(Received for publication, March 6, 1995; and in revised form, May 22, 1995)
From the
Thylakoid membranes of chloroplasts in higher plants harbor
different pathways for the translocation of proteins. One of these
routes is related to the prokaryotic Sec pathway, which mediates the
secretion of particular proteins into the periplasmic space and
involves the SecA protein as an essential component. We have isolated a
full size cDNA of 3739 nucleotides encoding the SecA homologue from
spinach. It contains an open reading frame of 1036 codons corresponding
to a polypeptide with a calculated mass of 117 kDa. The deduced amino
acid sequence shows between 43 and 49% identity to SecA proteins from
bacteria and lower algae and 62% identity to SecA of the cyanobacterium Synechococcus sp. PCC7942. Compared with the Escherichia
coli protein, spinach SecA carries an amino-terminal extension of
approximately 80 residues. In organello experiments performed
with the protein made in vitro by transcription of the cDNA
and cell-free translation of the resulting RNA showed that this
extension comprises a transit peptide that mediates the import of the
protein into the chloroplast. The processed product of approximately
107 kDa accumulates predominantly in the stroma and to a lower extent
associates with the thylakoid membrane. Comparably to E. coli, in which SecA activity can be inhibited by sodium azide, thylakoid
translocation of a subset of lumenal proteins is sensitive to sodium
azide in pea but not in spinach chloroplasts, suggesting that the
latter contain an azide-resistant SecA variant.
Nuclear-encoded proteins that are destined to the thylakoid
membranes, the inner membrane system of chloroplasts, are synthesized
in the cytosol as precursors with NH In spite of this similarity, the thylakoid translocation
of proteins carrying bipartite transit peptides does not proceed with a
single, common mechanism. Instead, three different pathways have been
characterized as yet that represent independent routes across or into
the thylakoid membrane, each specific for a subset of thylakoid
proteins (Cline et al.(1993); Hulford et al. (1994);
Michl et al.(1994); Robinson et al. (1994); for a
recent review see Robinson and Klsgen(1994)). One
of them is represented by the integration pathway for CFo-II, a
nuclear-encoded integral subunit of the plastid ATP synthase that
appears to insert spontaneously into the thylakoid membrane (Michl et al., 1994). A second pathway, which is independent of
stromal factors, depends strictly and apparently solely on the proton
gradient across this membrane, a unique feature among membrane
translocation pathways known to date. It appears not to be present in
prokaryotes, suggesting that it was developed at the eukaryotic level.
This idea is supported by the fact that none of the proteins
transported by this mechanism, i.e. the 16- and 23-kDa
subunits of the oxygen-evolving complex, subunit N of photosystem I,
and subunit T of photosystem II (Mould and Robinson, 1991; Mould et
al., 1991; Cline et al., 1992;
Klsgen et al., 1992; Henry et
al., 1994; Nielsen et al., 1994), have been found in
cyanobacteria. In contrast, a third translocation route for proteins
with bipartite transit peptides appears to be specific for proteins
that are present also in cyanobacteria, notably plastocyanin, the
33-kDa subunit of the oxygen-evolving complex and subunit F (or 3) of
photosystem I. This route possesses characteristics that are typical
for the secretion of proteins into the periplasmic space of Escherichia coli, i.e. it is strictly dependent on
nucleoside triphosphates as well as on soluble factor(s) in the matrix
but does not require a transmembrane potential, although this potential
can have a stimulatory effect in some instances (Theg et al.,
1989; Cline et al., 1992; Hulford et al., 1994;
Robinson et al., 1994; Karnauchov et al., 1994; Mant et al., 1994; Henry et al., 1994). Moreover,
experiments performed with isolated pea chloroplasts have shown that
this pathway involves a compound that is sensitive to sodium azide
(Henry et al., 1994; Karnauchov et al., 1994; Knott
and Robinson, 1994), in analogy to the azide sensitivity described for
the protein secretory pathway in E. coli (Oliver et
al., 1990; Pugsley, 1993). Although sodium azide can affect
ATP-binding proteins in general, in E. coli the toxic effects
are largely due to inhibition of the SecA protein, an essential
component of this pathway. Likewise, the plastid homologue to the
prokaryotic SecA protein, which has recently been characterized from
pea (Nakai et al., 1994b; Yuan et al. 1994), was
shown to be specifically involved in the thylakoid translocation of
plastocyanin and the 33-kDa subunit of the oxygen-evolving complex, two
proteins that are known to depend on an azide-sensitive component for
their thylakoid transfer. In addition, the thylakoid-targeting domains
of these two transit peptides can functionally replace bacterial signal
peptides (Seidler and Michel, 1990; Haehnel et al., 1994), and
protein secretion in bacteria remains sensitive to sodium azide under
these conditions. ( In order to further characterize this
translocation pathway, we have initiated experiments to isolate
constituents of the thylakoidal translocation apparatus in higher
plants. Here we describe the isolation and characterization of a full
size cDNA encoding the SecA homologue from spinach chloroplasts.
Enzymes were purchased from Boehringer Mannheim
(restriction endonucleases, S1-nuclease, and RNase inhibitor);
Eurogentec (Seraing, Belgium) (GoldStar DNA polymerase); Stratagene (La
Jolla, CA) (T3 RNA polymerase); New England Biolabs, Inc. (Bad
Schwalbach, Germany) (T7 RNA polymerase and T4 DNA ligase); Life
Technologies, Inc. (Eggenstein, Germany) (exonuclease III), and U. S.
Biochemical Corp. (Cleveland, OH) (Sequenase 2.0). Rabbit reticulocyte
lysates and [
Figure 1:
Effect of
translocation inhibitors on chloroplasts of different species. In
vitro translated precursors (lanes t) of plastocyanin (A) or the 16-kDa protein of the oxygen-evolving system (B) from spinach were incubated with intact chloroplasts
isolated from spinach (upper panels) and pea (lower
panels) in the presence or the absence of either 10 mM sodium azide (A) or 2 µM nigericin (B). After the import reaction, the organelles were
fractionated into stroma (lanes s) and thylakoids, which were
subsequently treated with either thermolysin (lanes +) or
mock treated (lanes -). Stoichiometric amounts of each
chloroplast fraction, corresponding to 25 µg of chlorophyll, were
loaded onto 10-17.5% SDS-polyacrylamide gradient gels (Laemmli,
1970) and visualized by fluorography. The positions of precursors (p), intermediates (i), and mature proteins (m) are indicated.
This observation can be interpreted in
two ways. The compound that exhibits azide sensitivity during protein
translocation in pea, presumably the SecA protein, assumes a structure
in spinach chloroplasts that makes it azide-insensitive. Alternatively,
the pathway including this compound may be lacking in spinach, which
would suggest that proteins that are usually transported along this
route utilize a different pathway, i.e. the
Figure 2:
Two independent thylakoid translocation
pathways for hydrophilic lumen proteins exist also in spinach
chloroplasts. In organello experiments were performed with
radiolabeled precursors of plastocyanin (upper panel) and the
23-kDa protein of the oxygen-evolving system (lower panel) in
the presence of increasing amounts of the 23-kDa precursor protein
obtained by overexpression in E. coli. In each lane, lysates
of total chloroplasts corresponding to 25 µg of chlorophyll were
loaded. The amount of competitor protein (in µM) present
in each assay is indicated above the lanes. For further
details see the legend to Fig. 1.
The insert of
Figure 3:
Nucleotide sequence and deduced amino acid
sequence of the cDNA encoding SecA from spinach. The deduced start and
stop codons and a possible polyadenylation signal are overlined.
Figure 4:
Alignment of the amino acid sequence of
the E. coli SecA protein (ec) with that deduced from
the spinach cDNA (so). Identical residues are marked by a vertical line and conserved residues by a colon. Stars indicate the two residues that are known to contribute
to azide sensitivity of the E. coli protein. The overlined
residues correspond to the positions of the degenerated
oligonucleotides that were used for cloning of the cDNA from
spinach.
In order to
determine the target organelle of the protein, SecA protein that was
generated in vitro by transcription of the cDNA and
translation of the resulting RNA was studied in in organello experiments with isolated spinach chloroplasts. After incubation
for 30 min in the light and subsequent proteolytic removal of proteins
attached to the organelle envelopes, the SecA protein could be
co-purified with intact chloroplasts that were reisolated from the
assays (Fig. 5). Generally approximately 10% of the SecA
precursor is imported under these conditions, a rate that is comparable
with that of other chloroplast proteins in such assays. The apparent
molecular mass of the internalized SecA protein is approximately 10 kDa
smaller than that of the in vitro translation product, which
correlates well with the expected size difference after removal of the
putative transit peptide. Within the chloroplast, the protein
accumulates predominantly in the stroma, although a small fraction is
often found associated with the thylakoid membrane ( Fig. 5and
data not shown), which indicates that the SecA protein may reversibly
bind to this membrane.
Figure 5:
In organello import of spinach
SecA into isolated chloroplasts. SecA precursor was synthesized in
vitro and analyzed as detailed in the legend to Fig. 1.
In organello experiments that were
performed in the presence of saturating amounts of precursors for the
23- or the 33-kDa protein of the oxygen-evolving complex showed that
both precursors compete with SecA for import into the organelle (data
not shown). This implies that all three proteins utilize at least one
factor in common during the transport process, which is a further
indication for a general translocase for most, if not all,
nuclear-encoded chloroplast proteins. The major goal of this study was to isolate and characterize
the cDNA for SecA of spinach chloroplasts. The cDNA selected is the
first example of a constituent of one of the protein transport
machineries operating at or in the thylakoid membranes of higher
plants. It encodes a precursor protein that is efficiently imported
into isolated intact chloroplasts. After import, SecA shows a
distribution within the organelle that is identical to that determined
for the authentic SecA of pea chloroplasts using Western analyses
(Nakai et al., 1994b; Yuan et al., 1994), i.e. it accumulates predominantly in the stroma, whereas a minor
fraction is associated with the thylakoid membrane. This is in
agreement with both the expected function of SecA and with findings in E. coli in which the homologous protein was shown to
reversibly interact with the inner membrane to facilitate the secretion
of proteins into the periplasmic space (Economou and Wickner, 1994; Kim et al., 1994). This analogy provides additional support for
the prokaryotic origin of the Sec-dependent translocation pathway in
chloroplasts. Interestingly, a comparable pathway has not been
described for mitochondria as yet, although these organelles are of
endosymbiotic origin as well. Our experiments, both Southern analysis
of genomic DNA and sequencing of the isolated cDNAs that are identical
even in their untranslated regions, gave no hint for the existence of
an additional nuclear gene that would encode a mitochondrial SecA
homologue (data not shown). Sec-dependent protein translocation is
generally sensitive to sodium azide, and azide resistance in E.
coli was shown to be correlated to mutations in the SecA protein
(Oliver et al., 1990). The lack of an inhibitory effect of
sodium azide on protein translocation across the thylakoid membrane of
spinach suggests, therefore, that these chloroplasts harbor an
azide-tolerant variant of the SecA protein. The structural basis for
this tolerance remains unclear. Comparison of the amino acid sequences
showed that the two residues known to confer azide sensitivity to SecA
in E. coli are conserved in the spinach protein (Fig. 4). Pairwise comparison of the SecA sequences known to
date shows a moderate degree of homology (Fig. 6). With only few
exceptions, identity between 40 and 50% is found, even for distant
species. Interestingly, in most cases the homology is also not
significantly higher for relatively closely related species such as E. coli and B. subtilis, which are 53% identical in
their amino acid sequences. The spinach protein is only 43-48%
identical to its plastid-encoded homologues from the chlorophyll a/c- and a/d-lineages of organisms (e.g. chromophyceae or rhodophyceae) but is 62% identical to
that of cyanobacteria, the presumed progenitors of chlorophycean
chloroplasts, which indicates that the gene for SecA in higher plants
was probably derived from the endosymbiotic progenitor of chloroplasts.
Figure 6:
Homology between SecA proteins of
different species. Identical residues between the amino acid sequences
of SecA proteins isolated from E. coli (ec; Schmidt et al.(1988)), Bacillus subtilis (bs; Sadaie et al.(1991)), Listeria monocytogenes (lm;
M. U. Owens, M. N. Berkaw, and M. G. Schmidt, EMBL data base accession
number L32090), Staphylococcus carnosus (sc; R.
Freudl, EMBL data base accession number X79725), Caulobacter
crescentus (cc; P. Kang and L. Shapiro, EMBL data base
accession number P38380), Antithamnion sp. (as;
Valentin(1993)), Olistodiscus luteus (ol; K. U.
Valentin, S. Fischer, and H. Vogel, EMBL data base accession number
Z35718), Pavlova lutherii (pl; Scaramuzzi et
al.(1992)), Synechococcus sp. PCC7942 (ss; Nakai et al. (1994a)), and S. oleracea (so; this
paper) were determined by pairwise alignment as exemplified in Fig. 4. The degrees of identity are given in
percentages.
Alignment of amino acid sequences shows that a basic level of
homology is found almost along the entire length of the polypeptide
chain and that the homology among SecA proteins is not restricted to
one or a few regions of the protein ( Fig. 4and data not shown).
Exceptions are the very COOH terminus that differs both in length and
sequence and, remarkably, insertions of approximately 85-135
residues in size, which are found in all SecA proteins from
photosynthetic organelles and organisms at a position corresponding to
residue 520 in the E. coli sequence ( Fig. 4and data
not shown). The sequences of these insertions do not exhibit
significant homology to each other. This suggests that they are either
of independent origin and/or of different function or that these
segments contribute to the specificity of targeting processes.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank®/EMBL Data Bank with accession number(s)
Z49124[GenBank].
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-terminal transit
peptides. According to their targeting properties, these transit
peptides are grouped into two major classes. One class consists of
envelope transfer signals that mediate the translocation of proteins
only into the chloroplast stroma, where they are completely removed by
a stromal endopeptidase. Presequences of this type are common to
stromal and many, though not all, integral thylakoid proteins such as
LHCPII, the apoprotein of the light-harvesting complex associated with
photosystem II, or the 20-kDa apoprotein of the CP24 complex (Lamppa,
1988; Viitanen et al., 1988; Cai et al., 1993). In
the latter case, they are not required for intraorganellar targeting of
the proteins into or across the thylakoid membrane, which in these
instances is mediated by hydrophobic signals in the respective mature
proteins. In contrast, the hydrophilic, lumenal proteins, such as
plastocyanin or the extrinsic subunits of the oxygen-evolving system,
depend on hydrophobic signals present in their transit peptides for
translocation across the thylakoid membrane (Smeekens et al.,
1986; James et al., 1989; Ko and Cashmore, 1989; Hageman et al., 1990; Clausmeyer et al., 1993). These transit
peptides are bipartite, carrying two targeting signals in tandem, an
NH
-terminal envelope transfer domain, which is generally
removed by a stromal endopeptidase, followed by the thylakoid-targeting
and -translocating domain (von Heijne et al., 1989). According
to structure predictions, this thylakoid transfer signal shows in all
bipartite transit peptides analyzed to date remarkable similarity to
bacterial signal peptides, which are responsible for the transport of
proteins into the periplasmic space across the bacterial inner
membrane.
)Collectively, these results suggest
strongly that the soluble factors involved in protein transport across
the inner bacterial membrane and the thylakoid membrane are closely
related and that this thylakoid translocation route therefore
originates in prokaryotes.
Materials
Spinach (Spinacia oleracea cv. Monatol) was grown under constant temperature (18-22
°C) and light regime (cycles of 10 h of light and 14 h of dark) and
harvested 3 weeks after sowing. Pea seedlings (Pisum sativum var. Feltham First) were grown for 8-9 days under 12 h
photoperiods.S]methionine were obtained from
Amersham (Braunschweig, Germany), and wheat germ extract was from
Promega (Madison, WI).
Polymerase Chain Reaction Amplification and cDNA
Cloning of a cDNA Encoding SecA of Spinach
For first strand
cDNA synthesis and PCR ()amplification, four degenerated
oligonucleotides were utilized: SecA-1
(5`-AT(A/C/T)GCNGA(A/G)ATG(A/C)(A/G)(A/G)ACNGGNGA(A/G)GGNAA(A/G)AC-3`),
SecA-2 (5`-CA(C/T)CA(A/G)GCN(A/G)TNGA(A/G)GCNAA(A/G)GA-3`), SecA-3
(5`-TC(C/T)TTNGC(C/T)TCNA(C/T)NGC(C/T)TG(A/G)TG-3`), and SecA-4
(5`-AT(A/G)TCNGTNCCNC(G/T)NCCNGCCAT(A/G)TT-3`), which correspond to
residues 99-109 (SecA-1), 354-360 (SecA-2 and -3), and
505-512 (SecA-4) of the E. coli SecA protein. First
strand cDNA was generated according to the protocol of Krolkiewicz et al.(1994) from poly(A)
RNA isolated from
spinach leaves using primer SecA-4. This cDNA was utilized as a
template for PCR amplification (30 cycles of denaturation for 60 s at
95 °C, annealing for 90 s at 37 °C, and polymerization for 60 s
at 72 °C) with primers SecA-1 and SecA-4. Products from
1.0-1.6 kilobase pairs were recovered from agarose gels and used
as templates in subsequent PCR reactions (conditions like above) in the
presence of either SecA-1 and SecA-3 or SecA-2 and SecA-4. The
resulting PCR fragments of 730 and 484 base pairs, respectively, were
inserted into the pCRII-vector (Invitrogen, Leek, Netherlands) and
utilized to screen a cDNA library prepared from poly(A)
RNA from spinach. The cDNAs isolated were subcloned with
pBSCM13
(Stratagene). The nucleotide sequence was
determined according to the protocol of Sanger et al.(1977)
after generating successive deletions from both sides by exonuclease
III treatment (Henikoff, 1984).
Miscellaneous
In vitro transcription, in vitro translation in cell-free lysates, and in
organello experiments were performed as described (Cai et
al., 1993; Clausmeyer et al., 1993). Competition
experiments with saturating amounts of precursor protein were performed
as detailed by Michl et al.(1994). Gel electrophoresis of
proteins under denaturing conditions was carried out according to
Laemmli(1970). The gels were processed by fluorography using 16% sodium
salicylate (Chamberlain, 1979). All other methods followed protocols of
Sambrook et al.(1989).
The Sec Pathway for Protein Transport across Thylakoid
Membranes of Spinach Is Resistant to Sodium Azide
The different
translocation pathways for lumenal proteins carrying bipartite transit
peptides can be distinguished by their sensitivities to translocation
inhibitors. The pH route can be inhibited by protonophors such as
nigericin, whereas sodium azide is a common inhibitor for protein
transport along the Sec-dependent pathway (see the introduction). Such
effects are generally analyzed with chloroplasts or thylakoid membranes
isolated from pea. If the inhibitors are applied to spinach
chloroplasts, it turns out that sodium azide does not result in the
expected inhibition of protein translocation. Although in pea
chloroplasts 2.5 mM sodium azide is sufficient to seriously
impair the thylakoid translocation of plastocyanin, the thylakoid
transfer of this protein in spinach chloroplasts is not affected even
at an inhibitor concentration of 10 mM (Fig. 1A and data not shown). On the other hand, nigericin exerts similar
effects on protein translocation in both pea and spinach chloroplasts.
In the presence of this protonophor, the 16-kDa subunit of the
oxygen-evolving complex accumulates in the stroma as an intermediate (Fig. 1B).
pH-dependent
route. We have therefore analyzed whether spinach chloroplasts still
have two independent translocation pathways for hydrophilic lumen
proteins. Competition experiments performed in the presence of
saturating amounts of the precursor for the 23-kDa protein showed that,
like in pea chloroplasts, the protein competes specifically with itself
and the 16-kDa protein but not with plastocyanin for thylakoid
translocation, as judged from the lack of any detectable stromal
intermediate even in overexposed gels and when utilizing densitometric
scanning procedures for detection ( Fig. 2and data not shown).
These results prove the existence of the two thylakoid translocation
pathways also in spinach. We assume that one of them is homologous to
the prokaryotic Sec pathway but that it involves a compound that is
less sensitive to sodium azide than the corresponding one in pea.
Molecular Characterization of a cDNA Encoding SecA from
Spinach
As a first step in the characterization of components of
the putative Sec pathway, we focussed on the isolation of a cDNA for
SecA from spinach. Degenerated oligonucleotides deduced from segments
that are conserved in all SecA proteins known to date were utilized in
a series of PCR experiments using first strand cDNA derived from
poly(A) RNA of spinach leaves as a template. Two
fragments of 730 and 484 base pairs, respectively, were amplified, both
of which contain open reading frames with significant homology to SecA
from E. coli. These fragments were subsequently used to screen
a
gt11-based cDNA library made from poly(A)
RNA
of spinach leaves. Among 1
10
recombinant phages,
three positives (
SocCpSecA-1, -2, and -3) carrying inserts of
approximately 3.7, 3.2, and 3.0 kilobase pairs, respectively, were
identified. Sequence analysis showed that all three exhibit homology to secA of E. coli (data not shown). Phage
SocCpSecA-1, which contains a single EcoRI fragment of
3739 base pairs, was chosen for further analysis.
SocCpSecA-1 was cloned with pBSC M13
, and the
nucleotide sequence of both strands was determined. Starting with the
first ATG, which is found at position 140 (Fig. 3), the cDNA
harbors an open reading frame of 1036 residues that corresponds to a
protein with a calculated mass of 117 kDa. The assumed start codon is
preceded by an in-frame stop codon at -139 and surrounded by a
nucleotide sequence that correlates well with the canonical sequence
deduced for the translation start in eukaryotes
(Ltcke et al., 1987; Kozak, 1991). The
3`-untranslated region of 492 nucleotides contains several stop codons
in all reading frames and ends with three adenine residues that might
represent the residual part of a poly(A) tail. Consistent with this is
a nucleotide sequence (TATAAA) at positions -46 to -41 from
the 3` end that matches the consensus motif for eukaryotic
polyadenylation signals (Proudfoot and Brownlee, 1976). We conclude
that
SocCpSecA-1 carries the complete coding sequence for the SecA
protein from spinach.
The SecA Protein of Spinach Is Imported into Isolated
Intact Chloroplasts
Comparison of the amino acid sequence
deduced from the cDNA with that of the SecA protein of E. coli shows that the spinach protein carries an amino-terminal extension
of approximately 80 amino acid residues (Fig. 4). It is likely
that this extension comprises a transit peptide that mediates the
transport of the cytosolically synthesized protein into its target
organelle, which we assumed to be the chloroplast in this instance.
However, the putative transit peptide deviates from typical plastid
import signals (von Heijne et al., 1989). For instance, it
does not start with a MA motif and carries a glutamate residue at
position 2 instead, which was noted for only few plastid transit
peptides including those of the triose phosphate translocator
(Flgge et al., 1989; Willey et
al., 1991) and of a plastid homologue to the 54-kDa subunit of the
signal recognition particle (Franklin and Hoffman, 1993). Also, the
number of hydroxylated residues (16%, all serine) is significantly
lower than found on average (28%), and there are numerous charged amino
acids (23%), including five acidic residues that are rare in plastid
transit peptides (von Heijne et al., 1989).
We thank Annette Meurs for skillful technical
assistance and Dr. Himadri Pakrasi (St. Louis, Missouri) for critical
reading of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.