©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A SecY Homolog in Arabidopsis thaliana
SEQUENCE OF A FULL-LENGTH cDNA CLONE AND IMPORT OF THE PRECURSOR PROTEIN INTO CHLOROPLASTS (*)

Vanessa Laidler Alison M. Chaddock Tracy G. Knott Denise Walker Colin Robinson (§)

From the Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 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.

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, 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.


EXPERIMENTAL PROCEDURES

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()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.

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, 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.


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.




RESULTS AND DISCUSSION

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.


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.

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.

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/CysAla)(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.

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,()but it should now be possible to generate antibodies to the Arabidopsis protein and probe the role of cpSecY in some detail.


FOOTNOTES

*
This work was supported by Biotechnology and Biological Sciences Research Council Grants C04270 and P02474. 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.

§
To whom correspondence and reprint requests should be addressed. Tel.: 44-1203-523-557; Fax: 44-1203-523-701.

The abbreviations used are: bp, base pair(s); kb, kilobase(s).

V. Laidler, A. M. Chaddock, T. G. Knott, D. Walker, and C. Robinson, unpublished data.


ACKNOWLEDGEMENTS

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.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.