From the Institut für Biologie III der
Rheinisch-Westfälische Technische Hochschule Aachen,
Worringerweg 1, 52074 Aachen, Germany, the § Department
d'Ecophysiologie Vegetale et de Microbiologie, Cadarache, F-13108, St.
Paul lez Durance Cedex, France, and ¶ Paradigm Genetics, Research
Triangle Park, North Carolina 27709
Received for publication, January 8, 2001, and in revised form, April 11, 2001
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ABSTRACT |
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The chloroplast signal recognition particle
(cpSRP) consists of an evolutionarily conserved 54-kDa subunit
(cpSRP54) and a dimer of a unique 43-kDa subunit (cpSRP43).
cpSRP binds light-harvesting chlorophyll proteins (LHCPs) to form a
cpSRP/LHCP transit complex, which targets LHCP to the thylakoid
membrane. Previous studies showed that transit complex formation is
mediated through the binding of the L18 domain of LHCP to
cpSRP43. cpSRP43 is characterized by a four-ankyrin repeat
domain at the N terminus and two chromodomains at the C terminus. In
the present study we used the yeast two-hybrid system and in
vitro binding assays to analyze the function of different domains
of cpSRP43 in protein complex formation. We report here that the first
ankyrin repeat binds to the 18-amino acid domain on LHCP that binds to
cpSRP43, whereas the third and fourth ankyrin repeats are
involved in the dimerization of cpSRP43. We show further that the
interaction of cpSRP43 with cpSRP54 is mediated via binding of the
methionine-rich domain of cpSRP54 to the C-terminally located
chromodomains of cpSRP43. Both chromodomains contain essential elements
for binding cpSRP54, indicating that the closely spaced chromodomains
together create a single binding site for cpSRP54. In addition, our
data demonstrate that the interaction of cpSRP54 with the chromodomains
of cpSRP43 is enhanced indirectly by the dimerization motif of cpSRP43.
The signal recognition particle
(SRP)1 is found in the
cytoplasm of most if not all eukaryotes and prokaryotes in which it plays a central role in the cotranslational insertion of membrane proteins into the endoplasmic reticulum and plasma membrane,
respectively (reviewed in Refs. 1 and 2). SRP is a ribonucleoprotein consisting of an RNA and at least one polypeptide of ~54 kDa (SRP54). The particle binds to the signal sequence of proteins as they emerge
from the translating ribosome and mediates targeting to the membrane.
SRP54 is composed of two domains: an N-terminal domain encoding a
GTP-binding site (G domain) and a C-terminal domain characterized
by an unusually high content of methionine (M domain) (3, 4).
Recently, a novel kind of SRP was found in the chloroplast (5, 6).
Chloroplast SRP (cpSRP) contains an SRP54 homologue (7) but differs
strikingly from the cytosolic SRP in that it contains a novel 43-kDa
subunit (cpSRP43) and lacks an RNA. It was shown that cpSRP is required
for the post-translational targeting of members of the light-harvesting
chlorophyll protein (LHCP) family to the thylakoid membrane (5, 8-11).
After import, the nuclear-encoded LHCP is bound by cpSRP to form the
soluble targeting intermediate of LHCP, which is designated "transit
complex" (12). Along with cpSRP, the insertion of LHCP into the
thylakoid membrane requires GTP (13), a chloroplast homologue of the
SRP receptor, chloroplast FtsY (14, 15), and the integral membrane
protein ALB3 (16).
Studies on the subunit stoichiometry and structure of cpSRP have
demonstrated that cpSRP is a trimer composed of one cpSRP43 dimer and
one cpSRP54 monomer (15). Analysis of the post-translational interaction between cpSRP and LHCP have shown that this interaction is
mediated through binding between cpSRP43 and the L18 domain of LHCP, an
18-amino acid peptide located between the second and third
transmembrane domains (17, 18). It is presently unknown whether one or
two subunits of LHCP are present in the transit complex.
The cpSRP43 sequence is characterized by the presence of two types of
motifs mediating protein-protein interactions (9). The N-terminal
region of cpSRP43 contains four ankyrin repeats, and the C-terminal
region contains two closely spaced chromodomains (9). The ankyrin
repeat, an ~33-amino acid-long motif, is found in a very large number
of proteins (reviewed in Refs. 19 and 20). The diversity of biological
roles of ankyrin repeat-containing proteins indicates that the ankyrin
repeat serves as a versatile module generating the dimerization
interface for a variety of different protein substrates. The
chromodomain is a 30-70-amino acid motif that was identified first in
the Drosophila chromatin-binding proteins polycomb and
heterochromatin protein (21, 22). Subsequently, chromodomains were
found in various proteins involved in the regulation of chromatin
structure by protein-protein interactions (23, 24). Interestingly,
cpSRP43 is the first example of a nonnuclear chromoprotein. In the
present work, we used the yeast two-hybrid system to analyze the
function of the ankyrin repeats and the chromodomains of cpSRP43 in
self-dimerization and heterodimerization with cpSRP54 and LHCP. The
binding properties of the minimal interaction domains identified by the
yeast two-hybrid system were confirmed using an in vitro
binding assay.
Plasmid Construction for the Yeast Two-hybrid
System--
Plasmid pAS2 (CLONTECH) was used to
construct the bait plasmids encoding the Gal4 DNA binding domain hybrid
protein. Plasmid pACT2 (CLONTECH) was used to
construct the prey plasmids encoding the Gal4 activation domain hybrid
protein. All constructs encode an additional hemagglutinin epitope tag.
All cDNAs encoding the L33 domain of LHCP (LHCP-(189-222)), the
mature form or truncations of cpSRP43 and cpSRP54, were obtained by PCR
amplification with Pfu polymerase (Stratagene) or
Pwo polymerase (Peqlab) using the plasmids pAB80 (25),
pSPUTKGSTchaos (15), and pAF1 (6) as templates. The cDNA
coding for mature cpSRP43 (residues 61-376) was obtained by using
primers 3 and 17 (Table I). Coding
sequences for the truncations of cpSRP43 were constructed by
using the primer combinations: 3/18 to yield cpSRP43-(61-258),
4/17 to yield cpSRP43-(259-376), 5/17 to yield cpSRP43-(125-376),
6/17 to yield cpSRP43-(156-376), 7/17 to yield cpSRP43-(190-376),
8/17 to yield cpSRP43-(223-376), 3/20 to yield cpSRP43-(61-227), 3/21
to yield cpSRP43-(61-193), 3/22 to yield cpSRP43-(61-159), 9/17 to
yield cpSRP43-(283-376), 10/17 to yield cpSRP43-(296-376), 4/25 to
yield cpSRP43-(259-350), 4/24 to yield cpSRP43-(259-339), and 4/23 to
yield cpSRP43-(259-320) (Table I).
The cDNA encoding mature cpSRP54 (residues 76-564), the G domain
(residues 76-370), and M domain (residues 371-564) of cpSRP54 were
obtained by using the primer combinations 1/15, 1/16, and 2/15,
respectively. For the amplification of a 33-amino acid-long region of
LHCP (residues 189-222) containing the L18 segment (17), primers 11 and 19 were used. The PCR products were digested with the restriction
enzymes NcoI or PagI and BamHI
and cloned into the NcoI-BamHI site of pAS2 or
pACT2. For each construct, the correct insert orientation and reading
frame were confirmed by sequencing.
Yeast Two-hybrid Assay--
Yeast strain Y190 was cotransformed
with the indicated combinations of the pAS2 and pACT2 constructs
according to Ref. 26. One half of the cells was plated on synthetic
medium lacking leucine and tryptophan, and the other half was plated on
synthetic medium lacking leucine, tryptophan, and histidine
( Plasmid Construction for Protein Binding Assays--
The
cDNAs coding for cpSRP43-(190-258), cpSRP43-(259-350),
cpSRP43-(61-159), and the M domain of cpSRP54 (residues 371-564) were
obtained by PCR amplification as described above. Primer combinations
12/26 and 7/27 were used to yield cpSRP43-(190-258)/1 and
cpSRP43-(190-258)/2, 13/28 to yield cpSRP43-(259-350), 14/29 to yield
cpSRP43-(61-159), and 2/30 to yield cpSRP54-(371-564) (Table I). The
reverse primer for the amplification of cpSRP43-(190-258)/2 was
designed to introduce two additional methionine residues at the C
terminus. The PCR products encoding cpSRP43-(190-258)/1, cpSRP43(259-350), and cpSRP43-(61-159) were digested with the restriction enzymes BamHI and ClaI and cloned
into the vector fragment from the in vitro translation
vector pSPUTK-GST-chaos (15) digested with
BamHI-ClaI. The resulting constructs encode cpSRP43-(190-258), cpSRP43-(259-350), and cpSRP43-(61-159) as GST
fusion proteins. The in vitro translation vector pSPUTK-GST was created by a blunt-end ligation of the vector fragment from pSPUTK-GST-chaos (15) digested with BamHI-ClaI
followed by a fill-in reaction by Klenow.
The PCR products encoding cpSRP43-(190-258)/2 and cpSRP54-(371-564)
were digested with NcoI and HindIII and cloned
into the NcoI-HindIII site of the in
vitro translation vector pGem4SS6.5Nco1 (7). For each construct
the correct insert orientation and reading frame were confirmed by
sequencing. The translation vector encoding the L18 domain of LHCP
(LHCP-(189-206)) as a preprolactin fusion was described by DeLille
et al. (17).
Protein Pull-down Assays--
To test if GST-cpSRP43-(190-258)
dimerizes with cpSRP43-(190-258), both proteins were cotranslated in a
wheat germ extract (Promega) in the presence of
[35S]methionine. Control samples included a cotranslation
of GST with cpSRP43-(190-258) and single translations of
GST-cpSRP43-(190-258) and cpSRP43 (190). 50 µl of the two
cotranslation reactions and 25 µl of in vitro translated
GST-cpSRP43-(190-258) were diluted into an incubation buffer (20 mM Hepes-KOH, pH 8.0, 50 mM KOAc, and 10 mM MgCl2) in a total volume of 120 µl. The
precipitation of the GST fusion proteins with glutathione-Sepharose was
done as described by Tu et al. (18). The eluted samples were
analyzed by Tricine-SDS-polyacrylamide gel electrophoresis and detected by radioimaging on a PhosphorImager.
Radiolabeled in vitro translated GST-cpSRP43,
GST-cpSRP43-(259-350), GST-cpSRP43-(61-159), and GST were separated
on a 13% acrylamide gel, and radioactivity was quantitated by
radioimaging on a PhosphorImager. Equal picomoles of in
vitro translated GST-cpSRP43, GST-cpSRP43-(259-350),
GST-cpSRP43-(61-159), and GST were incubated with 20 µl of
radiolabeled in vitro translated cpSRP54-(371-564) or
LHCP-(189-206) as indicated. All binding reactions contained equal
amounts of wheat germ extract. The proteins were diluted into an
incubation buffer as described above, and the samples were incubated at
25 °C for 20 min. The precipitation of the GST fusion proteins with
glutathione-Sepharose was done as described by Tu et al.
(18). The eluted samples were analyzed by 15% acrylamide gels and
detected by radioimaging on a PhosphorImager.
Dimerization of cpSRP43 Is Mediated via the Ankyrin Repeat Domain
of cpSRP43--
In a previous study, it was demonstrated that cpSRP43
occurs as a homodimer (15). This result was based on the observation that cpSRP43, from the stroma of an Arabidopsis mutant that
lacks cpSRP54 (ffc1-2), elutes from a gel filtration column
at 70 kDa, which is twice the predicted monomer molecular mass.
In addition, homodimerization of cpSRP43 was shown by cross-linking
experiments with highly purified recombinant cpSRP43 (15). Sequence
analysis of cpSRP43 has shown that the protein contains four ankyrin
repeats at the N terminus and two chromodomains at the C terminus (9). A schematic presentation of the exact location of these sequence motifs
is shown in Fig. 1. To determine which
domains of cpSRP43 mediate its dimerization, we cloned the mature
protein and various deletion constructs (Fig.
2) into the bait plasmid pAS2 and the prey plasmid pACT2. Yeast cells were cotransformed with the indicated plasmid combinations. The ability of the cotransformed yeast cells to
grow on medium lacking histidine (
To define more clearly the region of the ankyrin repeat domain involved
in dimerization, a series of truncations of cpSRP43, removing the
N-terminal region outside the ankyrin repeats (cpSRP43-(125-376)), followed by successive removal of three ankyrin repeats
(cpSRP43-(156-376), cpSRP43-(190-376), and cpSRP43-(223-376)), was
constructed (Fig. 2). Table II shows that mature cpSRP43 and
cpSRP43-(125-376) both dimerized with cpSRP43-(125-376),
cpSRP43-(156-376), cpSRP43-(190-376), and cpSRP43-(223-376) without
any obvious loss of interaction strength. These data suggest that the
fourth ankyrin repeat by itself is sufficient to mediate dimerization.
This result was corroborated by the finding that cpSRP43-(223-376)
interacts strongly with its corresponding prey construct. The third
ankyrin repeat is also capable of mediating dimerization as evidenced
by the strong interaction of cpSRP43-(61-227), lacking the fourth
ankyrin repeat, with cpSRP43 (Table II). When the third ankyrin repeat is also removed (cpSRP43-(61-193)), dimerization is essentially abolished. No binding was detected between cpSRP43 and
cpSRP43-(61-159) containing the first ankyrin repeat only. These data
together with the results from Table II indicate that the third and
fourth ankyrin repeats each are capable of mediating the dimerization of cpSRP43.
The M Domain of cpSRP54 Interacts with cpSRP43--
Like
cytoplasmic SRP54, chloroplast SRP54 is also composed of an N-terminal
G domain (residues 76-370) and a C-terminal M domain (residues
371-564). To analyze which domain of cpSRP54 mediates binding to
cpSRP43, mature cpSRP54 and its G and M domain were cloned in pAS2, and
the interaction with cpSRP43 as prey was tested. The results show that
cpSRP54 interacts strongly with cpSRP43 in the yeast two-hybrid system
and that the same strength of interaction is obtained between the M
domain of cpSRP54 and cpSRP43 (Table
III). No binding was observed between the
G domain of cpSRP54 and cpSRP43. These data show clearly that the
interaction between cpSRP43 and cpSRP54 is mediated via the M domain of
cpSRP54.
The Chromodomains of cpSRP43 Mediate Binding to cpSRP54--
To
characterize the binding site of cpSRP43 for cpSRP54, we tested the
binding of cpSRP54 to a series of N-terminal deletion constructs of
cpSRP43. No obvious change in binding intensity was observed between
cpSRP54 and mature cpSRP43, cpSRP43-(125-376), cpSRP43-(156-376),
cpSRP43-(190-376), or cpSRP43-(223-376) (Table IV). A pronounced lesser, but still
significant, binding was detected between cpSRP54 and the
chromodomains-containing C-terminal part of cpSRP43
(cpSRP43C-(259-376)) (Table IV). If the loss of binding activity
between cpSRP54 and cpSRP43C-(259-376) versus cpSRP54 and
cpSRP43-(223-376) is caused by a direct interaction of cpSRP54 with
the fourth ankyrin repeat, we reasoned that cpSRP54 should interact
with the ankyrin repeat-containing N-terminal part of cpSRP43.
However, binding of cpSRP54 to cpSRP43A-(61-258) or even cpSRP43-(61-305) was not observed. Together with the above-mentioned observation that cpSRP43-(223-376) forms a dimer (Table II), these binding data indicate that cpSRP54 binds cpSRP43 via the chromodomains and suggest that binding is promoted by the dimerization of cpSRP43. Alternatively, the dimerization motif of cpSRP43 may stabilize or
enhance cpSRP54 binding independently of dimerization.
To define the binding site of cpSRP43 for cpSRP54 in more detail, N-
and C-terminal truncations of cpSRP43C-(259-376) were constructed, and
the interaction with cpSRP54 was tested. Removal of amino acids
259-282 (cpSRP43-(283-376)), containing the first conserved region of
the first chromodomain, completely abolished binding to cpSRP54 (Table
IV). The C-terminal deletion of amino acid 351-376
(cpSRP43-(259-350)) had no effect on cpSRP54 binding, but additional
removal of the second strongly conserved region of the second
chromodomain (cpSRP43-(259-339)) resulted in a complete loss of
binding activity. These data demonstrate clearly that both
chromodomains contain essential elements for binding cpSRP54.
The L18 Domain of LHCP Binds to the First Ankyrin Repeat of
cpSRP43--
Recent studies have shown that the L18 domain, a
hydrophilic region located between the second and third transmembrane
domain of LHCP (Fig. 2), can efficiently bind cpSRP43 (17, 18). We observed a strong interaction between a 33-amino acid-long region containing L18 (LHCP-(189-222)) and cpSRP43 by using the yeast two-hybrid assay (Table V). Further
binding tests showed that LHCP-(189-222) interacts with the ankyrin
repeat-containing region of cpSRP43 (cpSRP43A-(61-258)), but no
binding was detected between LHCP-(189-222) and the C-terminal
chromodomains-containing domain of cpSRP43 (cpSRP43C-(259-376)) (Table
V). To map the binding site of cpSRP43A-(61-258) for LHCP-(189-222)
more precisely, we tested the interaction of LHCP-(189-222) with the
indicated N-terminal deletion constructs of cpSRP43 (Table V).
LHCP-(189-222) binds cpSRP43-(125-376) with the same
efficiency as cpSRP43, indicating that the N-terminal region outside
the ankyrin repeat domain is not involved in binding (Table V). No
binding was detected between LHCP-(189-222) and the constructs
cpSRP43-(156-376), cpSRP43-(190-376), and cpSRP43-(223-376) (Table
V). These data suggest that the first ankyrin domain mediates binding
to LHCP-(189-222). This assumption was confirmed by the observation
that LHCP-(189-222) interacts with the cpSRP43-(61-159) containing
the extreme N terminus and the first ankyrin repeat only. This
interaction was weaker than the interaction with the complete ankyrin
repeat domain. However, the differences in binding strength were not
distinctive enough to decide clearly whether LHCP binding needs a
dimeric state of cpSRP43.
The Results of the Yeast Two-hybrid System Are Confirmed by in
Vitro Protein Binding Assays--
We next sought to confirm the
binding properties of the minimal identified interaction domains
characterized by the yeast two-hybrid system by in vitro
fusion protein binding assays. First, we verified that the region of
cpSRP43 containing the third and fourth ankyrin repeat
(cpSRP43-(190-258)) is able to form homodimers. To test this notion we
cotranslated a GST-cpSRP43-(190-258) fusion protein together with
cpSRP43-(190-258) and checked whether cpSRP-(190-258) is
co-precipitated in a pull-down assay using glutathione-Sepharose. Control samples contained a cotranslation of GST and cpSRP43-(190-258) and a single translation of GST-cpSRP43-(190-258). Results of the
binding reaction show that cpSRP43-(190-258) is specifically co-precipitated by GST-cpSRP-(190-258) and not by the unfused GST
protein (Fig. 3A). The sample
containing the precipitation of GST-cpSRP43-(190-258) by itself did
not contain a radiolabeled protein of the size of cpSRP43-(190-258).
Therefore it can be ruled out that the radiolabeled protein in the
co-precipitation of GST-cpSRP43-(190-258) and cpSRP43-(190-258) is
generated by the degradation of GST-cpSRP43-(190-258).
Second, we wanted to confirm that the interaction of cpSRP54 with
cpSRP43 is mediated via binding of the M domain of cpSRP54 (cpSRP54-(371-564)) to cpSRP43-(259-350). To test that, in
vitro translated GST-cpSRP43-(259-350), GST-cpSRP43, or GST were
incubated with radiolabeled in vitro translated
cpSRP54-(371-564), and binding was analyzed using a pull-down assay.
Fig. 3B shows that cpSRP54-(371-564) is specifically
co-precipitated by GST-cpSRP43 and GST-cpSRP43-(259-350) and not by
the unfused GST protein. The binding of cpSRP54-(371-564) to
GST-cpSRP43-(259-350) is significantly weaker than the binding to
full-length GST-cpSRP43, reflecting very well the results obtained with
the yeast two-hybrid system.
Third, we confirmed that the first ankyrin domain of cpSRP43 binds to
the L18 domain of LHCP. In vitro translated
GST-cpSRP43-(61-159), GST-cpSRP43, or GST was incubated with
radiolabeled in vitro translated LHCP-(189-206). Fig.
3C shows that LHCP-(189-206) binds to
GST-cpSRP43-(61-159), supporting the result obtained by the yeast
two-hybrid system. As in the in vivo system, this
interaction was slightly weaker than the binding to the full-length
GST-cpSRP43.
In the present study we used the yeast two-hybrid system and
in vitro binding assays to define the interacting domains
among cpSRP43, cpSRP54, and LHCP. These proteins comprise the transit complex, an intermediate in the integration of LHCP into the thylakoid membrane. Although it was shown previously that the L18 domain of LHCP
binds to cpSRP43 (17, 18), neither the interacting domains of cpSRP43
that bind LHCP nor the interacting domains mediating homodimerization
of cpSRP43 or heterodimerization of cpSRP were identified previously.
We tested whether these interactions occurred through the predicted
protein interaction motifs within cpSRP43, namely the four ankyrin
repeats and two chromodomains. Four clear conclusions emerge from these
studies. First, the binding between LHCP and cpSRP43 occurs through the
first ankyrin repeat of cpSRP43 and the L18 domain. Second,
homodimerization of cpSRP43 occurs through both the third and fourth
ankyrin repeats. Third, heterodimerization between cpSRP43 and cpSRP54
is mediated via the M domain of cpSRP54 and requires both chromodomains
of cpSRP43. Fourth, the ankyrin repeats comprise at least two types of
nonoverlapping binding sites. One mediates homodimerization of cpSRP43,
and the other mediates the binding of cpSRP43 with LHCP.
Ankyrin repeats have been described to be involved in binding
heterologous proteins (19) as well as in mediating homodimerization (28, 29). The ankyrin repeats of cpSRP43 exhibit both types of
binding. Interestingly, repeats 3 and 4 are involved in forming cpSRP43
homodimers and seem fundamentally different from repeat 1, which cannot
homodimerize but can bind the L18 domain. The molecular basis for these
different binding properties of the two kinds of domains is an
interesting question for future studies. Our results do not specify a
role for the ankyrin repeat 2. This domain may be involved in binding
other factors or may have subtle influences over binding that are not
recognized with the present assay.
The chromodomain of cpSRP43 is distinctive from previously
characterized chromodomain proteins. The latter fall into three subgroups: those containing just a single chromodomain, those containing two chromodomains, and those containing a chromodomain and a
shadow chromodomain (23, 24). Shadow chromodomains are closely related
to chromodomains but form a distinct subgroup (23). Chromodomains found
in pairs are typically separated by a flexible linker and function
independently. For example, the chromodomain of heterochromatin
protein-1 Like cytosolic SRP54, cpSRP54 consists of two separate compact domains:
an N-terminal G domain and a C-terminal M domain. The G domain of
cpSRP54 is 70% similar to the prokaryotic protein, indicating that
both domains have an analogous function. The similarity between the
prokaryotic and chloroplast M domain (50%) (7) is less pronounced.
In cytosolic SRP54, the M domain mediates the binding of the RNA
component of SRP and also recognizes the signal sequence of the protein
being translated (3, 4). Although not explicitly shown, the M domain of
cpSRP54 is also likely to bind to substrate because direct interaction
between cpSRP54 and LHCP has been shown to occur via chemical
cross-linking (8), and the hydrophobic sequence in LHCP is required for
transit complex formation (18). In this work, we show that the
interaction between cpSRP54 and cpSRP43 is mediated via the M domain of
cpSRP54. Thus binding of cpSRP54 to cpSRP43 and cytosolic SRP54 to
SRP-RNA both occur through their respective M domains. In a previous
report we showed that cpSRP54 does not bind prokaryotic SRP-RNA and
that prokaryotic SRP54 can not bind to cpSRP43 (6). Together these data
support the idea that the M domain of cpSRP54 has evolved a
fundamentally different binding function from its cytosolic predecessor. The exact nature of the motif mediating the binding to
cpSRP43 remains to be resolved.
It had been determined previously that each complex of cpSRP contains
two cpSRP43 subunits/subunit of cpSRP54 (15). However, it was
not known whether the dimerization of cpSRP43 promoted binding to
cpSRP54. In the current work we observed that constructs of cpSRP43
lacking the dimerization domain bound cpSRP54 with strongly reduced
efficiency. Thus it seems that dimerization of cpSRP43 indeed
stabilizes cpSRP. In the current work we also observed that constructs
of cpSRP43 lacking the dimerization domain also bound LHCP less
effectively. Thus the dimerization of cpSRP43 may also promote the
binding of substrate. From these observations, we predict that
mutations preventing cpSRP43 dimerization will lack activity in LHCP
integration. This hypothesis is currently being tested.
Our data suggest that in the transit complex, cpSRP43 acts as a
molecular adaptor with the contact sites for dimerization in the middle
of the protein and the N- and C-terminal flexible arms providing
independent binding sites for LHCP and cpSRP54. The arms may form a
cradle around LHCP where the N terminus of cpSRP43 binds to the
hydrophilic part of LHCP, and the C terminus covers the hydrophobic
portion of LHCP via mutual binding to cpSRP54. A diagram depicting this
putative arrangement is shown in Fig. 4.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Primers used for PCR amplification
Leu/
Trp/
His) and containing 25 mM 3-aminotriazole.
Yeast cells were incubated for 6 days. Activation of the
his/lacZ reporter was scored by
measuring the growth and assessing the
-galactosidase activity by
using the filter-lift assay (27). Growth was classified in (+++), (++),
(+), and (
), where (+++) means that most colonies have a diameter of
>1.5 mm and (
) indicates normal background growth (white-ish
colonies <0.6 mm). The filter lifts were incubated for at least
1.5 h to develop a blue color ((+++), strong blue color; (++),
medium blue color; (+), slight blue color; (
), no blue color
development). As a positive control we cotransformed yeast cells with
the control vectors pVA3 and pTD1 (CLONTECH). These
cells showed strong growth on
Leu/
Trp/
His medium and developed a
strong blue color after a 1.5-h incubation. As negative controls, each
protein hybrid construct was cotransformed with either pAS2 or pACT2.
Yeast cells cotransformed with pAS2 and the constructs cloned into
pACT2 showed only background growth on
Leu/
Trp/
His plates and had
no
-galactosidase activity even after 24 h of color
development. Constructs encoding either deletions of cpSRP43 or mature
cpSRP43 and cloned into pAS2 showed slight self-activation of the
his reporter, which led to some cell growth (+) on
Leu/
Trp/
His medium. However, all of these yeast clones showed no
blue color development after 1.5 h. Yeast cells cotransformed with
pACT2 and the pAS2 constructs encoding mature or deletions of cpSRP54
showed only background growth on
Leu/
Trp/
His plates and had no
-galactosidase activity even after 24 h of color development. LHCP-(189-222) cloned into pAS2 showed strong self-activation of the
his-lacZ reporter, such that only the pACT2 version of this construct was used. For fusion proteins that showed only weak or
no interaction in the yeast two-hybrid experiments, an expression level
comparable with full-length cpSRP43 or cpSRP54 was verified by Western
blot analysis using antibodies against the hemagglutinin tag (Roche
Molecular Biochemicals). All experiments were performed at least
in duplicate.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
His) and to express
-galactosidase indicates an interaction of the tested proteins.
Table II shows that bait cpSRP43
interacts with prey cpSRP43, confirming our previous conclusion that
cpSRP43 forms a homodimer (15). As a first step toward analyzing which
domain of cpSRP43 mediates dimerization, the interaction between mature
cpSRP43 and the ankyrin repeat-containing N-terminal part of cpSRP43
(cpSRP43A-(61-258)) versus the chromodomains-containing
C-terminal part of cpSRP43 (cpSRP43C-(259-376)) was tested.
Table II shows that mature cpSRP43 interacts with cpSRP43A-(61-258)
but not with cpSRP43C-(259-376). The notion that the self-dimerization
of cpSRP43 is mediated via the ankyrin repeat domain was further
confirmed by the observation that bait and prey cpSRP43A-(61-258)
interact but that no interaction was seen between the two
cpSRP43C-(259-376) constructs.
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Fig. 1.
Scheme of the domain organization of
cpSRP43. The N-terminal region of cpSRP43 contains an ankyrin
repeat domain with four ankyrin repeats (residues 130-254). The
C-terminal region of cpSRP43 contains two closely spaced chromodomains
(first chromodomain, residues 271-320; second chromodomain, residues
321-368). The black boxes within the chromodomains indicate
conserved regions common in chromodomains and shadow chromodomains. The
gray box within the second chromodomain indicates a
conserved region common in chromodomains only (9, 23, 31). The
numbers refer to the amino acid position.
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Fig. 2.
Schematic representation of the constructs of
cpSRP43, cpSRP54, and LHCP used in the yeast two-hybrid assay.
A, constructs of cpSRP43 (a detailed description of the
shaded boxes is given in the Fig. 1 legend). B,
constructs of cpSRP54. The protein consists of an N-terminal
GTPase-containing domain (G domain) and a C-terminal methionine-rich
domain (M domain). C, the position of the L33 domain of LHCP
within the second and third transmembrane domain (TM) of the
full-length protein (dashed lines) is indicated. The
abbreviations of all constructs are given.
The ankyrin repeat domain of cpSRP43 mediates its self-dimerization
his-lacZ
reporter was measured as described under "Experimental Procedures."
A schematic presentation of the used constructs is given in Fig. 2. /;
not measured.
The M domain of cpSRP54 binds cpSRP43
his-lacZ reporter was measured as described
under "Experimental Procedures."
Both chromodomains of cpSRP43 are involved in the binding of cpSRP54
his-lacZ reporter was
measured as described under "Experimental Procedures."
The L18 domain of LHCP binds to the first ankyrin repeat of cpSRP43
his-lacZ reporter was measured as described
under "Experimental Procedures."
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Fig. 3.
In vitro protein binding assays
confirm the results obtained by the yeast two-hybrid system.
A, first and second lanes,
radiolabeled cotranslation products (Co-TP) of
cpSRP43-(190-258) and GST-cpSRP43-(190-258) or cpSRP43-(190-258) and
GST. Third-fifth lanes, results of a pull-down assay in
which the cotranslation products or radiolabeled in vitro
translated GST-cpSRP43-(190-258) were used as described under
"Experimental Procedures." B, first lane,
radiolabeled in vitro translated cpSRP54-(371-564)
(54-M). Second-fourth lanes, results of a
pull-down assay in which radiolabeled in vitro translated
cpSRP54-(371-564) was incubated with equal picomoles of in
vitro translated GST-cpSRP43, GST-cpSRP43-(259-350), or GST as
described under "Experimental Procedures." C,
first lane, radiolabeled in vitro translated
LHCP-(189-206)-preprolactin (L-18).
Second-fourth lanes, results of a pull-down assay in which
radiolabeled in vitro translated
LHCP-(189-206)-preprolactin was incubated with equal picomoles of
in vitro translated GST-cpSRP43, GST-cpSRP43-(61-159), or
GST as described under "Experimental Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
functions as a protein interaction motif for heterologous
proteins, whereas the shadow domain of heterochromatin protein-1
mediates homodimerization (30-32). Interestingly, the chromodomains of
cpSRP43 are not separated by a linker region, which led to the
speculation that the two domains comprise a single protein binding site
(9). We now provide two lines of evidence to support this assumption.
First, the cpSRP43 chromodomains only seem to interact with cpSRP54; these domains do not mediate homodimerization nor do they promote binding to LHCP, chloroplast
FtsY,2 and
ALB3.2 No binding between
cpSRP43 and chloroplast FtsY was also noted by Tu et al.
(18) using pull-down assays. Second, with regard to the binding between
cpSRP54 and the chromodomains, the interaction was abolished after the
deletion of a conserved portion of a single chromodomain, indicating
that both chromodomains contain essential regions for binding cpSRP54.
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Fig. 4.
Model of the transit complex. cpSRP
consists of one cpSRP43 dimer and one cpSRP54 monomer. The
homodimerization of cpSRP43 is mediated via the third and fourth
ankyrin repeat of cpSRP43. The heterodimerization between cpSRP43 and
cpSRP54 occurs between the M domain of cpSRP54 and both chromodomains
of cpSRP43. The cpSRP complex is presumed to interact with a single
molecule of LHCP to form the transit complex. In this complex, the L18
domain of LHCP interacts with the first ankyrin repeat of cpSRP43, and
a hydrophobic domain of LHCP contacts cpSRP54, presumably within
the M domain. It can be speculated that cpSRP forms a cavity to shield
LHCP from the aqueous phase. tm1, tm2,
tm3, transmembrane domains 1, 2, and 3, respectively.
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ACKNOWLEDGEMENTS |
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We are grateful to Alan Slusarenko for giving us the opportunity to perform the experiments described in this study in his laboratory and for critical reading of the manuscript. We thank Nikolaus Schlaich for advice concerning the yeast two-hybrid technique.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grant SCHU 1163/2.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
49-241-805871; Fax: 49-241-8888181; E-mail:
schuenemann@bio3.rwth-aachen.de.
Published, JBC Papers in Press, April 16, 2001, DOI 10.1074/jbc.M100153200
2 E. Klostermann, E. Jonas-Straube, and D. Schünemann, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: SRP, signal recognition particle; G domain, N-terminal domain encoding a GTP-binding site of cpSRP54; M domain, C-terminal methionine-rich domain of cpSRP54; cpSRP, chloroplast SRP; LHCP, light-harvesting chlorophyll protein; L18, the 18-amino acid domain on LHCP that binds to cpSRP43; PCR, polymerase chain reaction; GST, glutathione S-transferase; Tricine, N-(2-hydroxy-1,1-bis(hydroxy- methyl)ethyl)glycine.
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REFERENCES |
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