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J Biol Chem, Vol. 274, Issue 17, 12177-12182, April 23, 1999
From the Department of Plant Biology, Carnegie Institution of
Washington, Stanford, California 94305 and the SecY is a component of the protein-conducting
channel for protein transport across the cytoplasmic membrane of
prokaryotes. It is intimately associated with a second integral
membrane protein, SecE, and together with SecA forms the minimal core
of the preprotein translocase. A chloroplast homologue of SecY (cpSecY)
has previously been identified and determined to be localized to the
thylakoid membrane. In the present work, we demonstrate that a SecE
homologue is localized to the thylakoid membrane, where it forms a
complex with cpSecY. Digitonin solubilization of thylakoid membranes
releases the SecY/E complex in a 180-kDa form, indicating that other
components are present and/or the complex is a higher order oligomer of
the cpSecY/E dimer. To test whether cpSecY forms the protein-conducting channel of the thylakoid membrane, translocation assays were conducted with the SecA-dependent substrate OE33 and the
SecA-independent substrate OE23, in the presence and absence of
antibodies raised against cpSecY. The antibodies inhibited
translocation of OE33 but not OE23, indicating that cpSecY comprises
the protein-conducting channel used in the SecA-dependent
pathway, whereas a distinct protein conducting channel is used to
translocate OE23.
Thylakoid membranes consist of proteins synthesized by both
nuclear and chloroplast genomes. Nuclear encoded thylakoid proteins are
first targeted to the chloroplast by means of the transit peptide,
which initiates the translocation of the protein across the envelope
membranes into the stroma (1). Translation initiation of chloroplast
encoded thylakoid proteins appears to occur in the stroma (2, 3), and
then synthesis appears to continue on thylakoid bound ribosomes through
a co-translational targeting mechanism (4). Considerable progress has
been made in defining mechanisms by which nuclear encoded thylakoid
proteins insert or translocate posttranslationally across the membrane.
One class of proteins insert into the membrane in the absence of an
energy supply, soluble factors, or membrane components (5-8). A second class of proteins does not require any soluble factors but requires a
trans-thylakoid pH gradient and the membrane protein encoded by the
gene hcf106 (9-13). A third class of proteins requires ATP
and a chloroplast homologue of the bacterial protein, SecA (14, 15).
Finally, a fourth class of proteins requires GTP (16), chloroplast
homologues of the bacterial proteins
SRP541 (17, 18) and
FtsY,2 and a novel protein,
cpSRP43 (19, 20). Little is known about the targeting of chloroplast
encoded proteins; however, it is likely that they share many of the
same translocation components described above (21-24). In two of the
cases mentioned above, no soluble factors are required (6, 10); in two
other cases, reconstitution has been achieved in the presence of
purified soluble components instead of stroma (14),2
thereby defining the minimum soluble-factor requirements. However, membrane components remain to be elucidated for the Protein export across the bacterial inner membrane is catalyzed by a
membrane-embedded translocation apparatus consisting of SecY, SecE,
SecG, SecF, SecD, and YajC in conjunction with the peripheral protein,
SecA (25, 26). The essential core of the translocase is SecY/SecE and
SecA (27, 28). SecY/E form a transmembrane channel through which the
exported protein is threaded (29). SecA is thought to act like a
piston, pushing the protein through the membrane channel (30, 31).
Bacteria also contain an SRP, and recently it was shown that polytopic membrane proteins are dependent on this complex for insertion into the
cytoplasmic membrane (32). Furthermore, it was shown that the Sec
translocase is utilized in the bacterial SRP pathway. Thus, the SRP-
and SecY-dependent pathways converge at SecY (33).
Bacteria also contain a pathway for exporting proteins across the inner
membrane independently of SecY (34). Proteins that utilize this pathway
have a twin arginine motif at the N terminus (34, 35), as do proteins
that utilize the In addition to SecA, chloroplasts contain a thylakoid protein related
to SecY (39). However, the cpSec translocase is largely uncharacterized. It has been assumed, although it remains to be shown,
that cpSecY is part of the translocase that translocates SecA-dependent substrates. Furthermore, it is not known
whether there is convergence of the SRP- and
Arabidopsis thaliana (ecotype Columbia) was grown in
a growth chamber in a 16 h light/22 °C temperature
versus 8 h dark/18 °C temperature cycle. The light
intensity during the light period was 60 µE m Radiolabeled Arabidopsis cpSecE precursor (pcpSecE), the
intermediate form of wheat OE33 (iOE33), and wheat OE23 precursor (pOE23) were prepared by in vitro transcription and
translation by using SP6 polymerase and [35S]methionine
as described (41).
CpSecE Cloning--
The forward primer
5'-CCACATGTCACTAACCGCACAATTC-3' and the reverse primer
5'-CCCAAGCTTCACATCATGCTGAAGAAGTCTTGAAC-3' were used to amplify the gene
encoding cpSecE (Cse) from Arabidopsis genomic DNA by PCR using Pfu polymerase (Stratagene). To enhance
radiolabeling, the amplified Cse PCR product was designed to
contain two additional methionine residues at the C terminus. The
PCR product was digested with AflIII and HindIII
and cloned into the NcoI-HindIII site of the
translation vector pGem4SS6.5NcoI (17), resulting in the plasmid
pGem4SS6.5NcoIcpSecE.
For overexpression of cpSecE, a N-terminal hexahistidine-tagged
version was constructed. Cse was amplified from
pGem4SS6.5NcoIcpSecE by using the forward primer
5'-CCACATGTCACTAACCGCACAATTC-3' and the reverse primer
5'-CGGGATCCATGTCACTAACCGCACAATTC-3'. The PCR product was digested with
HindIII and BamHI and cloned into the HindIII-BamHI site of the expression vector pQE30
(Qiagen). The resulting plasmid (pQE30cpSecE) was transformed into
SG13009 cells.
Antibodies and Immunoblot Analysis--
SG13009 cells containing
pQE30cpSecE were grown to an A600 of ~0.6 and
incubated with 1 mM
isopropyl-
Antibodies against cpSecY were raised in rabbits injected with the
synthetic peptide CYKNIEFYELDKYDP, corresponding to the C terminus of
Arabidopsis cpSecY, fused to keyhole limpet hemocyanin.
Immunoblot analysis was done as described in Ref. 42. For cpSecE
detection, crude antiserum was used at a dilution of 1:750. For cpSecY
detection, the IgG fraction purified by ammonium sulfate precipitation
and diethyl aminoethyl (DEAE)-Sephadex (43) was used at a dilution of
1:3500. Proteins were detected by enhanced chemiluminescence (42).
Thylakoid Isolation--
Arabidopsis leaf
tissue (10 g of fresh weight, 4-6 weeks old) was ground in 400 ml of
50 mM Hepes-KOH, pH 8.0, 330 mM sorbitol, 10 mM EDTA, 5 mM sodium ascorbate, 0.05% bovine
serum albumin in a polytron (Calbiochem) and the homogenate was
filtered through two layers of Miracloth. The filtrate was centrifuged
for 5 min at 2600 × g. The pellets were resuspended in
30 ml of 50 mM Hepes-KOH, pH 8.0, 330 mM
sorbitol and centrifuged for 5 min at 2600 × g. Afterward, the pellet was resuspended in 10 ml of 10 mM
Hepes-KOH, pH 8.0, 5 mM MgCl2 (HM buffer) and
kept on ice for 10 min. Thylakoids were pelleted by centrifuging for 5 min at 2600 × g and washed two times in HM buffer.
Finally the thylakoids were resuspended at 1 mg of chl/ml in HM buffer
for translocation experiments or at 2 mg of chl/ml in 20 mM
Hepes, pH 8.0, for other experiments, respectively.
Immunoprecipitation--
150 µl of thylakoids (2 mg of chl/ml
in 20 mM Hepes-KOH, pH 8.0) and an equal volume of
detergent solution (4% digitonin in 20 mM Hepes-KOH, pH
8.0, 400 mM NaCl, 2 mM phenylmethylsulfonyl fluoride) were mixed and incubated on ice for 30 min. Solubilized thylakoid membrane proteins were separated from membranes by
centrifuging for 10 min at 100,000 × g. The
supernatant was incubated with 1.6 mg of anti-cpSecY IgGs cross-linked
to 10 mg of protein A-Sepharose beads (43) for 2 h at 4 °C. The
beads were transferred into Wizard minicolumns (Promega) and washed
with 1 ml of 1% digitonin in 20 mM Hepes-KOH, pH 8.0, 200 mM NaCl followed by 4 ml of 20 mM Hepes-KOH, pH
8.0, 200 mM NaCl. Excess fluid was removed by centrifugation in a microcentrifuge, and the proteins were eluted with
30 µl of 8 M urea in 2× sample buffer.
Import and Translocation Assays--
Import of cpSecE into pea
chloroplasts, treatment of intact chloroplasts with 0.1 mg/ml
thermolysin, and fractionation of chloroplasts into thylakoids and
stroma were done according to Ref. 44. Translocation of iOE33 and pOE23
was essentially done as described (45). Arabidopsis
thylakoids were prepared as described above. For inhibition of the
translocation with anti-SecY antibodies, thylakoids (45 µg chl) were
incubated with the indicated amounts of purified total IgGs (12 µg
protein/µl) for 1.5 h at 4 °C. The membranes were washed one
time in HM buffer and resuspended at 1 mg of chl/ml in HM buffer (for
pOE23) or pea stroma (for iOE33). Pea stroma was prepared as described
(44) by lysing chloroplasts containing 2 mg of chl in 1 ml of HM
buffer. 45 µl of the thylakoid suspension were incubated with 5 µl
in vitro translated iOE33 or pOE23 and incubated for 30 min
at 25 °C under illumination (100 µmol m ArabidopsisContains a Homologue of Bacterial
SecE--
SecE is an essential protein in bacteria (47, 48). Most
forms of SecE contain a single transmembrane domain at the C terminus, unlike Escherichia coli, which contains three transmembrane
domains (49). The highest sequence conservation between homologues
occurs at the cytoplasmic domain just preceding the transmembrane
domain (49, 50). Mutational analysis in E. coli has revealed
that the conserved region followed by a generic transmembrane domain is
essential for SecE function (50, 51). Recently, Bevan et al.
(52) deposited into the GenBankTM data base, 1.9 mB of
contiguous sequence from chromosome 4 of Arabidopsis. They
noted that one of the hypothetical open reading frames has similarity
to SecE preprotein translocase (GenBankTM accession number,
Z97337). Fig. 1 shows an alignment of the Arabidopsis hypothetical protein and bacterial SecE
sequences. The putative protein most resembles SecE from
Thermotoga maritimus, in which the overall similarity is
28%, and similarity is 69% between residues 111 and 173. Like other
SecE proteins, the Arabidopsis sequence predicts a protein
with a single transmembrane domain at the C terminus with type II
topology.
ArabidopsisSecE Is a Chloroplast Protein--
The
putative SecE protein is predicted to have a chloroplast transit
peptide with a processing site between residues 38 and 39 based on the
ChloroP transit peptide prediction program (53). To test this
prediction, radiolabeled putative SecE protein (Fig. 2, lane 1) was incubated with
isolated pea chloroplasts for 30 min. Nonimported protein was degraded
by protease treatment, and chloroplasts were fractionated into stroma
and thylakoids. As shown in Fig. 2, lane 3, a smaller,
protease-resistant 16-kDa protein was present in the thylakoid fraction
consistent with the size of the product predicted by ChloroP (15 kDa).
That the putative SecE clone encodes a chloroplast protein was further
established by immunoblot analysis. Antibodies that were raised against
recombinant protein expressed from the putative SecE clone
cross-reacted with a 16-kDa thylakoid protein that had the same
apparent molecular weight as the imported protein (Fig. 2, lanes
3 and 4). The protein could not be extracted from the
thylakoid membrane by incubation of the membranes with 0.1 N NaOH (Fig. 2, lanes 5 and 6),
indicating that cpSecE is an integral membrane protein, as predicted
from the sequence analysis (Fig. 1). Together, these experiments
indicate that the putative SecE protein is encoded as a precursor
containing a functional chloroplast transit peptide and the mature
protein is localized in the thylakoid membrane.
cpSecE Is Bound to cpSecY--
To test whether cpSecE forms a
complex with cpSecY, we examined whether the two proteins
co-chromatographed and co-immunoprecipitated after detergent
solubilization of thylakoid membranes. To facilitate this analysis,
polyclonal antibodies were raised against a C-terminal peptide of
Arabidopsis cpSecY. These antibodies reacted with a single
protein in wheat germ translation extracts containing cpSecY precursor
(Fig. 3) but did not cross-react with any
proteins in wheat germ extract (data not shown) and reacted with a
single 44-kDa protein found in the thylakoid membrane fraction after alkali extraction (Fig. 3), consistent with the fact that SecY is an
integral membrane protein with 10 putative transmembrane helices
(54).
Thylakoid membrane proteins were solubilized with 2% digitonin, at
approximately 70% efficiency, and the extracted proteins were
separated by gel filtration chromatography. The relative amount of
cpSecY and cpSecE in the various fractions was determined by immunoblot
analysis using antibodies against cpSecY and cpSecE, respectively. As
shown in Fig. 4, both proteins co-eluted
in a single peak as higher molecular mass species of approximately 180 kDa. These data suggest either that other subunits are present or
multiple copies of SecY and SecE are present in each complex. Furthermore, these data indicate that most, if not all, cpSecY and
cpSecE are associated.
A similar conclusion is reached by the co-immunoprecipitation
experiment. Antibodies raised against cpSecY were used to
immunoprecipitate the digitonin-solubilized complex, and cpSecY and
cpSecE in the supernatant and precipitate were detected by immunoblot
analysis. As shown in Fig. 5, cpSecY and
cpSecE were quantitatively removed from the solubilized thylakoid
proteins by the anti-cpSecY antibody and were recovered in the
immunoprecipitate, whereas none of the proteins were precipitated by an
irrelevant antiserum. cpSecY/E complex was not stable in 1%
octylglucoside/dodecylmaltoside (1:1) or 1% Triton X-100 (data not
shown). Where reconstitution of the translocase has been successful,
digitonin has also been the detergent of choice (25, 55).
cpSecY Is Sensitive to Trypsin--
It is well established that
trypsin treatment of thylakoid membranes inhibits the translocation of
proteins across the thylakoid membrane (6, 56). A likely target of
trypsin action is the Sec translocase. To examine whether trypsin
cleaves SecY, we performed immunoblot analysis on trypsin treated
thylakoids. Fig. 6 reveals that SecY is
indeed cleaved by levels of trypsin that efficiently inactivate
translocation or integration of proteins into the thylakoid membrane.
CpSecY Translocates OE33 but Not OE23 across the Thylakoid
Membrane--
To address whether cpSecY is a component of the
translocon mediating the translocation of substrates on the Sec or
The substrate for the This work clearly establishes the existence of a chloroplast
localized SecE protein that is tightly associated with cpSecY. Thus, we
can conclude that at a minimum, chloroplasts contain all the core
elements of the Sec translocase: SecY, SecA, and SecE. The core
elements of the Sec related translocase of the ER include Sec61 Several lines of evidence have indicated that there are multiple
pathways for targeting proteins to the thylakoid membrane (reviewed in
Refs. 1, 59, and 60). First, in vitro studies indicated that
substrates fall into distinct classes with regard to their ability to
act as competitors of protein targeting to the thylakoid membrane (11).
Second, each of these classes has distinct energetic requirements for
protein targeting (10, 16). Third, genetic studies largely corroborate
the in vitro studies; loss of Hcf106, SecA, or cpSRP43
resulted in selective reductions in the proteins shown to be substrates
for the To test whether convergence occurs for the cpSRP pathway, considerable
effort was made to reconstitute LHCP integration in Arabidopsis thylakoids supplemented with pea stroma.
Unfortunately, thylakoids that translocated OE33 and OE23 failed to
integrate LHCP. Arabidopsis thylakoids added to pea
thylakoids efficiently inhibited LHCP integration into the pea
thylakoids, and the inhibition could be overcome by treatment of the
Arabidopsis thylakoids with alkylating agents. Thus, it
appears that the Arabidopsis thylakoids possess an
inhibitory activity that may act on either the pea stroma or thylakoids
to prevent LHCP integration.
Plants that lack cpSRP are viable and contain elevated levels of
cpSecY,3 suggesting the
possibility that the increases observed in the mutant compensate for
the loss of targeting efficiency resulting from the absence of cpSRP.
Alternatively, the elevated level of cpSecY could indicate that cpSecY
forms an alternative pathway for the cpSRP-dependent
substrates. However, if the cpSRP delivers its substrate to cpSecY, it
must use the translocase independently of SecA, as LHCP integration is
not inhibited by azide, which inhibits SecA activity (11), LHCP
integration is not competed by SecA-dependent substrates
(11), and LHCP levels are not reduced in SecA mutants (12). These
observations suggest the possibility that the SecY/E core has activity
in the absence of SecA. Whereas loss of either SecA and SecY is lethal,
the phenotype of the SecY mutant is more severe than the SecA mutant or
even the SecA/Hcf106 double mutant (40). This observation is also
consistent with the notion that cpSecY/E has a residual activity in the
absence of SecA.
We thank Colin Robinson and Ken Cline for
providing the wheat pOE23 and iOE33 clones and Alexandra Mant for
advice concerning the translocation assays.
*
This work was supported by grants from the United States
Department of Agriculture (to N. E. H.) and Deutsche
Forschungsgemeinschaft (to D. S.). This article is Carnegie
Institution of Washington Publication No. 1410.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: Dept. of Plant Biology,
Carnegie Institution of Washington, 260 Panama St., Stanford, CA 94305. Tel.: 650-325-1521, ext. 214; Fax: 650-325-6857; E-mail: hoffman{at}andrew2.stanford.edu.
2
C.-J. Tu, D. Schuenemann, and N. E. Hoffman, manuscript in preparation.
3
P. Amin, D. Sy, M. Pilgrim, D. Parry, L. Nussaume, and N. E. Hoffman, submitted for publication.
The abbreviations used are:
SRP, signal
recognition particle;
cpSRP, chloroplast SRP;
iOE33, intermediate form
of the 33-kDa oxygen evolving protein;
pOE23, precursor of the 23-kDa
oxygen evolving protein;
chl, chlorophyll;
HM buffer, 10 mM
Hepes-KOH, pH 8.0, 5 mM MgCl2.
Chloroplast SecY Is Complexed to SecE and Involved in the
Translocation of the 33-kDa but Not the 23-kDa Subunit of the
Oxygen-evolving Complex*
, and Zentrum
Biochemie und Molekulare Zellbiologie, Gosslerstrasse 12d,
D-37073 Goettingen, Germany
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
pH, cpSec, and
cpSRP pathways.
pH pathway in chloroplasts (36). After Hcf106 was
identified as a membrane component of the pH pathway (13, 21), it
became clear that two bacterial homologues, now designated TatA and
TatE, also exist (13, 37, 38). Deletion of tatA/E
inactivates transport of proteins containing a twin arginine motif but
has no affect on Sec-dependent proteins (37). Thus, it was
shown that the pH pathway first described in chloroplasts also
exists in bacteria (13, 34, 37, 38).
pH-dependent targeting pathways at the level of SecY.
Genetic experiments with maize mutants support the idea of pathway
convergence, as SecY mutants have a more severe phenotype than
SecA/Hcf106 double mutants (40). In this report, we have characterized
a putative cpSecE homologue, and we establish that this protein indeed
is a chloroplast protein that forms a complex with cpSecY. Furthermore,
we have raised antibodies against cpSecY and have used these antibodies
to address whether SecA and pH-dependent proteins both
utilize SecY for their translocation across thylakoid membranes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
s2. Digitonin (Calbiochem, high purity) was solubilized
as a 10% stock solution in boiling ddH2O and kept at
95 °C for 15 min. After cooling the solution was spun for 10 min in
a microcentrifuge, and the supernatant was used as stock solution.
-D-thiogalactoside overnight. Cells were
harvested, frozen and lysed in Buffer B (8 M urea, 100 mM
Na2HPO4/NaH2PO4, 10 mM Tris, pH 8.0) + 1 mM phenylmethylsulfonyl fluoride. The histidine-tagged cpSecE was bound to Ni2+-NTA
agarose; the column was washed three times with Buffer B and one time
with Buffer C (8 M urea, 100 mM
Na2HPO4/NaH2PO4, 10 mM Tris, pH 6.3) and eluted with Buffer C + 250 mM imidazole. The eluted cpSecE was further purified by
SDS-polyacrylamide gel electrophoresis. The major 26-kDa band was
excised from acrylamide gels and used to raise antibodies in chicken
(Cocalico Biologicals, Inc., Reamstown, PA).
2
s2). Assays for iOE33 translocation additionally
contained 4 mM ATP. After incubation samples were digested
with 0.2 mg/ml thermolysin for 1 h on ice. Thylakoids were washed
with 1 ml of HM buffer and resuspended in 25 µl of 4× SDS sample
buffer. For documentation, gels were developed by fluorography using
either autofluor (National Diagnostics, Manville, NJ) or 20%
2,5-diphenyloxazole in acetic acid (46). For quantification gels were
scanned by a PhosphorImager and quantitated using Imagequant software
from Molecular Dynamics.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Alignment of A. thaliana
cpSecE with bacterial SecE sequences. Sequences of SecE
homologues from Thermotoga maritimus, Bacillus subtilis,
Synechocystis sp. strain PCC 6803, and Staphylococcus
aureus were aligned to A. thaliana cpSecE sequence
using Clustal W 1.7 and shaded with Boxshade. Residues conserved in all
sequences are shaded in black, and residues conserved in
four or five sequences are shaded in gray. Predicted
transmembrane domains are underlined. The putative cleavage
site for the stromal processing peptidase is indicated by an
arrow.
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Fig. 2.
CpSecE is an integral membrane protein of the
thylakoid membrane. A, in vitro translated
[35S]methionine-labeled cpSecE precursor
(pcpsecE) (lane 1) was imported into isolated pea
chloroplasts. After the import reaction, chloroplasts were
thermolysin-treated, repurified by centrifugation over a Percoll
cushion, and separated into the stromal compartment (str.)
(lane 2) and thylakoid membranes (thyl.)
(lane 3). B, Arabidopsis thylakoid
membrane proteins (equivalent to 20 µg of chl) were separated by
SDS-polyacrylamide gel electrophoresis (15% acrylamide) and subjected
to immunoblot analysis with anti-cpSecE antibodies (lanes
4-6) and preimmune serum (PI-serum) (lanes
7-9). Integral membrane proteins (P) (lanes
5 and 8) were separated from peripheral membrane
proteins (S) (lanes 6 and 9) by
incubating thylakoid membranes with 0.1 N NaOH for 15 min
at 4 °C followed by a 5 min centrifugation in a
microcentrifuge.
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Fig. 3.
Antibodies against cpSecY recognize a 44-kDa
integral membrane protein of the thylakoid membrane. In
vitro translated cpSecY precursor (pcpSecY) or
Arabidopsis thylakoid membrane proteins (equivalent to 2 µg of chl. were separated by SDS-polyacrylamide gel electrophoresis
(12% acrylamide) and subjected to immunoblot analysis with anti-cpSecY
antibodies. Integral membrane proteins (P) were separated
from peripheral membrane proteins (S) by incubating
thylakoid membranes (thyl.) with 0.1 N NaOH for
15 min at 4 °C, followed by a 5-min centrifugation in a
microcentrifuge.
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Fig. 4.
CpSecY and cpSecE cofractionate during gel
filtration analysis. 100 µl of solubilized thylakoid membrane
proteins (equivalent to 200 µg of chlorophyll; see under
"Experimental Procedures" for details) were fractionated on a
Superose 6HR column (Amersham Pharmacia Biotech) in 20 mM
Hepes-KOH, pH 8.0, 0.1% digitonin, 200 mM NaCl at a flow
rate of 0.5 ml/min. The column was calibrated using the following
proteins as standards: ferritin dimer (1, 880 kDa), bovine
thyroglobulin (2, 670 kDa), ferritin monomer (3, 440 kDa), sweet potato amylase (4, 200 kDa), bovine serum
albumin (5, 66 kDa), and cytochrome c
(6, 14 kDa). The fractions were precipitated with 10%
trichloroacetic acid, and cpSecY (closed circles) and cpSecE
(open triangles) were detected by immunoblot analysis. Films
were scanned and quantitated using Imagequant software from Molecular
Dynamics. Vo, void volume.
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Fig. 5.
Coimmunoprecipitation of cpSecY and
cpSecE. Thylakoid membrane proteins were solubilized with 2%
digitonin, and an immunoprecipitation was performed using anti-cpSecY
antibodies or antibodies against an irrelevant protein as a control
(see under "Experimental Procedures" for details). The solubilized
thylakoid proteins (L), the flow through (FT),
and the immunoprecipitate (IP) were subjected to immunoblot
analysis with anti-cpSecY and anti-cpSecE antibodies.
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Fig. 6.
CpSecY is sensitive to trypsin
digestion. Thylakoid membranes (2.5 µg of chlorophyll) were
digested with the indicated amounts of trypsin for 30 min on ice in 50 mM Hepes-KOH, pH 8.0, 330 mM sorbitol. After
stopping the reaction by the addition of 2 mM
phenylmethylsulfonyl fluoride, the membranes were washed in 50 mM Hepes-KOH, pH 8.0, 330 mM sorbitol, 25 mM EDTA. The membrane proteins were solubilized in 4×
sample buffer for 30 min at 37 °C and analyzed by immunoblot using
antibodies against cpSecY.
pH pathways, we took advantage of the fact that the anti-cpSecY
antibody recognized the native cpSecY (Fig. 5). Assuming that the C
terminus of SecY faces the stroma, these antibodies should bind and
conceivably inhibit SecY activity. Antibodies against cpSecY and cpSecE
failed to recognize the corresponding proteins in pea and spinach,
necessitating the use of Arabidopsis thylakoids in the
assays. To improve yields, Arabidopsis thylakoids were
isolated directly from leaf tissue and not from intact chloroplasts.
Pea stroma was used as the source of SecA. The substrate for the sec
pathway, wheat iOE33, was efficiently translocated into the lumen of
Arabidopsis thylakoids and processed to the mature size
(Fig. 7A, lanes 1 and
2). Little to no translocation occurred in the absence of
stroma, as shown in lane 3. When thylakoids were
preincubated with increasing amounts of anti-cpSecY antibodies, translocation was progressively inhibited (Fig. 7A, lanes
4-7). However, the inhibition could be relieved by adding an
excess of cpSecY peptide antigen during the antibody pretreatment (Fig. 7A, lane 8). Furthermore, antibodies against an irrelevant
protein, cpSRP54, were not inhibitory (Fig. 7A, lane 9).
Thus, the antibody effect was specific for cpSecY. These data provide
the first direct demonstration that the Sec pathway substrate, iOE33,
utilizes cpSecY for translocation. These data also suggest that cpSecY has a similar topology as the bacterial homologue, where the N and C
termini are in the stroma (or the cis side of the membrane).
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Fig. 7.
Anti-cpSecY antibodies inhibit the
translocation of iOE33 but not the translocation of pOE23.
Arabidopsis thylakoids were incubated with iOE33 (lane
1) (A) or pOE23 (lane 1) (B) in
the absence (lane 2) or presence of 0.05, 0.1, 0.3, and 1 µl of anti-cpSecY antibodies (12 µg of IgGs/µl) (lanes
4-7, respectively) as detailed under "Experimental
Procedures." Control assays (lane 3) were performed in the
absence of stroma for iOE33 or the presence of 15 µM CCCP
for pOE23. Controls shown in lane 8 and 9 were
done in presence of 1 µl of anti-cpSecY antibodies (12 µg of IgGs)
and 100 ng of cpSecY-peptide (lane 8) or anti-cpSRP54
antibodies (12 µg of IgGs) as an irrelevant antiserum (lane
9). TP, translation product; mOE33 and
mOE23, mature OE33 and OE23.
pH pathway, wheat pOE23, was also efficiently
translocated into the thylakoid lumen and was processed to the mature
form (Fig. 7B, lanes 1 and 2). The translocation of pOE23 was dependent on the pH, as demonstrated by the complete inhibition resulting from CCCP addition (Fig. 7B, lane 3).
In contrast to the results seen with the Sec substrate, preincubation of the thylakoids with increasing amounts of anti-cpSecY antibodies had
no affect on pOE23 translocation (Fig. 7B, lanes 4-7). This result demonstrates that pOE23 and probably other substrates of the
pH pathway are translocated via a translocon lacking cpSecY. Thus,
the pH and Sec pathways are parallel and do not converge at cpSecY
in the thylakoid membrane.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(a
homologue of SecY), Sec61
(a homologue of SecE), and Sec61 (49).
Three to four of these heterotrimers form a pore-like structure in the
ER (57, 58) that remains stable after solubilization with digitonin.
Solubilization of the thylakoid membrane using the same detergent
releases a 180-kDa complex that contains SecY and SecE. This complex
may consist of multiple copies of SecY/E dimers forming a ring-like
structure, like those seen after purification of the ER-complex, and
may also include additional subunits, e.g. a SecG homologue.
An important goal for future work will be to establish the subunit
composition and stoichiometry of the proteins in the complex.
pH, Sec, and cpSRP pathways, respectively (12, 20, 61, 62).
However, these studies did not exclude the possibility that SecY was
common to all pathways. It has been observed that the targeting
information specifying the pH versus the Sec pathway is
present in the transit peptide (36, 63-66), and when a Sec transit
peptide is used to direct a pH protein to the Sec translocase, the
protein fails to be translocated across the thylakoid membrane (63,
65). Based on these findings, it has been postulated that substrates using the pH pathway are unable to translocate through the Sec system, and hence a distinct translocase may be employed by the pH
pathway (65). The results from this paper clearly establish the
validity of this hypothesis, as convergence at the level of the Sec
translocase does not occur for the pH pathway.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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