©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation and Characterization of a cDNA Encoding the SecA Protein from Spinach Chloroplasts
EVIDENCE FOR AZIDE RESISTANCE OF Sec-DEPENDENT PROTEIN TRANSLOCATION ACROSS THYLAKOID MEMBRANES IN SPINACH (*)

(Received for publication, March 6, 1995; and in revised form, May 22, 1995)

Jrgen Berghfer Ivan Karnauchov Reinhold G. Herrmann Ralf Bernd Klsgen (§)

From the Botanisches Institut der Ludwig-Maximilians-Universitt, Menzinger Strasse 67, D-80638 Mnchen, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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(2)-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(2)-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.

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. (^1)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.

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.


EXPERIMENTAL PROCEDURES

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.

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 [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 (^2)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).


RESULTS

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 DeltapH 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).


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 DeltapH-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.


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.



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 10^6 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.

The insert of 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.


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.



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).


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.


DISCUSSION

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.


FOOTNOTES

*
This work was supported by Grant SFB 184 from the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) Z49124[GenBank].

§
To whom correspondence should be addressed: Botanisches Institut, Menzinger Strasse 67, D-80638 Mnchen, Germany. Tel.: 49-89-17-861-218; Fax: 49-89-17-82-274.

^1
R. B. Klsgen, unpublished results.

^2
The abbreviation used is: PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank Annette Meurs for skillful technical assistance and Dr. Himadri Pakrasi (St. Louis, Missouri) for critical reading of the manuscript.


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