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The Plant Cell, Vol. 1, 1223-1230, December 1989 O 1989 American Society of Plant Physiologists
Severa1 Proteins lmported into Chloroplasts Form Stable
Complexes with the GroEL-Related Chloroplast
Molecular Chaperone
Thomas H. Lubben, Gail K. Donaldson, Paul V. Viitanen, and Anthony A. Gatenby'
Molecular Biology Division, Central Research and Development Department, Experimental Station, E.I. DuPont de
Nemours & Co., Wilmington, Delaware 19880-0402
Nine different proteins were imported into isolated pea chloroplasts in vitro. For seven of these [the large and small
subunits of ribulose-l,5bisphosphate carboxylase/oxygenase (Rubisco), B-subunit of ATP synthase, glutamine
synthetase, the light-harvesting chlorophyll a / b binding protein, chloramphenicol acetyltransferase, and pre-8lactamase], a fraction was found to migrate as a stable high-molecular-weight complex during nondenaturing gel
electrophoresis. This complex contained the mature forms of the imported proteins and the groEL-related chloroplast
chaperonin 60 (previously known as Rubisco subonit binding protein). Thus, the stable association of imported
proteins with this molecular chaperone is widespread and not necessarily restricted to Rubisco subunits or to
chloroplast proteins. With two of the imported proteins (ferredoxin and superoxide dismutase), such complexes
were not observed. It seems likely that, in addition to its proposed role in assembly of Rubisco, the chloroplast
chaperonin 60 is involved in the assembly or folding of a wide range of proteins in chloroplasts.
INTRODUCTION
An abundant chloroplast stromal protein has been implicated in the assembly of the photosynthetic enzyme ribulose-1,.Ç-bisphosphate carboxylase/oxygenase (Rubisco)
(Barraclough and Ellis, 1980; Roy et al., 1982; Cannon,
Wang, and Roy, 1986; Gatenby et al., 1988), and has been
referred to as either the large subunit binding protein
(Barraclough and Ellis, 1980), Rubisco subunit binding
protein (Ellis and van der Vies, 1988), or the chloroplast
chaperonin (Hemmingsen, et al., 1988). Chloroplast chaperonin is related to the groEL protein of Escherichia coli
(Hemmingsen et al., 1988), a heat-shock protein that is
essential for cell growth (Fayet, Ziegelhoffer, and Georgopoulos, 1989) and bacteriophage assembly (Georgopoulos et al., 1973; Sternberg, 1973; Zweig and Cummings, 1973; Kochan and Murialdo, 1983). In addition,
groEL is required for assembly of prokaryotic Rubisco
synthesized in E. coli (Goloubinoff, Gatenby, and Lorimer,
1989), and may also be involved in protein secretion (Bochkareva, Lissin, and Girshovich, 1988; Lecker et al., 1989).
To establish a uniform nomenclature for this ubiquitous
class of proteins found in organelles and bacteria, we shall
refer to these polypeptides of an approximate molecular
weight of 60,000 as chaperonin 60 (cpn60).
It has been proposed that heat-shock proteins of two
distinct classes, the hsp7O-immunoglobulin heavy chain
' To whom correspondence should be addressed.
binding protein family (Pelham, 1986, 1988; Chirico,
Waters, and Blobel, 1988; Deshaies et al., 1988; Zimmermann et al., 1988; Flynn, Chappell, and Rothman, 1989),
and the cpn60 family (Bochkareva et al., 1988; Hemmingsen et al., 1988; Cheng et al., 1989; Goloubinoff et al.,
1989; Lecker et al., 1989; Ostermann et al., 1989), assist
other polypeptides to maintain, or assume, a conformation
required for their correct assembly or localization. How
this is achieved has not been resolved, but a feature of
proteins in both families is that they hydrolyze ATP (Pelham, 1988) and interact with incorrectly or partially folded
proteins (Gething, McCammon, and Sambrook, 1986;
Bochkareva et al., 1988; Kassenbrock et al., 1988; Lecker
et al., 1989). It has been demonstrated that mitochondria
contain a protein, hsp60, that is related to bacterial cpn60
(McMullin and Hallberg, 1988), and that assembly and
intraorganellar targeting of some proteins in mitochondria
involve this chaperonin (Cheng et al., 1989; Ostermann et
al., 1989).
To determine whether chloroplast cpn60 may be involved in interactionswith proteins other than Rubisco, we
imported nine different proteins into isolated chloroplasts
in vitro. This selection of proteins includes severa1 polypeptides that are subunits of oligomeric structures, one
monomeric chloroplast protein and one monomeric secretory protein. For seven of these proteins, a fraction of the
imported protein was found in a high-molecular-weight
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The Plant Cell
complex. This complex contained the importedprotein and
cpn6O. The complex was stable during electrophoresis on
nondenaturing gels but was disruptedeither,by illumination
of chloroplasts or by addition of ATP to soluble extracts.
One monomeric protein (ferredoxin) and one polypeptide
that forms a dimer (superoxide dismutase) did not form
such stable complexes with cpn60.
RESULTS
lmport of Proteins into Chloroplasts
The proteins chosen for this study were the bacterial
proteins pre-P-lactamase (preBLA, monomeric) and chloramphenicol acetyltransferase (CAT, trimeric), and the plant
enzymes Silene pratensis ferredoxin (FD, monomeric), pea
(Pisum sativum) superoxide dismutase (SOD, homodimeric), and alfalfa (Medicago sativa) glutamine synthetase
(GS, octomeric). In addition, the P-subunit of the thylakoid
ATP synthase from maize (Zea mays) chloroplasts (CF,-p)
and the light-harvesting chlorophyll a/b protein (LHCP)
from Petunia hybrida were imported; the Anacystis nidulans large subunit (LS) and pea small subunit (SS) of
Rubisco (hexadecameric) were imported as controls. Plasmids were constructed that fused the appropriate genes
to a soybean (Glycine max) chloroplast Rubisco SS transit
peptide (TP) coding sequence, except for ferredoxin, superoxide dismutase, and preLHCP, which were imported
using their naturally occurring transit peptides. Precursor
proteins, shown in Figure 1, were producedas radiolabeled
polypeptides by in vitro transcription and translation, and
were post-translationallyimported into isolated pea chloroplasts. Figure 2 shows radiolabeled proteins from translation reactions and from import reactions after analysis
by electrophoresis on SDS-polyacrylamide gels and fluorography. It should be noted that, although the size of
preLHCP is given as 28 kD (predictedfrom DNA sequence)
in Figure 1, on SDS-polyacrylamide gels (Figure 2) it migrates as a protein slightly larger than the 30-kD molecular
mass marker (Viitanen, Doran, and Dunsmuir, 1988). Import reactions were conducted in subdued light (less than
1 pEinstein/cm*) to preserve the cpn6O-imported protein
complexes (see below), and externa1ATP providedenergy
for import. As shown in Figure 2, all precursor proteins,
except for TPpreBLA, were imported and processed to
the size expected for proteins from which the transit
peptide had been removed. Following import of TPpreBLA,
three polypeptides were found inside chloroplasts (Figure
2). These were the correct size for TPpreBLA, preBLA,
and BLA. This suggested that (1) the import of some
TPpreBLA occurred unaccompanied by cleavage of the
chloroplast transit peptide and (2) the bacterial presequence may be recognized and cleaved in the chloroplast
stroma. Regardless, all proteins examined were imported
well enough to undertake the binding studies described
below. This required that we be able to determine whether
17'0 of the imported protein were bound to cpn60.
Binding of lmported Proteins to cpn6O
Chloroplast cpn60, like E. coli cpn60 (groEL), can form
oligomers composed of 14 subunits, each of apparent
molecular weight (M,)
of 60,000 to 61,000 (Hemmingsen
et al., 1988). These cpn6O oligomers, together with associated proteins, can be resolved on nondenaturing gels as
a sharp band, which migrates more slowly than Rubisco
holoenzyme. Both the cpn6O and Rubisco bands are
clearly visible upon staining (Barracloughand Ellis, 1980;
Roy et al., 1982; Gatenby et al., 1988). Furthermore, cpn60
oligomers dissociate under certain conditions (Bloom,
Milos, and Roy, 1983; Hemmingsen and Ellis, 1986; Roy,
Hubbs, and Cannon, 1988). To determine whether imported proteins were bound to cpn60, import reactions
were analyzed by electrophoresis on low-percentage polyacrylamide gels at 4°C in the absence of denaturants. As
shown in Table 1, neither imported SOD nor FD associated
with cpn6O. Consistent with an earlier study (Gatenby et
al., 1988), a small but significant portion of imported SS
(about 5%) formed a stable complex with cpn60. This was
also observed for LHCP, regardless of whether it was
Precursor Proteins
2kíLml
20
62
59
31
38
52
17
24
28
31
Figure 1. Precursor Proteins.
Precursor proteins were made by in vitro transcription and translation of plasmids that contained promoters for SP6 or T2 RNA
polymerase upstream of the coding regions (Lubben and Keegstra, 1986). mRNA from the transcription reactions was translated
in vitro in a system derived from either reticulocytelysate or wheat
germ. The resulting proteins are shown diagramatically.
Chloroplast Chaperone Protein Complexes
A B
A B
A
B
A
B
LTPLS
/PpreBLA
-praBLA
A
30
B
A
B
A
B
i
A
B
A
sented stable complexes of cpn60 with imported proteins,
or merely artifacts of co-migration during electrophoresis.
To help make this distinction, we treated Chloroplasts that
contained imported proteins and stromal extracts from
these Chloroplasts as described below. Results from these
studies are shown in Figure 3.
Similar results were obtained for all the proteins that comigrated with cpn60 oligomers (Figure 3). When either
buffer, a glucose/hexokinase mixture or MgCI2 alone (Figure 3), or the sulfhydryl-reducing agent dithiothreitol (data
not shown) were added to chloroplast extracts, no change
was observed in the radiolabeled band that co-migrated
with cpn60. When intact Chloroplasts that contained imported proteins were incubated in light, either in the absence (Figure 3, lane A) or presence (Figure 3, lane B) of
chloramphenicol to inhibit intraorganellar protein synthesis,
the radiolabeled band decreased to about one-third its
control level (Figure 3, lane C). The stained protein band
of cpn60 decreased similarly (data not shown). As anticipated, cpnGO oligomers dissociated when MgCI2 and ATP
were added to extracts made from Chloroplasts that contained imported proteins (Figure 3, lane F). Following this
treatment, some of the radiolabeled protein migrated near
the top of nondenaturing gels as a high-/Wr complex (Figure
3, lane F). In contrast, no dissociation was observed when
the nonhydrolyzable ATP analog /i.-y-methyleneadenosine
5'-triphosphate was used instead of ATP (Figure 3, lane
G). From these studies, we concluded that (1) the observed
band was indeed cpn60 and (2) those proteins that comigrated with the cpn60 band were associated with cpn60.
I
A
-p>FD
1225
B
prSOD
B
Figure 2. SDS-Polyacrylamide Gel Electrophoresis of Proteins
Imported into Chloroplasts.
Precursor proteins were incubated with intact pea Chloroplasts.
After import, Chloroplasts were treated with protease (thermolysin)
to remove external proteins, and intact Chloroplasts were reisolated, lysed, and analyzed by electrophoresis on SDS-polyacrylamide gels. Radiolabeled bands were then visualized by
fluorography. Lanes A, translation products; lanes B, imported,
protease-protected proteins. The identity of the precursor protein
is indicated beside each panel; the migration of 14C-labeled molecular mass standards is shown: myosin, 200 kD; phosphorylase b,
92 kD; bovine serum albumin, 68 kD; ovalbumin, 46 kD; carbonic
anhydrase, 30 kD; lysozyme, 14 kD.
imported using its own transit peptide (as preLHCP) or the
transit peptide of TPSS (as TPLHCP) (Table 1). In contrast,
significantly larger amounts of some of the radiolabeled
imported proteins (BLA, CAT, SS, LS, CF,-,cf, and GS)
were found to co-migrate with the cpn60 band. For these
proteins, further studies were done to determine the nature
of their interaction with cpn60.
Cpn60 oligomers dissociate in the presence of ATP in a
reaction that requires ATP hydrolysis (Bloom et al., 1983;
Hemmingsen and Ellis, 1986; Roy et al., 1988). It is also
known that partial dissociation of cpn60 oligomers and
associated LS occurs when intact Chloroplasts are incubated in light (Roy et al., 1988). These characteristics
provided a convenient way to determine whether the radioactive bands observed in nondenaturing gels repre-
Table 1. Association of Imported Proteins with Chloropiast
cpn60
Precursor Protein
% in cpn60 ± so
TPCF,-/}
TPLS
TPGS
TPCAT
38 ±5
34 12
24 11
17 2.8
6 2.6
5 0.6
3 0.6
3 ±0.6
TPpreBLA
TPSS
TPLHCP
preLHCP
prFD
prSOD
a
ND"
NO
ND, not detectable. The limit of detection was 1%.
Bands that contained cpn60 were excised from nondenaturing
gels similar to those shown in Figure 3 and counted using liquid
scintillation methods. Similarly, bands that contained imported
proteins were also excised from SDS-polyacrylamide gels such
as those in Figure 2. The amount of protein in the cpn60 band
was compared with that found in the band from the SDS gel.
These percentages represent the fraction of total imported protein
that was found associated with cpn60. Values are the average of
at least three experiments.
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The Plant Cell
TPpreBLA
A B C D E F G
till!*}
TPCAT
A B C D E F G
-cpn601
RubisCO
TPSS
A B C D E F G
TPLS
A B C D E F G
— cpn60 —
> RubisCO'1
TPCF-P
TPGS
A B C D E F G
A B C D E F G
To determine whether the imported proteins in the cpn60
complex contained a transit peptide or only mature, processed proteins, these complexes were examined further.
Portions of nondenaturing gels that contained cpn60 and
imported proteins were excised from nondenaturing gels,
equilibrated in SDS sample buffer, and analyzed by SDS
gel electrophoresis. For technical reasons, only proteins
that showed a high level of association with cpn60 were
analyzed. Only mature proteins, without transit peptides,
were found (data not shown). We anticipated this result
because the SDS gels of total imported proteins (Figure 2)
showed that, except for imported TPpreBLA, only processed, mature proteins were present.
To quantitate the interaction of the various imported
proteins with cpn60, the radiolabeled cpnGO oligomer
bands were excised from the nondenaturing gels and
counted using liquid scintillation methods. Similarly, bands
that contained imported proteins were also excised from
SDS-polyacrylamide gels (Figure 2). We preferred this
method over densitometry because stained protein bands
significantly quench any associated label during fluorography but not during liquid scintillation counting of excised
bands. The amount of radiolabeled protein in the cpnGO
bands was compared with that found in the bands from
the SDS gels. These values are shown in Table 1 and
represent the fraction of total imported protein that was
found associated with cpn60. These ranged from 38 ± 5%
for imported CF,-0 to undetectable levels (less than 1%)
for imported SOD and FD.
DISCUSSION
Figure 3. Effect of Treatments on Association of cpnGO with
Imported Proteins.
Intact chloroplasts from the reactions in Figure 2 were subjected
to various treatments, followed by analysis by nondenaturing
polyacrylamide gel electrophoresis and fluorography. Conditions
for electrophoresis were essentially as described (Gatenby et al.,
1988), except that electrophoresis was done at 4°C. The migration positions of the chloroplast chaperonin (cpn60) and Rubisco
holoenzyme (RubisCO) were determined by lightly staining with
bromphenol blue. This staining quenched the radioactive fluorescence of these bands so that the association of imported LS with
Rubisco holoenzyme is not visible. However, radioactivity in Rubisco was readily determined by the use of liquid scintillation
methods to count excised bands. The precursor proteins used in
each reaction are indicated above each panel. For lanes A and B,
intact chloroplasts were incubated in light at 25°C for 10 min. The
reaction for lane B contained 100 Mg/mL chloamphenicol to inhibit
intraorganellar protein synthesis. For lanes C to G, chloroplasts
were lysed hypotonically in 10 mM HEPES-KOH, pH 7.5, and
centrifuged to remove membranes. Various additions were made
to fractions of the supernatant, and these reaction were incubated
for 10 min at 25°C. The additions resulted in these final concentrations in the various reactions: lane C, 10 mM HEPES-KOH, pH
7.5 (control); lane D, 0.08 units/mL hexokinase, 1 mM glucose;
lane E, 10 mM MgCI2; lane F, 10 mM MgCI2/5 mM ATP; lane G,
10 mM MgCI2/5 mM /3,7-methyleneadenosine5'-triphosphate.
In this report we show that a wide variety of imported
proteins are found associated with cpn60. Although binding of intraorganellar-synthesized LS (Barraclough and
Ellis, 1980; Roy et al., 1982) and imported LS (Gatenby et
al., 1988) and SS (Ellis and van der Vies, 1988; Gatenby
et al., 1988) to cpnBO has been reported, the association
of nonRubisco proteins with cpn60 in chloroplasts has not
been described before.
The interaction of several disparate imported proteins
with cpn60 may indicate a general role for this oligomer at
some point in the folding or assembly pathway of imported
chloroplast proteins. The folding and assembly of proteins
to their final conformation has previously been thought to
be an intrinsic feature of the amino acid sequence of a
protein (Anfinsen, 1973). However, in some instances, it is
now known that this process requires the presence of
auxiliary proteins (Cheng et al., 1989; Goloubinoff et al.,
1989; Ostermann et al., 1989). One such class of proteins
are chaperonins (Hemmingsen et al., 1988), which are
involved in protein folding (Ostermann et al., 1989; Goloubinoff et al., 1990) and are required for the assembly of
some oligomeric structures (Georgopoulos et al., 1973;
Sternberg, 1973; Zweig and Cummings, 1973; Kochan
Chloroplast Chaperone Protein Complexes
and Murialdo, 1983; Cheng et al., 1989; Goloubinoff et al.,
1989). Therefore, binding of imported proteins to cpn60,
as observed in the present study, may be an early event
in the folding of chloroplast proteins. It should be noted
that a stable association was observed between cpn60
and the monomeric protein BLA, which obviously does not
become assembled into an oligomeric structure. Apparently, monomeric polypeptides, as well as those that are
components of oligomeric structures, can form complexes
with cpn60 upon import. The product from the association
of nascent or imported polypeptides with cpn60 might be
folded monomers, or assembly-competent subunits of oligomeric proteins, instead of assembled oligomers, thus
invoking a role for cpn60 in protein folding directly. However, both the recognition determinants and the exact role
of these molecular chaperones in protein folding and assembly pathways remain to be determined before a functional model can be established.
If binding of these imported proteins represents an initial
event in protein folding, then the interaction of such a
diverse group of imported proteins with cpn60 indicates
that a common recognition motif may be present in these
proteins. Because chaperonins are not considered to be
part of the final structure (Hemmingsen et al., 1988) and
apparently do not bind to completely folded proteins (Goloubinoff et al., 1990), this motif may be accessible only in
polypeptides entering the chloroplast in an incompletely
folded state. This ability to recognize common sequential
or transiently exposed structural features of the partially
folded imported protein would account for the observed
broad specificity of cpn6O. This type of protein interaction
could also explain the association of cpn60 with Rubisco
LS, synthesized within the chloroplast (Barraclough and
Ellis, 1980; Roy et al., 1982). Elongating polypeptide
chains, emerging from ribosomes, may present features
during translationor after release in an unfolded or partially
folded form that are available for interaction with cpn60 in
a manner analogous to the interaction with polypeptides
entering the organelle.
Two proteins, prSOD and prFD, did not form stable
complexes with cpn60. These proteins were imported
using their own transit peptides rather than the transit
peptide of TPSS. This might lead one to conclude that the
interaction of proteins with cpn6O was mediated by the
transit peptide of TPSS. However, we think this is unlikely
for severa1reasons. First, the Same low level of association
of LHCP with cpn60 was observed whether LHCP import
was directed via its own transit peptide (as preLHCP) or
via the transit peptide of TPSS (as TPLHCP). Second,
when cpn60-imported protein complexes from nondenaturing gels were analyzed by SDS gel electrophoresis, only
mature proteins without transit peptides were found. Finally, a number of proteins without transit peptides have
been shown to associate with cpn60 or groEL. For example, chloroplast-synthesized LS, which contains no
transit peptide, strongly binds to cpn60 oligomers (Barraclough and Ellis, 1980, Roy et al., 1982, 1988), and two
1227
bacterial proteins, preBLA and CAT, which contain no
chloroplast transit peptide, associate transiently with the
heat-shock groEL protein in E. coli S30 extracts (Bochkareva et al., 1988). However, regardless of what determines whether a protein interacts with cpn60, the observation that a variety of proteins form stable complexes
with cpn6O provides a tool with which to examine further
the role of cpn60 in protein folding and import.
Because cpn6O proteins have been found in a wide
variety of organisms (McMullin and Hallberg, 1988) and
share a high level of amino acid sequence homology
(Hemmingsen et al., 1988), it is likely that they also exhibit
a broad specificity with regard to the proteins with which
they interact in the folding or assembly process. Indeed,
the data shown here, which demonstrate a stable association between pea chloroplast cpn60 and a variety of
heterologous proteins, support this. Thus, the chloroplast
chaperonin interacts with the prokaryotic BLA, CAT, and
LS proteins, in addition to binding to plant proteins from
different species (maize CF,-0, alfalfa GS, petunia LHCP).
Furthermore, it is noteworthy that a number of different
temperature-sensitive mutations can be suppressed in vivo
by overproduction of the E. coli groE proteins in bacterial
hosts (Van Dyk, Gatenby, and LaRossa, 1989). Other heatshock proteins also exhibit broad specificity. In vitro interactions betweena large number of short synthetic peptides
and another heat-shock protein (hsc70) have been demonstrated (Flynn et al., 1989). Our results suggest that
chloroplast cpn6O may be crucial in mediating the posttranslational folding and assembly of many imported or
organelle-encoded proteins. Indeed, a similar role has been
suggested recently for mitochondrial cpn60, which is involved in the assembly and intraorganellar targeting of
mitochondrial proteins (Cheng et al., 1989, Ostermann et
al., 1989).
METHODS
Enzymes and Reagents
Most biochemicals were from Sigma unless indicated otherwise.
Radioisotopes, EN3HANCE,and Econofluor were from DuPontNew England Nuclear; SP6 RNA polymerase and restriction enzymes were from Bethesda Research Laboratories; NCS tissue
solubilizerwas from Amersham; RNasin,and lysate for translation,
made from either rabbit reticulocyte or wheat germ, was from
Promega; acrylamide, bisacrylamide, and N,N,N',N'-tetramethylethylenediamine were from Bio-Rad.
Plasmid Construction
Plasmids that encode TPSS, TPLS, and TPCF-P (Lubben and
Keegstra,1986; Gatenby et al., 1988) have been described before.
Plasmids pSPFD22 (Smeekens, van Binsbergen, and Weisbeek,
1228
The Plant Cell
1985), which encodes prFD, pSPA2 (Scioli and Zalinskas, 1988),
which encodes prSOD, and M13mpl8/preLHCP (Viitanen et al.,
1988), which encodes preLHCP, have also been described before.
To make pTPBLA, which encodes TPpreBLA, pGMTP was cut
with Pstl, treated with T4 DNA polymerase, and cut with Ball.
This fragment encodes the transit peptide of TPSS, but with a
Ball site at the 5’ end. This was ligated into pTG2 (Kadonaga et
al., 1984), which had been cut with EcoRl and treated with the
Klenow fragment of DNA polymerase. pGMTP was made by
ligation of the small Hindlll/EcoRIfragment of pGM22/3 (Gatenby
et al., 1988), which encodes the same transit peptide found in
TPSS, into Hindlll/EcoRI-cut pGEMl /BAL. pGEMl/BAL was
made by cutting pGEM1 (Promega) with Sphl, treating with T4
DNA polymerase, and inserting Ball linkers (Bethesda Research
Laboratories, 5’-GTGGCCAC-3’). To make pTPCAT, which encodes TPCAT, a Bglll/partial Hincll fragment encoding CAT was
subcloned from pB3CA81 (French and Ahlquist, 1987) via a
pUC18 polylinker into pGM22/3 cut with Sphl/Pstl. To make
pTPGS, which encodes TPGS, pGSl O0 (DasSarma, Tischer, and
Goodman, 1986) was cut with Bglll, filled in with Klenow, and cut
with Stul. This 1.2-kb glutamine synthetase coding region was
ligated into pGM22/3, which had been cut with Sal1 and repaired
with Klenow. Plasmid pTPLHCP, which encodes TPLHCP (a
chimeric protein in which the transit protein of TPSS has been
fused to mature LHCP), was constructed from a modified version
of M13mpl8/preLHCP (Viitanen et al., 1988). Briefly, the region
encoding the first methionine of mature LHCP ( C C E A ) was
converted to an Sphl site ( G C E C ) , using oligonucleotide-directed mutagenesis as previously described (Viitanen et al., 1988).
A fragment that encoded mature LHCP was excised from this
modified plasmid with Sphl/Xbal. This fragment was ligated into
Sphl/Xbal-cut pGM22/3 (which contains the transit peptide of
TPSS) to produce pTPLHCP.
were subjected to seven different treatments. Each aliquot contained the equivalent of 30 pL (of a total of 220 pL) of the original
import reaction. For the first two treatments,two aliquots Of intact
chloroplasts were incubated in light at 25°C for 10 min. To one
of these chloramphenicol was added to a final concentration of
1O0 pg/mL to inhibit intraorganellar protein synthesis. After this
incubation, chloroplasts were collectedby centrifugationand lysed
hypotonically in 10 mM HEPES-KOH, pH 7.5, and centrifuged to
remove membranes. For the other five treatments, soluble extracts were prepared from chloroplasts that had been lysed hypotonically as described above. Various additions were made to
these extracts (as described in the legend to Figure 3), and these
were incubated for 10 min at 25°C. After incubation, portions of
these reactions and the extracts from the two light-treated chloroplasts were analyzed by SDS-PAGE (Laemmli, 1970) and by
nondenaturing gel electrophoresis through 6% polyacrylamide
gels as described (Laemmli, 1970) except that neither SDS nor 8mercaptoethanol was present (Gatenby et al., 1988). Radiolabeled
bands were visualized by fluorography after treatment of gels with
EN3HANCE.Quantitation of the number of imported molecules
was calculated from the number of leucine or methionine residues
per imported protein after liquid scintillationcounting of gel slices
that had been sdubilized in NCS tissue solubilizer (Walter, Ibrahimi, and Blobel, 1981).
ACKNOWLEDGMENTS
We would like to thank Roy French, Howard M. Goodman, Jeremy
R. Knowles, Janet Scioli, and Sjef Smeekens for providing plasmids, and Cathy Kalbach and Karen Bacot for excellent technical
assistance.
In Vitro Transcription and Translation and Protein lmport
Transcription of linearized plasmid DNA templates to produce
mRNA was performed as described previously (Lubben and
Keegstra, 1986). Translation of this mRNA, in lysate from wheat
germ or reticulocyte lysate, was done according to the manufacturer’s (Promega) protocol, using either 3H-le~~ine
or 35S-methionine. Protein import reactions were done as described previously
(Lubben and Keegstra, 1986), except that reactions were done in
subdued light (less than 1 pEinsteinlcm2)with 45 pL of translation
per reaction, and contained 10 mM ATP to provide energy for
import. After a 30-min incubationat room temperature, reactions
(220 pL) were centrifuged and chloroplasts were resuspended in
0.1 mg/mL thermolysin and incubated for 30 min to remove
externally bound proteins (Cline et al., 1985). lntact chloroplasts
were repurifiedby centrifugationthrough 40% Percoll (Cline et al.,
1985), and the chloroplastswere resuspended in 220 pL of impor?
buffer (Cline et al., 1985) and divided into aliquots for further
treatment.
Analysis of Binding of lmported Proteins to cpn6O Oligomers
For each precursor protein, protease-treatedchloroplastscontaining imported proteins or soluble extracts from these chloroplasts
Received August 1O, 1989; revised October 16, 1989.
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T H Lubben, G K Donaldson, P V Viitanen and A A Gatenby
Plant Cell 1989;1;1223-1230
DOI 10.1105/tpc.1.12.1223
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