From the
Proteins are translocated across the thylakoid membrane by two
distinct pathways in higher plant chloroplasts, one of which is related
to prokaryotic Sec-dependent translocation mechanisms. SecY is an
essential, hydrophobic component of the membrane-bound translocase
complex in bacteria, and we report here the nucleotide sequence of a
full-length cDNA encoding a homolog of SecY from Arabidopsis
thaliana. The predicted protein of 551 residues includes an
amino-terminal extension of approximately 120 residues when compared
with other SecY proteins. The deduced sequence of the mature protein,
cpSecY, is 41% identical with SecY from Synechococcus and 33%
identical with the Escherichia coli protein. The extension
serves to target the protein into chloroplasts;
transcription-translation of the cDNA yields a 58-kDa precursor protein
which is imported into pea chloroplasts, processed to a product of 46
kDa, and targeted into the thylakoid membrane. Genetic and biochemical studies have identified several
proteins, encoded by sec genes, that are required for the
translocation of proteins across the plasma membrane in bacteria
(reviewed in (1) ). These proteins include soluble/peripheral
components (SecB and SecA) and a membrane-bound translocase complex
that includes SecY and
SecE(2, 3, 4, 5) . Other integral
membrane proteins, SecD and SecF, appear to participate in the later
stages of the translocation process(6) . SecY is a particularly
interesting protein that spans the membrane 10 times (7) and is
homologous to the SEC61 Several lines of evidence have
indicated that a Sec-type mechanism also operates for the transport of
proteins across the thylakoid membrane. Nuclear-encoded thylakoid lumen
proteins are synthesized with bipartite presequences containing
``envelope transit'' and ``thylakoid transfer''
signals in tandem; the transfer signals are similar in several
structural respects to bacterial export, or ``signal''
peptides(9, 10, 11, 12) . SecY
homologs have also been found in the plastid genomes of red algae and
cyanophytes(13, 14) , and recent studies have shown
the involvement of SecA in the transport of proteins across the
chloroplast thylakoid membrane(15, 16) . A major
surprise, however, has been the demonstration of a second,
Clone 107N18T7 was obtained from the Arabidopsis biological resource center in Columbus, OH, after partial sequence
analysis of random clones in the MSU Arabidopsis thaliana Expressed Sequence Tag (EST) programme indicated that this clone
contained regions homologous to known secY genes in the data
base; approximately 420 bp Protocols for transcription-translation and chloroplast import have
been described elsewhere(9) . Preparation of enriched envelope
and thylakoid membrane fractions was carried out using a method
modified from that of Flgge et
al.(18) . Chloroplasts from an import experiment were
lysed on ice for 5 min in 10 mM Hepes-KOH, 5 mM
MgCl
Figure 3:
Import of pre-cpSecY into pea chloroplasts
and localization of the processed product. A, pre-cpSecY was
synthesized in vitro by transcription-translation of psecY17
as detailed under ``Experimental Procedures,'' and the
radiolabeled translation product (lane T) was incubated with
intact pea chloroplasts. After incubation, samples of the chloroplasts
were analyzed directly (lane C) or after protease treatment of
the organelles (lane Cp). B, pre-cpSecY was imported
into chloroplasts as in A, after which the organelles were
fractionated as detailed under ``Experimental Procedures'' to
generate samples of envelope membranes (E), stromal phase (S), thylakoid membranes (T), and protease-treated
thylakoids (Tp). Samples were immunoblotted using a mixture of
antisera against spinach phosphate translocator (PT) and the
extrinsic 23-kDa photosystem II protein (23K). A second gel
was fluorographed, and mature-size cpSecY was quantitated by laser
densitometry; the graph shows the percentage of imported
cpSecY present in each fraction.
Figure 1:
Nucleotide and deduced amino acid
sequence of a cDNA clone, psecY17, encoding a SecY homolog from Arabidopsis thaliana.
Figure 2:
Alignment of the A. thaliana SecY
protein (A.thal) with SecY sequences from Synechococcus (Synech) and E. coli. Identical residues are
indicated by asterisks, and conserved residues by dots. A putative cleavage site for the stromal processing
peptidase is indicated by an arrowhead. Transmembrane segments
in the E. coli protein are underlined and numbered in parentheses.
In overall structural terms,
the Arabidopsis protein is also similar to other SecY
proteins. Topology analysis of E. coli SecY strongly suggests
that the protein contains 10 transmembrane segments(7) , and
the hydropathy profile of the protein encoded by psecY17 is very
similar to those of other SecY proteins (not shown). The general
organization of the protein thus appears to have been highly conserved
in various genera of bacteria, in the plastid genomes of eukaryotic
algae, and now also in higher plants. It remains to be determined
whether Sec-dependent protein translocation is similar in mechanistic
terms in these disparate organisms.
Most integral
thylakoid membrane proteins are synthesized with stroma-targeting
presequences, with information in the mature proteins specifying
localization in the thylakoid (rather than the envelope) membrane. The
cpSecY presequence appears to fulfill a similar function, because it
does not resemble the bipartite presequences which usually target
lumenal proteins across the thylakoid membrane. The latter peptides
contain a hydrophobic region followed by an Ala-X-Ala (or very
similar) motif specifying processing by the thylakoidal processing
peptidase, and there is no evidence for this type of peptide. The
presequence is, however, relatively long for a stroma-targeting signal
if the mature cpSecY protein is of a similar size to other SecY
proteins. The start of the mature protein cannot be identified
empirically because there is no strict consensus cleavage site for the
stromal processing peptidase. Nevertheless, many stroma-targeting
peptides end with a loosely conserved motif
(Val/Ile-X-Ala/Cys Given that cpSecY is almost
certainly synthesized with a stroma-targeting presequence, the
implication is that information in the mature protein specifies
integration into the thylakoid membrane. One or more of the 10
transmembrane segments may therefore serve as a thylakoid integration
signal, and it will be interesting to determine mechanistic details of
the integration process. cpSecY is probably the most hydrophobic
protein known to be imported into chloroplasts, and it will be equally
interesting to determine how this protein is prevented from aggregating
during passage through the stromal phase. Most importantly, however,
the sequence data reported here will enable more detailed studies to be
carried out on the membrane-bound proteins of the Sec apparatus. Due to
the extreme hydrophobicity of SecY, biochemical studies on its role
have relied on the use of antibodies to block the action of the
protein(23) , and it has only proved possible to raise
antibodies to peptide fragments corresponding to hydrophilic, exposed
areas of the SecY protein. Antibodies raised against E. coli SecY do not cross-react with cpSecY from pea or spinach,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
protein involved in protein transport
across the endoplasmic reticulum (8) . This observation
suggests that these translocation mechanisms are fundamentally similar
and derived from a common ancestor.
pH-driven thylakoidal protein translocase which has very different
features (reviewed in (17) ). To date, no sec genes
have been cloned from higher plants, and it is unclear whether the
Sec-dependent translocation system of thylakoids is similar in
structural terms to those of prokaryotes. In this report we describe
the sequence of an Arabidopsis thaliana cDNA encoding a SecY
homolog, cpSecY, which is synthesized with a presequence and imported
into the thylakoid membrane of pea chloroplasts.
(
)was sequenced in
the laboratory of T. Newman (Michigan). The insert was excised using SalI and NotI, end-labeled, and used to screen an Arabidopsis cDNA library (
ZipLox
(Life
Technologies Inc.) that was also obtained from the Ohio stock center.
One of the clones isolated, psecY17, contained a 1.8-kb insert; in
vitro transcription-translation of this clone generated a 58-kDa
protein which was found to import into chloroplasts and is thus
full-length (see text). Both strands of psecY17 were fully sequenced.
, pH 8.0, to a concentration of 0.5 mg/ml chlorophyll.
The lysate was centrifuged for 30 s at 3700
g to
pellet thylakoid membranes, and the supernatant was recentrifuged twice
at 3700
g to recover remaining thylakoids; these
pellets were pooled and washed once in the same buffer. The final
pellet corresponds to the ``thylakoid'' fraction described in Fig. 3B. A second sample of thylakoid fraction was
incubated with thermolysin (0.2 mg/ml) for 30 min on ice (=
protease-treated thylakoid fraction). The supernatants from the three
30-s centrifugations were centrifuged at 100,000
g for
15 min to recover envelope membranes; the supernatant (= stromal
fraction) was removed and the envelope membranes were washed once to
generate the ``envelope'' fraction. Samples of each fraction
were analyzed by fluorography to visualize the imported cpSecY
polypeptides, and by immunoblotting using an Amersham International plc
kit for enhanced chemiluminescence.
Characterization of an Arabidopsis thaliana cDNA
Encoding a SecY Homolog
The MSU Arabidopsis EST
programme carries out the partial sequencing of random cDNA clones in
order to identify, by comparison with DNA and protein data bases,
putative clones encoding proteins of interest. One of the clones was
reported to be homologous to secY genes, and we have
characterized the clone and related gene product to determine the full
sequence and location of the encoded protein. The 1.1-kilobase pair
insert of clone 107N18T7 was used to screen an Arabidopsis cDNA library, and one of the positives, psecY17, contained a
1.8-kilobase pair insert which was fully sequenced. The cDNA sequence
predicts a protein of 551 residues (Fig. 1). Comparison with the secY genes from Synechococcus(19) and Escherichia coli(20) shows that psecY17 is highly
homologous to these genes but contains approximately 120 additional
residues at the amino terminus (Fig. 2); this putative
presequence is similar in terms of amino acid composition to the
presequences of numerous stromal proteins, suggesting a
plastid-targeting function. As would be expected for a chloroplast
protein, given the strong evidence for the evolution of chloroplasts
from cyanobacteria, the Arabidopsis SecY protein is more
homologous to Synechococcus SecY than to the E. coli protein (41% and 33% identity, respectively, at the amino acid
level if the amino-terminal extension is ignored). Areas of homology
are found over most of the ``mature'' protein sequence but
are particularly pronounced in the second transmembrane segment and the
hydrophilic sections on either side.
The SecY Homolog Is Targeted into the Chloroplast and
Inserted into the Thylakoid Membrane
Although the sequence of
the Arabidopsis secY cDNA is consistent with the presence of a
presequence in the initial translation product, it was deemed important
to determine experimentally whether this is the case, and whether the
protein is targeted into chloroplasts. There is no consensus sequence
for chloroplast-targeting signals, and mitochondrial targeting signals
are not dissimilar in terms of amino acid composition(21) .
This point was addressed by preparing an in vitro transcription-translation product of psecY17 and incubating the
labeled protein with intact pea chloroplasts (Fig. 3A).
The data show that the SecY homolog is synthesized as a 58-kDa
precursor protein, efficiently imported into the organelles and
processed to a mature size of 46 kDa which is protected from
proteolysis (confirming import). These data demonstrate that psecY17
does indeed encode a chloroplast SecY homolog, which we have termed
cpSecY. Fractionation studies were carried out to determine the
location of the imported protein, and the results are shown in Fig. 3B. Following import, the chloroplasts were lysed
and separated into a stromal fraction, and fractions enriched in
envelope and thylakoid membranes. The purity of the membrane fractions
was assessed using antisera raised against the 30-kDa phosphate
translocator of the inner envelope membrane (18) and the 23-kDa
protein of the oxygen-evolving complex (a thylakoid lumen protein). The
immunoblot in Fig. 3B shows that the envelope fraction
does in fact contain the vast majority of envelope membranes, since
virtually all of the phosphate translocator is found in this fraction.
The envelope fraction also contains a significant proportion of the
23-kDa protein, indicating contamination with thylakoid membranes. The
thylakoid fraction (T) is less contaminated, containing most
of the 23-kDa protein and very little phosphate translocator. The
lumenal 23-kDa protein in the thylakoid fraction is resistant to
proteolysis (lane Tp), as expected of a thylakoid lumen
protein. Quantitation of the imported cpSecY in these fractions (graph in Fig. 3B) shows that most of the
protein (44%) is found in the thylakoid membrane, where it is sensitive
to proteolysis. 33% of the cpSecY is found in the envelope fraction,
but the immunoblot data indicate that most (if not all) of this protein
is present in contaminating thylakoid membranes: if the cpSecY were
targeted into the envelope, the vast majority of protein would be
localized in the envelope fraction, as is the case with the phosphate
translocator. The remainder of the imported cpSecY is found in the
stromal fraction. We conclude from this experiment that cpSecY is
targeted to the thylakoid membrane, although we cannot formally rule
out the possibility that some protein resides in the envelope
membranes. The stromal fraction presumably contains cpSecY molecules
which are en route to the thylakoid membrane.Ala)(22) , and a motif of this
description can be found in the pre-cpSecY sequence as indicated in Fig. 2. The cleavage point would then occur after 121 residues
of presequence, leaving a mature protein of 430 residues. As with most
stroma-targeting presequences(21) , the presequence is highly
enriched in hydroxylated residues (40 out of 121 if cleavage is where
indicated), and overall positively charged, although it also contains
several acidic residues (probably 5 or 6) which are uncommon in
chloroplast protein presequences.
(
)but it should now be possible to generate antibodies
to the Arabidopsis protein and probe the role of cpSecY in
some detail.
We gratefully acknowledge the help of Dr. Malcolm
Bennett in screening cDNA libraries, and we thank Dr. Susanne Brink
for help in preparing envelope membranes.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.