, with a parallel loss of the 14-kDpeptide (data not shown). We initially observed completeconversion to the modified form upon the addition of CoAto a final concentration of 0.5 mM (data not shown),indicating that, although Mg2* ions stimulate modification,the level of CoA in the assay appears to be a limiting factorfor this step. To establish more clearly the CoA-dependentmodification of ACP in these reactions, CoA was added ata final concentration of 0.1 nM to 500 ^M. When pre-ACPwas used as the substrate, the 14-kD peptide appearedas the primary product at low levels (<0.5 /iM). However,remarkably at only 10 ^M CoA, complete conversion ofthe 14-kD peptide to the modified form was observed, asillustrated in Figures 6A and 6B. Because the organelle-free reactions without added CoA show some modifyingactivity, the soluble chloroplast extract or the wheat germtranslation lysate are undoubtedly contributing a small butlimiting amount of CoA, and, therefore, the final concentra-tion of CoA may actually be somewhat greater than 10nM. When pre-ACPASer was used as the substrate, itwas proteolytically processed, releasing a single peptideboth without and upon addition of CoA at all concentra-tions (Figures 6A and 6B). These results clearly demon-strate that CoA does not inhibit the removal of the transitpeptide but rather stimulates a modification reaction thatrequires serine 38.

The products of the CoA-stimulated reaction were alsoanalyzed on conformationally sensitive gels in parallel with

the products of a standard organelle-free assay. As shownin Figure 5C (lane 4), the CoA-dependent, modified formco-migrates with the major product of the import reactionand ahead of the 14-kD peptide. Once again, the migrationof the mutant peptide released from pre-ACPASer, withthe serine-to-alanine substitution, was uneffected by theaddition of CoA (Figure 5D, lane 4).

100

e-5J> u

U

80 -

60 -

0.2 0.4 0.6 0.8 1.0

CoA concentration

BACP wild type ACPASer

0 0.1 0.5 1.0 10 0 0.1 0.5 1.0 10

[CoA] in u.M

Figure 6. Modification Reaction Is Dependent on CoA in theOrganelle-Free Assay.

(A) A graphic representation of the results shown in (B), wherepercentage conversion to the modified form (as determined fromdensitometry scan of gel shown below) is plotted against the CoAconcentration in the organelle-free assay. Pre-ACP (Q) and pre-ACPASer (O) were analyzed.(B) Organelle-free reactions were carried out using wheat germlysate translation products of pre-ACP and pre-ACPASer as sub-strates with the addition of CoA ranging from 0.1 uM to 10 ^M asindicated below each lane. The positions of apo-ACP (open arrow)and modified form (filled arrow) are indicated.

Chloroplast Import of ACP 201

We have noted that the wheat germ lysate possesses alow level of the Chloroplast processing enzyme capable ofcleaving various precursors, suggesting that it may alsocontain other Chloroplast enzymes. In fact, mock reactionsusing 5 mM Hepes-KOH, pH 8.0, instead of the spinachextract have shown that the wheat germ lysate indeedcontributes to the modification reaction converting the 14-kD peptide to the 18-kD form. However, when the wheatgerm lysate was boiled to inactivate its enzymatic activity,the CoA-dependent modification in the organelle-freeassay still occurred with the spinach extract (data notshown). To establish unequivocally that the spinach extractis capable of carrying out the modification independent ofthe components of the wheat germ lysate, reactions withand without the addition of CoA were performed using thetranslation products of a reticulocyte lysate. Neither theprocessing nor the modification activities were found inmock reactions containing the reticulocyte products and 5mM Hepes-KOH, pH 8.0, substituted for the spinach ex-tract. When the spinach extract was added without CoA,pre-ACP gave rise to only the 14-kD peptide. Upon additionof CoA, however, at least 40% of the 14-kD peptide wasconverted to the modified form, as shown in Figure 7. Boththe processing and modification activities were lost whenthe spinach extract was boiled (data not shown).

1 2 3 4 5 6 7

4

Figure 7. Organelle-Free Processing and CoA-Dependent Modi-fication of Reticulocyte Translation Products.

Translation products of pre-ACP (lane 1) were incubated in orga-nelle-free reactions without (lanes 2, 4, and 6) and with (lanes 3,5, and 7) the addition of CoA to 100 ^M, as indicated by minusand plus signs below each lane. The reactions contained either 5mM Hepes-KOH, pH 8.0, instead of spinach Chloroplast extract(lanes 2 and 3), spinach Chloroplast extract clarified by ultracen-trifugation (lanes 4 and 5), or crude soluble extract from lysedchloroplasts (lanes 6 and 7). The positions of apo-ACP andmodified form are indicated by open and filled arrows, respectively.

Figure 8. E. co/i Phosphodiesterase Reverses the CoA-Dependent Modification of the 14-kD Peptide.

An organelle-free assay was carried out using wheat germ trans-lation products and 10 p.M CoA to convert all of the 14-kD peptide(open arrow) to the modified form (closed arrow). The products(lane 1) were then dialyzed, subdivided into four fractions, andemployed in a phosphodiesterase reaction (see Methods andtext). Lanes 2 through 5 show a mock reaction containing noenzyme (lane 2), and reactions using 20 units (lane 3), 40 units(lane 4), or 400 units (lane 5) of E. coli phosphodiesterase.

E. coli ACP Phosphodiesterase Regenerates the14-kD peptide from the Modified Form

To investigate further the identity of the modified formgenerated in the CoA-dependent reaction, the products ofan organelle-free assay were treated with ACP phospho-diesterase purified from E. coli (Therisod and Kennedy,1987). This enzyme is specific for holo-ACP, catalyzingthe cleavage of the phosphodiester bond linking the phos-phopantetheine to the special serine, and does not reactwith other substrates such as CoA (Vagelos and Larrabee,1967). An organelle-free reaction containing pre-ACP syn-thesized in a wheat germ lysate and 10 nM CoA wasperformed as described above, in which the 14-kD peptidewas completely converted to the 18 kD product, as illus-trated in Figure 8, lane 1. The products of this reactionwere dialyzed against 50 mM Tris-HCI, pH 8.5, to removeCoA and used as the substrate for the ACP phosphodi-esterase enzyme.

Phosphodiesterase reactions were carried out using 20units (Figure 8, lane 3), 40 units (lane 4), and 400 units(lane 5) of the enzyme. The units of activity are based onthe ability of this enzyme to remove the phosphopante-theine from the homologous substrate, bacterial holo-ACP

202 The Plant Cell

(Therisod and Kennedy, 1987), and are given to provide a relative measure of the efficiency of the bacterial enzyme using the spinach ACP substrate (see Methods). A mock reaction, containing no enzyme, was included to demon- strate that the phosphodiester bond remains stable under the reaction conditions in the absence of the phosphodi- esterase (lane 2). In the reaction containing 400 units of the E. coli phosphodiesterase, approximately 30% of the modified ACP product was converted back to the 14-kD unmodified form. The sensitivity of the CoA-dependem modification to the phosphodiesterase, i.e., the conversion of the 18-kD peptide back to the 14-kD form, indicates that the modified form is holo-ACP. Hence, the results of the organelle-free assay, coupled with those from the import reactions, strongly support the conclusion that the chloroplast contains an enzyme capable of transferring the phosphopantetheine group of COA to ACP, producing hOlO-ACP.

DISCUSSION

We have demonstrated in this study that the transport of ACP into the chloroplasts does not require the attachment of its phosphopantetheine prosthetic group. Substitution of serine 38, to which the phosphopantetheine is usually attached, does not prevent efficient import. Significantly, our results indicate that the chloroplast itself contains an enzyme, i.e., holo-ACP synthase, capable of adding the functional prosthetic group. Evidence has recently been presented that plant cells contain a cytoplasmic holo-ACP synthase that transfers the phosphopantetheine group from COA. However, it is not known where this reaction normally occurs in vivo, and a chloroplast enzyme could not be excluded (Elhussein et al., 1988). The addition of prosthetic groups to various proteins targeted to organ- elles can occur both before and after import. Among proteins destined for the mitochondria, for example, pyri- doxal phosphate is added to aspartate aminotransferase in'the mitochondrial matrix after its translocation and pro- teolytic processing (Sharma and Gehring, 1986). Similarly, ferredoxin is imported and processed to its mature form by chloroplasts before incorporation of the iron-sulfur com- plex (Takahashi et al., 1986). On the other hand, heme appears to be added to cytochrome c at the inner face of the mitochondrial outer membrane before its localization to the intermembrane space (Nicholson and Neupert, 1989). and addition of FAD to ferredoxin-NADP+ oxidore- ductase, which is destined for the chloroplast thylakoids, has also been reported to occur in the cytoplasm (Carrillo, 1985). One might predict that the addition of the prosthetic group to ACP confers on the precursor a conformation that favors efficient import into the chloroplast. Current evidence indicates that features that stabilize precursor tertiary structure, such as ligand binding, block protein transport into the mitochondria (Eilers and Schatz, 1986),

whereas destabilization by mutation or precursor unfolding by cytosolic factors (Vestweber and Schatz, 1988; Cheng et al., 1989) promote the import process. Although the phosphopantetheine apparently stabilizes the conforma- tion of mature ACP (Rock et al., 1981), it is difficult to predict the structural changes that the precursor, contain- ing a 56-amino acid transit peptide with a basic composi- tion, would undergo upon phosphopantetheine addition. In any case, if the phosphopantetheine interacts with the protein moiety to confer a unique tertiary structure, the conformation is not essential to mediate pre-ACP import into the chloroplast.

Pre-ACP is processed to two forms upon import into the chloroplast. Our results indicate that the 14-kD peptide arises from the removal of the ACP transit peptide by the chloroplast processing enzyme. The slower migrating pep- tide, however, is a derivative of the 14-kD peptide pro- duced by a highly specific modification reaction. Four criteria indicate that the modification is due to the attach- ment of the phosphopantetheine to apo-ACP by a chloro- plast holo-ACP synthase. First, the production of the mod- ified form depends on the presence of serine 38, to which the prosthetic group is normally attached. Second, com- pared with denaturing gels, conformationally sensitive gels show a reverse in the relative mobility of the 14-kD peptide and modified form as predicted for the relationship be- tween apo-ACP and holo-ACP. Third, COA rapidly stimu- lates the modification reaction in an organelle-free assay. Fourth, most convincingly, the modified peptide is con- verted back to the 14-kD peptide by E. coli phosphodies- terase. In addition, the conformationally sensitive gels in- dicate that the newly imported and proteolytically proc- essed ACP does not carry the phosphopantetheine based on the mobilities of apo-ACP generated from pre-ACPASer and the 14-kD peptide cleaved from pre-ACP. That is, the 14-kD peptide does not migrate ahead of apo-ACP, as predicted if it carriad the prosthetic group, thereby main- taining a more stable conformation. Finally, it has been demonstrated that the transfer of acyl groups such as malonyl from malonyl-COA, for example, to holo-ACP by an ACP transacylase is not affected by the addition of COA at low concentrations (less than 10 gM), and, at higher concentrations, the transfer is inhibited (Guerra and Ohl- rogge, 1986). It is, therefore, improbable that the modified form is the result of a two-step process requiring first the synthesis of malonyl-COA and then the transfer reaction. Based on these considerations, we conclude that a holo- ACP synthase performs the modification reaction.

The question arises, what would be the function of two holo-ACP synthases-one cytoplasmic and the other chlo- roplastic-in the plant cell? What are their substrates and respective roles in terms of converting ACP to its active form? If the phosphopantetheine is usually added in the cytoplasm immediately upon pre-apo-ACP synthesis, then what is the function of the chloroplast enzyme? One possibility is that there is considerable turnover of the

Chloroplast lmport of ACP 203

prosthetic group regenerating pools of apo-ACP in the chloroplast under certain conditions, which would then be available as a substrate for holo-ACP synthase. This would be one way of regulating de novo fatty acid biosynthesis. In E. coli, the turnover of the phosphopantetheine appears to be low (Jackowski and Rock, 1984), although reported rates have differed by an order of magnitude (Powell, Bauza, and Larrabee, 1973), and the cells do not appear to maintain a steady-state amount of apo-ACP (Jackowski and Rock, 1983). Regulation of fatty acid biosynthesis by the alteration of the apo-ACP to holo-ACP ratio, then, may not be a strategy employed in E. coli (see Jackowski and Rock, 1984). However, it is interesting to note that recently a new function for ACP in the synthesis of membrane- derived oligosaccharides in E. coli has been described in which the phosphopantetheine is not required, suggesting an alternative physiological role for apo-ACP (Therisod and Kennedy, 1987). In higher plants, the levels of apo-ACP and holo-ACP have not been extensively examined, and the possibility that prosthetic group turnover represents another mechanism for control of fatty acid biosynthesis cannot be discounted.

We have been able to reconstitute the COA-dependent modification reaction in an organelle-free assay. The chlo- roplast extract typically employed was clarified by ultra- centrifugation to remove all membranes, and, thus, we conclude that the modifying activity, i.e., holo-ACP syn- thase, is most likely located in the soluble stromal phase of the chloroplast or, perhaps, the space separating the outer and inner membranes. The conditions of the orga- nelle-free assay were originally optimized for removal of the transit peptide (Lamppa and Abad, 1987; Abad et al., 1989). Thus, apo-ACP, the 14-kD peptide, is the predom- inant product of the reaction when pre-ACP is the sub- strate. The COA concentration curve suggests that the 14- kD peptide can be converted to the modified form, i.e., the mature form of ACP, lacking a transit peptide, may serve as a substrate for phosphopantetheine addition in the chloroplast. Therefore, the following pathway for the syn- thesis and import of ACP should be considered: (1) pre- apo-ACP is synthesized in the cytoplasm, (2) pre-apo-ACP is imported and proteolytically processed to apo-ACP, and (3) the prosthetic group is transferred from COA to apo- ACP to produce holo-ACP within the chloroplast.

The import reactions show that ACP is found predomi- nantly in the stroma of the chloroplast. In a recent report, using immunogold labeling and electron microscopy, ACP was localized to the thylakoid membranes (Slabas and Smith, 1988). We conclude from our results that, if ACP does become membrane-associated, it must be a very transient association that is easily disrupted because we have observed essentially no radiolabeled protein in the membrane fractions from thermolysin-treated organelles after import. By using the same methods in other studies, however, we have shown that mutant polypeptides of the light-harvesting chlorophyll a/b-binding protein that be-

come peripherally associated with the thylakoids, but can- not insert, still fractionate with the membranes after chloroplast lysis (Clark, Abad, and Lamppa, 1989).

A comparison of the spinach and E. coli ACPs shows that about one-third of their residues are identical, i.e., 29 of 82 amino acids of the spinach ACP. The most conserved domain extends almost continuously for 17 amino acids surrounding serine 38. This degree of relatedness is ap- parently sufficient for the bacterial phosphopantetheine to recognize spinach ACP, albeit less efficiently than the homologous substrate, and remove the phosphopante- theine. Systematic substitution of the conserved residues could now be employed to identify the minimum determi- nants necessary for phosphodiesterase activity.

In summary, we have identified a modification activity in the soluble fraction of the chloroplast that converts a 14- kD peptide to a slower migrating form that is dependent on the addition of COA. This modification reaction is re- versible by treatment with E. coli ACP phosphodiesterase. Taken together, our results provide convincing evidence that the enzyme involved is a holo-ACP synthase. By using the organelle-free assay, we can now characterize the properties of this enzyme, unequivocally establish its sub- strate specificity, and begin its purification.

METHODS

Plant Growth and Chloroplast lsolation

Spinach (Spinacea oleracea, Melody) plants were grown in a greenhouse (Department of Ecology and Evolution, University of Chicago) and transferred to a growth chamber (26OC, cool flu- orescent lights) 3 weeks to 4 weeks after planting and 1 week to 2 weeks before harvesting. A typical chloroplast isolation used 25 g of leaf tissue, fresh weight, homogenized with a Polytron ho- mogenizer at 4OC in 2 mM EDTA, 1 mM MgC12, 1 mM MnCI2, 50 mM Hepes-KOH, pH 8.0, 0.33 M sorbitol, 5 mM sodium ascor- bate, 0.25% BSA at a ratio of 20 mL/g of tissue, fresh weight. lntact chloroplasts were isolated on Percoll gradients (Bartlett, Grossman, and Chua, 1982).

Construction of Plasmids

A 500-bp EcoRI-Hindlll fragment of a cDNA sequence (pCGNlSOL, kindly provided by Vic Knauf, Calgene, Davis, CA) coding for spinach ACP I (Scherer and Knauf, 1987) was inserted into the transcription vector plB131 (International Biotechnologies Inc., New Haven, CT) downstream from the T7 promoter, yielding the plasmid plBI-ACP500. The 500-bp insert contains the se- quence coding for the mature ACP I protein as well as a 56-amino acid putative transit peptide and 36 bp of the 5'- and 53 bp of the 3'-nontranslated sequences.

Single-Stranded DNA lsolation

Single-stranded DNA was isolated by inoculating 3 mL of 2 X YT media (1.6% tryptone, 17'0 yeast extract, 8.5 mM NaCI) containing

204 The Plant Cell

150 pg/mL ampicillin with a single colony of E. coli strain MV1193 containing the plBI-ACP500 plasmid. After 2 hr of growth at 37OC, the culture was inoculated with the helper phage M13K07 at a multiplicity of infection of 20, and growth was continued at 37°C for 45 min, at which time kanamycin was added to 80 pg/mL final concentration. After continued incubation at 37OC for 45 min, 1 O0 pL of the culture was used to inoculate 1 O mL of 2 x YT media (75 pg/mL kanamycin, 150 pg/mL ampicillin), which was incubated at 37°C overnight. The cells were removed by centrifugation at 30009 for 1 O min, and the phage particles were precipitated out of the supernatant at room temperature for 2 hr by the addition of 3% PEG, 0.25 M NaCI. The precipitated phage particles were collected by centrifugation at 77009 for 20 min, resuspended in 10 mM Tris, 0.1 mM EDTA, and extracted with phenol and chloroform. Single-stranded DNA was collected by ethanol precip- itation and resuspended in 30 pL of 10 mM Tris, 0.1 mM EDTA.

In Vitro Mutagenesis

The ACPASer mutation was created using a protocol, based on the method of Eckstein and co-workers (Taylor, Ott, and Eckstein, 1985a), designed to give high yields of mutant plasmids (Amer- sham Corp., Arlington Heights, IL). Briefly, an oligonucleotide (sequence shown in Figure l) , complementary to the ACP cDNA sequence surrounding the sequence coding for the special serine but containing three nucleotide changes, was hybridized to the single-stranded plBI-ACP500 DNA and used as a primer for second-strand synthesis by the Klenow fragment of DNA polym- erase I. In the resulting heteroduplex, the newly synthesized mutant strand was chemically distinguished from the original wild- type strand by the incorporation of dCTP&. Certain restriction endonucleases (Ncil was used here) are unable to cut phospho- rothioate DNA, allowing selective nicking of the wild-type strand (Taylor et al., 1985b). Exonuclease 111 was then used to excise through the sequence on the wild-type strand complementary to the mutated sites. The gaps were repaired with DNA polymerase I, using the mutant strand as a template, creating the double- stranded mutant plasmid plBI-ACPASer. The incorporation of a silent, single-nucleotide change immediately 5' to the new alanine codon created a unique Mlul restriction site, allowing for rapid and accurate isolation of positive plasmids.

translation products. The chloroplasts were incubated with trans- lation products at 26°C with gentle shaking for 45 min (except where indicated in text), and then diluted fivefold with HSM, spun at 25009 for 1 min, and immediately resuspended in cold 1 mM phenylmethylsulfonyl fluoride (PMSF) for lysis. When chloroplasts were treated with thermolysin, half of the incubation reaction was mixed with an equal volume of 200 pg/mL thermolysin in HSM, 4 mM CaCI,. The reaction was stopped by making it 40 mM EGTA, 2 mM PMSF, and diluted threefold with HSM; the chloroplasts were pelleted at 25009 for 1 min and immediately lysed in 1 mM PMSF. To separate the membrane and soluble phases of the chloroplast, the samples were spun for 20 min in a microcentrifuge at 16000g. For SDS-PAGE analysis, the pellet was resuspended in 0.5 M Tris-HCI, pH 6.8, 5% 6-mercaptoethanol, 2% SDS, 10% glycerol. The protein from the supernatant was precipitated with 10% TCA, washed with 80% acetone, and resuspended in the same buffer. For nondenaturing gels, the samples were resus- pended in 0.5 M Tris-HCI, pH 6.8, 1 O mM DTT, 10% glycerol.

Organelle-Free Reactions

Chloroplast soluble extracts were prepared according to the protocol of Abad et al. (1989). lntact chloroplasts were resus- pended in HSM and centrifuged for 1 min at 25009. The pellet was gently resuspended in a volume of ice-cold 5 mM Hepes- KOH, pH 8.0, equal to the original HSM volume, and kept at 4°C for 30 min for lysis. The lysed chloroplasts were centrifuged at 160009 for 1 O min to remove the bulk membranes, and membrane vesicles were removed by recentrifuging the supernatant at 142,OOOg. A typical 25-pl processing reaction contained 20 mM Tris-HCI, pH 8.0, 3 pg/mL chloramphenicol, 5 pL of a 30-pL translation reaction (40,000 cpm to 50,000 cpm), and 15 pL of clarified soluble chloroplast extract, and was incubated at 26OC for 90 min. Both wheat germ and reticulocyte lysate translation products have been used; the wheat germ lysate generally trans- lated the pre-ACP RNA more efficiently. The reactions were stopped by bringing the products to 0.5 M Tris-HCI, pH 6.8, 5% p-mercaptoethanol, 2% SDS, 1 0% glycerol for SDS-PAGE analy- sis, or to 0.5 M Tris-HCI, pH 6.8, 10 mM DTT, 10% glycerol for analysis on nondenaturing polyacrylamide gels. For reactions containing COA (free acid; Sigma, St. Louis, MO), a sterile solution was added to a final concentration as indicated in the text.

In Vitro Transcription, Translation, and lmport Phosphodiesterase Treatment

The plBI-ACP500 and plBI-ACPASer plasmids were linearized with EcoRl and transcribed with T7 RNA polymerase (New Eng- land Biolabs, Beverly, MA). The RNA generated from a 50-pL reaction was resuspended in 25 pL of sterile water, and 2 pL to 4 pL (estimated 0.5 pg to 1 .O pg) were used in a standard 30-pL reticulocyte (Bethesda Research Laboratories, Bethesda, MD) or wheat germ (Amersham Corp.) translation reaction as prescribed by the vendor, using 35S-methionine (1 O00 Ci/mmol) as the labeled amino acid. Optimal translation was achieved using 33 mM K- acetate in both wheat germ and reticulocyte translation systems.

Procedures for the import reaction were essentially as de- scribed (Bartlett et al., 1982). lntact chloroplasts were resus- pended in HSM (50 mM Hepes-KOH, pH 8.0, 0.33 M Sorbitol, 8 mM methionine) at a concentration of 800 pg to 1000 pg of chlorophyll/mL, and 50 pL was added to a 300-pL reaction containing HSM, plus 10 mM ATP, 10 mM MgCI?, and 50 pL of

After organelle-free reactions containing wheat germ lysate trans- lation products and 10 pM COA, the samples were dialyzed against 50 mM Tris-HCI, pH 8.5, on microdialysis filters (Type SV, 0.025-pm pore size; Millipore Corp., Bedford, MA) for 1 hr at room temperature to remove COA. They were then treated with purified E. coli ACP phosphodiesterase (kindly provided by Eugene Ken- nedy and Anthony Fishl, Harvard University, Cambridge, MA) essentially as described. The reactions contained 50 mM Tris- HCI, pH 8.5, 25 mM MgCI?, 20 pM MnCI,, 1 mM DTT, and were incubated at 35'C for 2 hr with either 20 units, 40 units, 400 units or no enzyme as described in the text. Units of activity were expressed as picomoles per minute per milliliter on the basis of release of the TCA-soluble radiolabeled phosphopantetheine from the TCA-insoluble E. coli holo-ACP (Therisod and Kennedy, 1987). The reactions were stopped by bringing the products to 0.5 M

Chloroplast lmport of ACP 205

Tris-HCI, pH 6.8, 5% P-mercaptoethanol, 2% SDS, 10% glycerol, and analyzed by SDS-PAGE.

Gel Analysis

For SDS-PAGE analysis, samples were boiled for 3 min and immediately loaded on 15% polyacrylamide gels as described (Laemmli, 1970; Piccioni, Bellemere, and Chua, 1982). Nondena- turing gels were essentially as described (Rock and Cronan, 1981). The separating gel contained 20% acrylamide, 0.5% N,N- methylenebisacrylamide, 0.5% N,N,N’,N’-tetramethylethyldi- amine, 0.375 M Tris-HCI, pH 9.0. A stacking gel was poured over the separating gel and contained the same components except that the acrylamide concentration was 5% and the Tris-HCI buffer was 0.125 M, pH 6.8. The gels were run at 7 mA overnight at 37OC, using a running buffer of 50 mM Tris, 400 mM glycine, until the tracking dye reached the bottom of the gel. Under these conditions, the precursor proteins do not substantially enter the gel.

After electrophoresis, gels were stained with Coomassie Blue in 50% methanol, 7% acetic acid, destained, and prepared for autoradiography by soaking in DMSO for 1 hr, 2,5-diphenyloxa- zole-DMSO for 2.5 hr, water for 30 min, and then dried.

ACKNOWLEDGMENTS

We wish to thank Drs. Anthony Fischl and Eugene Kennedy for kindly providing the purified E. coli ACP phosphodiesterase. We are grateful to Roben Buell for her excellent technical assistance. This work was supported by U.S. Department of Agriculture Grant 89-3761-4471 (awarded to G.K.L.) and a Sigma Xi Grant-in-Aid of Research (to M.D.F.).

Received October 27, 1989; revised December 29, 1989.

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DOI 10.1105/tpc.2.3.195 1990;2;195-206Plant Cell

M D Fernandez and G K Lamppaevidence for a chloroplast holo-ACP synthase.

Acyl carrier protein (ACP) import into chloroplasts does not require the phosphopantetheine:

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