Download Acyl Carrier Protein (ACP) lmport into Chloroplasts Does not

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’
Department of Biochemistry and Molecular Biology, University of Chicago, 920 East 58th Street, Chicago, lllinois 60637
Department of Molecular Genetics and Cell Biology, University of Chicago, 920 East 58th Street, Chicago,
lllinois 60637
a
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‘-phosphopantetheine 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 polypeptide, the ACPs of plants and bacteria are small (9 kD to 1O
kD), nonassociated soluble proteins. The amino acid sequences 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 sequence 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; Ohlrogge 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 spinach 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 Keegstra, 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 understanding of these events, as well as confirm those more
general features of the import pathway. Furthermore, posttranslational 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 addition 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 special 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 responsible for the addition of the phosphopantetheine prosthetic
group. The implications of this are discussed in terms of
the function of ACP.
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 serineto-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 restriction site created immediately5' to the new alanine codon.
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, translation 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-
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 thermolysin to remove any proteins on the exterior of the chloroplasts, 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
A Spinach
B pea
I
2
3
1
2
T.P.
M
M
T
T.P.
M
3
4
5
M
S
S
T
197
was carried out in which spinach Chloroplasts were incubated with radiolabeled precursor protein for 1 min, 5 min,
and 15 min, as illustrated in Figure 3. We argued that, if
the upper band represented an intermediate form, it should
appear first and then gradually give rise to the lower, fully
processed approximately 14-kD form over time. We saw
the opposite effect where the lower band appeared in
greater abundance at the 1 min time point and then the
ratio rapidly shifted so that by 15 min the upper band was
the most prominent. This result indicated that the higher
molecular weight product was a derivative of the lower
molecular weight form, suggesting a covalent modification of the 14-kD product upon import of ACP into the
Chloroplasts.
T
Figure 2. in Vitro Import of Pre-ACP into Chloroplasts Yields Two
Products.
(A) The reticulocyte lysate translation products of pre-ACP (T.P.,
lane 1) were incubated with spinach Chloroplasts, and the total
membrane (M, lane 2), membrane after thermolysin treatment (M
+ T, lane 3), total soluble (S, lane 4), and soluble proteins after
thermolysin treatment (S + T, lane 5) were analyzed. The open
arrow indicates the position of the 14-kD product and the filled
arrow indicates the position of the 18-kD product.
(B) In vitro import of pre-ACP into pea Chloroplasts. The lanes are
numbered the same as (A).
soluble fraction, both lower in molecular weight and resistant to thermolysin treatment, indicating that the precursor
was proteolytically processed and sequestered to the
stromal compartment of the chloroplast during the import
reaction.
The size of the lower band, which we estimated as
about 14 kD, was consistent with the removal upon import
of the 56-amino acid putative transit peptide by the chloroplast processing activity, i.e., "transit peptidase." The
appearance of the upper band (about 18 kD), typically the
more abundant of the two products, was unexpected.
Significantly, the relative ratio of these two bands was
variable. Although not yet systematically analyzed in detail,
our results suggest that the ratio of these two products is
sensitive to seasonal changes affecting the physiological/
developmental state of the plant. In contrast to spinach,
Chloroplasts isolated from young pea leaves consistently
generated more of the lower molecular weight product
(see Figure 2B). The nature of these two products of preACP import is investigated in the experiments described
below.
The Larger Import Product Is not a Proteolytic
Processing Intermediate
To explore the possibility that the upper band was a
precursor processing intermediate, a time course of import
Conversion of the "Special" Serine to Alanine Does
not Prevent Import but Eliminates Production of the
Larger Import Product
Spinach ACP is known to be modified by the addition of a
4'-phosphopantetheine prosthetic group attached, via a
phosphodiester bond, exclusively at a serine 38 amino
acids from the start of the mature protein. Evidence has
been presented that the major site of this addition is the
cytoplasm (Elhussein et al., 1988), but it has not been
excluded that there is also addition of the prosthetic group
in the chloroplast itself. To determine whether the slower
migrating, apparently higher molecular weight product that
we observed upon import resulted from a modification of
ACP through the special serine, site-directed mutagenesis
1
2
3
4
T.P.
M
M
T
S
1 min
5
S
T
2
M
3
M
T
4
S
5 min
5
S
+
T
2
M
3
4
M
+
T
5
S
+
15 min
Figure 3. Time Course of Import Indicates That the Larger Product Is a Derivative of the 14-kD Peptide.
Reticulocyte lysate translation products are shown in lane 1, and
lanes 2 to 5 are the same as those described in Figure 2 and as
indicated below each lane. Translation products of pre-ACP were
incubated with Chloroplasts for 1 min, 5 min, and 15 min as
indicated below each reaction. The positions of the 14-kD and
modified form are indicated by open and filled arrows, respectively.
198
The Plant Cell
was used to eliminate the phosphopantetheine binding site
by converting serine 38 to alanine (see Figure 1 and
Methods). In addition to the serine-to-alanine conversion,
the mutation also included a silent single-nucleotide
change immediately 5' to the new alanine codon that
created a unique Mlul restriction site, providing a rapid
method of screening for mutated plasmids.
A restructured plasmid containing the desired substitution was transcribed and translated as previously described for wild-type pre-ACP. The radiolabeled mutant
precursor, referred to as pre-ACPASer, was used in an in
vitro import reaction. If imported and processed, it should,
by definition, give rise to apo-ACP with a single residue
change because the phosphopantetheine cannot be
added. The results of this experiment are shown in Figure
4. The radiolabeled ACPASer precursor protein co-migrated with wild-type pre-ACP during SDS-PAGE (Figure
4, lanes 1). The products of the import reaction, however,
were strikingly different. As before, the wild-type precursor
gave rise to two thermolysin-resistant products in the
soluble fraction. On the other hand, the pre-ACPASer
mutant produced only one band, migrating slightly faster
than the approximately 14-kD product of wild-type import.
Thus, the serine-to-alanine conversion resulted in a loss of
the higher molecular weight derivative, indicating that the
ACP wild type
1
T.P.
2
Organelle-Free Processing Yields Two Products That
Co-Migrate with Those from Import Reactions
ACP ASer
3
M M
4
5
1
S
T.P.
T
2 3 4
M
smaller form of pre-ACP import is apo-ACP. Two important
points emerged from this mutational analysis. First, the
larger import product arises from a modification that depends on the presence of serine 38. Second, the phosphopantetheine prosthetic group, which cannot be added to
pre-ACPASer, is not required for ACP translocation across
the chloroplast envelope or for proteolytic processing upon
import.
The minor difference in mobility of the lower molecular
weight product of wild-type ACP import and the product
of pre-ACPASer import is most likely due to an altered
conformation caused by the single serine-to-alanine mutation. A similar change in electrophoretic mobility has been
identified for a mutant ACP that has Ser 38 converted to
cysteine (Jaworski, Post-Beittenmiller, and Ohlrogge,
1989b). Predictions of E. coli ACP secondary and tertiary
structure (Rock and Cronan, 1979; Mayo, Tyrell, and Prestegard, 1983) suggest that the special serine resides at
the base of a central hydrophobic corridor formed by four
a-helices. Predictions of spinach ACP secondary structure
are consistent with this model (M. D. Fernandez, unpublished data; Kuo and Ohlrogge, 1984). The substitution of
the nonpolar alanine residue for the polar serine in this
critical region might, therefore, change the overall conformation and migration of the protein.
5
M
T
Figure 4. In Vitro Import of the Pre-ACPASer Mutant Produces
Only the 14-kD Form, Defined as Apo-ACP.
Parallel in vitro import reactions were carried out using radiolabeled pre-ACP and pre-ACPASer proteins from reticulocyte lysates. Translation products are shown in lanes 1, and lanes 2 to
5 are as described in Figure 2 and as indicated below each lane.
The positions of apo-ACP and the modified form are indicated by
open and filled arrows, respectively.
We have recently developed an assay for organelle-free
processing of the precursor of light-harvesting chlorophyll a/b-binding protein in which the transit peptide is
removed (Lamppa and Abad, 1987; Abad, Clark, and
Lamppa, 1989). In addition, the crude soluble extract
processes the small subunit of ribulose-1,5-bisphosphate
carboxylase/oxygenase precursor, as has been observed
in analogous experiments by Robinson and Ellis (1984).
Using a similar protocol, we have prepared chloroplast
soluble extracts from spinach that contain an active processing enzyme (see Methods). Our goal was to establish
whether direct removal of the transit peptide of pre-ACP,
independent of translocation across the chloroplast envelope, generated the same products as the in vitro import
reactions. Figures 5A and 5B show the products of organelle-free processing reactions, using the wild-type and
ACPASer precursors synthesized in a wheat germ lysate,
compared with the soluble products of import reactions,
analyzed on a denaturing SDS-polyacrylamide gel. The
organelle-free assay (lanes 2) generated products of the
same size as the import reactions (lanes 1) for both the
wild-type and mutant substrates. That is, pre-ACP was
cleaved and again gave two products, whereas preACPASer yielded only one peptide, the smaller form. In
contrast to the import reactions, however, the soluble
extracts always produced more of the 14-kD peptide than
of the higher molecular weight form from the wild-type
Chloroplast Import of ACP
A
B
1 2 3 4
1 2 3 4
D
Figure 5. An Organelle-Free Assay Reconstitutes Removal of the
Transit Peptide and the Modification Reaction.
Analysis by denaturing and conformationally sensitive gels.
(A) Analysis of the wild-type precursor products on denaturing
gels. The soluble products of an import reaction using reticulocyte
lysate translation products from thermolysin-treated chloroplasts
(lane 1), and the products of organelle-free processing reactions
using translation products from a wheat germ lysate and soluble
chloroplast extracts prepared from pea (lane 2) and spinach (lane
3) leaves were analyzed on a 15% SDS-polyacrylamide gel. Lane
4 shows an organelle-free reaction to which CoA was added at a
final concentration of 10 ^M. The positions of apo-ACP and the
modified 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 precursor
were carried out and the lanes are numbered the same.
(C) Analysis of the wild-type precursor products on nondenaturing, conformationally sensitive gels. Samples from parallel reactions to (A) were analyzed on 20% conformationally sensitive
gels. The lanes are numbered as above, and the positions of apoACP and the modified form are indicated by open and filled arrows,
respectively.
(D) Analysis of the pre-ACPASer products on conformationally
sensitive gels. Reactions identical to those described for the wildtype precursor were carried out and the lanes are numbered the
same.
199
substrate. These results support the conclusion that the
14-kD form is the primary product of the simple removal
of the transit peptide. In addition, the organelle-free assay
exhibits a modifying activity, yielding the 18-kD peptide,
but it is less active than in intact organelles. Parallel reactions were carried out in the presence of a soluble extract
from pea chloroplasts (Figures 5A and 5B, lanes 3), and
we usually found that, with a pea extract, less of the higher
molecular weight form was produced than upon addition
of the spinach extract. This difference in the ability of the
spinach and pea extracts to produce the modified form is
consistent with the results obtained using intact organelles
(see Figure 2B).
Conformationally Sensitive Gel Analysis of Import and
Processing Reaction Products
It has been well documented that modification of ACP
through serine 38 leads to bands of altered mobility during
electrophoresis. This phenomenon has been best characterized using E. coli ACP derivatives on nondenaturing or
"conformationally sensitive" gels (Rock and Cronan, 1981;
Rock, Cronan, and Armitage, 1981), and has also been
applied to the analysis of spinach ACP derivatives (Jaworski, Clough, and Barnum, 1989a). ACP undergoes a pHinduced conformational change upon transition from the
stacking gel (pH 6.8) to the separating gel (pH 9.0) that is
characterized by the loss of «-helical content (Schulz,
1975) and an expansion of the Stokes radius (Rock and
Cronan, 1979). Therefore, ACP derivatives that destabilize
the protein moiety migrate more slowly because of this
partial denaturation, and, conversely, ligands that stabilize
the protein migrate faster. These studies have shown that
the conformational changes induced by addition of the
phosphopantetheine prosthetic group to the unmodified
protein, apo-ACP, to produce holo-ACP and further addition of various acyl chains yielding acyl-ACP cause predictable shifts in mobility on nondenaturing gels. The order
of relative mobility of these conformers in this system is
acyl-ACP > holo-ACP > apo-ACP.
To explore the nature of the ACP modification, the
products of both the import and processing reactions were
analyzed on conformationally sensitive gels. Figures 5C
and 5D show an analysis of the same samples as in Figures
5A and 5B. As on the denaturing gels, we saw a single
band of identical mobility in both organelle-free processing
and import reactions using pre-ACPASer as the substrate
(see Figure 5D, lanes 1 to 3). Two products were again
detected from the wild-type precursor, but their relative
mobility was reversed on the conformationally sensitive
gel. The modified form of ACP, the slower migrating band
on denaturing gels, which is the major product of import
reactions (Figure 5C, lane 1) and the minor product of
processing reactions (Figure 5C, lanes 2 and 3), was now
seen 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 denaturing gels causes a conformational change that enhances
the mobility of the protein on nondenaturing gels. Importantly, the 14-kD peptide and apo-ACP (pre-ACPASer
product) have not changed their relative positions, as
would have been expected if the 14-kD peptide carried the
phosphopantetheinegroup, i.e., was holo-ACP, supporting
the conclusion that their different mobilities are due to the
serine-to-alanine substitution. Hence, the enhancement of
the mobility of the modified protein in relation to apo-ACP
on conformationally sensitive gels, its slower migration
during SDS-PAGE, and the observation that its presence
is dependent on serine 38 strongly suggested that the
modification of the 14-kD peptide is due to the addition of
the phosphopantetheine prosthetic group.
CoA-Dependent Modification of the 14-kD Peptide
The enzyme holo-ACP synthase catalyzes the transfer of
the phosphopantetheine group from CoA to apo-ACP in £.
co// (Alberts and Vagelos, 1966; Elovson and Vagelos,
1968) and higher plants (Elhussein et al., 1988), although
the properties of the latter reaction are not well understood. In addition to CoA as a substrate, the reaction also
requires divalent cations such as Mg2+. We examined the
effects of the addition of MgCI2 and CoA to the organellefree assays. MgCI2 stimulated production of the modified
form at as little as 1 mM, with a parallel loss of the 14-kD
peptide (data not shown). We initially observed complete
conversion to the modified form upon the addition of CoA
to 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 factor
for this step. To establish more clearly the CoA-dependent
modification of ACP in these reactions, CoA was added at
a final concentration of 0.1 nM to 500 ^M. When pre-ACP
was used as the substrate, the 14-kD peptide appeared
as the primary product at low levels (<0.5 /iM). However,
remarkably at only 10 ^M CoA, complete conversion of
the 14-kD peptide to the modified form was observed, as
illustrated in Figures 6A and 6B. Because the organellefree reactions without added CoA show some modifying
activity, the soluble chloroplast extract or the wheat germ
translation lysate are undoubtedly contributing a small but
limiting amount of CoA, and, therefore, the final concentration of CoA may actually be somewhat greater than 10
nM. When pre-ACPASer was used as the substrate, it
was proteolytically processed, releasing a single peptide
both without and upon addition of CoA at all concentrations (Figures 6A and 6B). These results clearly demonstrate that CoA does not inhibit the removal of the transit
peptide but rather stimulates a modification reaction that
requires serine 38.
The products of the CoA-stimulated reaction were also
analyzed on conformationally sensitive gels in parallel with
the products of a standard organelle-free assay. As shown
in Figure 5C (lane 4), the CoA-dependent, modified form
co-migrates with the major product of the import reaction
and ahead of the 14-kD peptide. Once again, the migration
of the mutant peptide released from pre-ACPASer, with
the serine-to-alanine substitution, was uneffected by the
addition of CoA (Figure 5D, lane 4).
100
80 -
e-5
J> u
60 -
U
0.2
0.4
0.6
0.8
1.0
CoA concentration
B
ACP wild type
0
0.1
0.5 1.0
ACPASer
10
0
0.1
0.5
1.0
10
[CoA] in u.M
Figure 6. Modification Reaction Is Dependent on CoA in the
Organelle-Free Assay.
(A) A graphic representation of the results shown in (B), where
percentage conversion to the modified form (as determined from
densitometry scan of gel shown below) is plotted against the CoA
concentration in the organelle-free assay. Pre-ACP (Q) and preACPASer (O) were analyzed.
(B) Organelle-free reactions were carried out using wheat germ
lysate translation products of pre-ACP and pre-ACPASer as substrates with the addition of CoA ranging from 0.1 uM to 10 ^M as
indicated below each lane. The positions of apo-ACP (open arrow)
and modified form (filled arrow) are indicated.
Chloroplast Import of ACP
We have noted that the wheat germ lysate possesses a
low level of the Chloroplast processing enzyme capable of
cleaving various precursors, suggesting that it may also
contain other Chloroplast enzymes. In fact, mock reactions
using 5 mM Hepes-KOH, pH 8.0, instead of the spinach
extract have shown that the wheat germ lysate indeed
contributes to the modification reaction converting the 14kD peptide to the 18-kD form. However, when the wheat
germ lysate was boiled to inactivate its enzymatic activity,
the CoA-dependent modification in the organelle-free
assay still occurred with the spinach extract (data not
shown). To establish unequivocally that the spinach extract
is capable of carrying out the modification independent of
the components of the wheat germ lysate, reactions with
and without the addition of CoA were performed using the
translation products of a reticulocyte lysate. Neither the
processing nor the modification activities were found in
mock reactions containing the reticulocyte products and 5
mM Hepes-KOH, pH 8.0, substituted for the spinach extract. When the spinach extract was added without CoA,
pre-ACP gave rise to only the 14-kD peptide. Upon addition
of CoA, however, at least 40% of the 14-kD peptide was
converted to the modified form, as shown in Figure 7. Both
the processing and modification activities were lost when
the spinach extract was boiled (data not shown).
1
2
3
4
5
6
7
201
Figure 8. E. co/i Phosphodiesterase Reverses the CoADependent Modification of the 14-kD Peptide.
An organelle-free assay was carried out using wheat germ translation 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, and
employed in a phosphodiesterase reaction (see Methods and
text). Lanes 2 through 5 show a mock reaction containing no
enzyme (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 the
14-kD peptide from the Modified Form
4
Figure 7. Organelle-Free Processing and CoA-Dependent Modification of Reticulocyte Translation Products.
Translation products of pre-ACP (lane 1) were incubated in organelle-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 minus
and plus signs below each lane. The reactions contained either 5
mM Hepes-KOH, pH 8.0, instead of spinach Chloroplast extract
(lanes 2 and 3), spinach Chloroplast extract clarified by ultracentrifugation (lanes 4 and 5), or crude soluble extract from lysed
chloroplasts (lanes 6 and 7). The positions of apo-ACP and
modified form are indicated by open and filled arrows, respectively.
To investigate further the identity of the modified form
generated in the CoA-dependent reaction, the products of
an organelle-free assay were treated with ACP phosphodiesterase purified from E. coli (Therisod and Kennedy,
1987). This enzyme is specific for holo-ACP, catalyzing
the cleavage of the phosphodiester bond linking the phosphopantetheine to the special serine, and does not react
with other substrates such as CoA (Vagelos and Larrabee,
1967). An organelle-free reaction containing pre-ACP synthesized in a wheat germ lysate and 10 nM CoA was
performed as described above, in which the 14-kD peptide
was completely converted to the 18 kD product, as illustrated in Figure 8, lane 1. The products of this reaction
were dialyzed against 50 mM Tris-HCI, pH 8.5, to remove
CoA and used as the substrate for the ACP phosphodiesterase enzyme.
Phosphodiesterase reactions were carried out using 20
units (Figure 8, lane 3), 40 units (lane 4), and 400 units
(lane 5) of the enzyme. The units of activity are based on
the ability of this enzyme to remove the phosphopantetheine 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 demonstrate that the phosphodiester bond remains stable under
the reaction conditions in the absence of the phosphodiesterase (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 organelles can occur both before and after import. Among
proteins destined for the mitochondria, for example, pyridoxal phosphate is added to aspartate aminotransferase
in'the mitochondrial matrix after its translocation and proteolytic processing (Sharma and Gehring, 1986). Similarly,
ferredoxin is imported and processed to its mature form
by chloroplasts before incorporation of the iron-sulfur complex (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+ oxidoreductase, 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 conformation of mature ACP (Rock et al., 1981), it is difficult to
predict the structural changes that the precursor, containing a 56-amino acid transit peptide with a basic composition, 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 peptide, however, is a derivative of the 14-kD peptide produced by a highly specific modification reaction. Four
criteria indicate that the modification is due to the attachment of the phosphopantetheine to apo-ACP by a chloroplast holo-ACP synthase. First, the production of the modified form depends on the presence of serine 38, to which
the prosthetic group is normally attached. Second, compared 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 between apo-ACP and holo-ACP. Third, COA rapidly stimulates the modification reaction in an organelle-free assay.
Fourth, most convincingly, the modified peptide is converted back to the 14-kD peptide by E. coli phosphodiesterase. In addition, the conformationally sensitive gels indicate that the newly imported and proteolytically processed 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 maintaining 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 Ohlrogge, 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 holoACP synthase performs the modification reaction.
The question arises, what would be the function of two
holo-ACP synthases-one cytoplasmic and the other chloroplastic-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
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 membranederived 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 chloroplast extract typically employed was clarified by ultracentrifugation to remove all membranes, and, thus, we
conclude that the modifying activity, i.e., holo-ACP synthase, 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 organelle-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 predominant product of the reaction when pre-ACP is the substrate. The COA concentration curve suggests that the 14kD 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 synthesis and import of ACP should be considered: (1) preapo-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 apoACP to produce holo-ACP within the chloroplast.
The import reactions show that ACP is found predominantly 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-
203
come peripherally associated with the thylakoids, but cannot 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 apparently sufficient for the bacterial phosphopantetheine to
recognize spinach ACP, albeit less efficiently than the
homologous substrate, and remove the phosphopantetheine. Systematic substitution of the conserved residues
could now be employed to identify the minimum determinants necessary for phosphodiesterase activity.
In summary, we have identified a modification activity in
the soluble fraction of the chloroplast that converts a 14kD peptide to a slower migrating form that is dependent
on the addition of COA. This modification reaction is reversible 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 substrate 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 fluorescent 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 homogenizer 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 ascorbate, 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
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 (InternationalBiotechnologies
Inc., New Haven, CT) downstream from the T7 promoter, yielding
the plasmid plBI-ACP500. The 500-bp insert contains the sequence 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.
A
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
multiplicityof 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, 1O0
pL of the culture was used to inoculate 1O mL of 2 x YT media
(75 pg/mL kanamycin, 150pg/mL ampicillin),which was incubated
at 37°C overnight. The cells were removed by centrifugation at
30009 for 1O 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-strandedDNA was collected by ethanol precipitation 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 methodof Ecksteinand co-workers(Taylor, Ott, and Eckstein,
1985a), designed to give high yields of mutant plasmids (Amersham 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 polymerase I. In the resulting heteroduplex, the newly synthesized
mutant strand was chemically distinguished from the original wildtype strand by the incorporation of dCTP&. Certain restriction
endonucleases (Ncil was used here) are unable to cut phosphorothioate 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 doublestranded mutant plasmid plBI-ACPASer. The incorporation of a
silent, single-nucleotidechange immediately5' to the new alanine
codon created a unique Mlul restrictionsite, allowing for rapid and
accurate isolation of positive plasmids.
translation products. The chloroplasts were incubated with translation 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
phenylmethylsulfonylfluoride (PMSF) for lysis. When chloroplasts
were treated with thermolysin, half of the incubationreaction 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 resuspended in 0.5 M Tris-HCI, pH 6.8, 1O mM DTT, 10% glycerol.
Organelle-Free Reactions
Chloroplast soluble extracts were prepared according to the
protocol of Abad et al. (1989). lntact chloroplasts were resuspended in HSM and centrifuged for 1 min at 25009. The pellet
was gently resuspended in a volume of ice-cold 5 mM HepesKOH, 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
160009for 1O 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 translated 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, 10% glycerol for SDS-PAGE analysis, 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
PhosphodiesteraseTreatment
The plBI-ACP500 and plBI-ACPASer plasmids were linearized
with EcoRl and transcribed with T7 RNA polymerase (New England 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(1O00 Ci/mmol) as the labeled
amino acid. Optimal translation was achieved using 33 mM Kacetate in both wheat germ and reticulocyte translation systems.
Procedures for the import reaction were essentially as described (Bartlett et al., 1982). lntact chloroplasts were resuspended 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-freereactions containing wheat germ lysate translation 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 Kennedy and Anthony Fishl, Harvard University, Cambridge, MA)
essentially as described. The reactions contained 50 mM TrisHCI, 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
releaseof the TCA-soluble radiolabeledphosphopantetheine from
the TCA-insolubleE. coli holo-ACP(Therisodand Kennedy, 1987).
The reactions were stopped by bringing the products to 0.5 M
Chloroplast lmport of ACP
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). Nondenaturing gels were essentially as described (Rock and Cronan,
1981). The separating gel contained 20% acrylamide, 0.5% N,Nmethylenebisacrylamide, 0.5% N,N,N’,N’-tetramethylethyldiamine, 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 concentrationwas 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-diphenyloxazole-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 supportedby U.S. Department of Agriculture Grant
89-3761-4471 (awarded to G.K.L.) and a Sigma Xi Grant-in-Aidof
Research (to M.D.F.).
Received October 27, 1989; revised December 29, 1989.
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