COMMUNICATION
A Signal Peptide That Directs Non-Sec Transport in Bacteria Also Directs Efficient and Exclusive Transport on the Thylakoid Delta pH Pathway*

Hiroki Mori and Kenneth ClineDagger

From the Horticultural Sciences Department and Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, Florida 32611-0690

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

Signal peptides that specifically direct precursor proteins to the thylakoid Delta pH pathway possess an N domain RR motif. Signal peptides that direct transport of bacterial proteins across a non-Sec export pathway possess an N domain RRXFLK consensus motif. Recent genetic studies suggest an evolutionary link between these two protein translocation pathways. To further explore this relationship, we examined the thylakoid targeting capability of the signal peptide for Escherichia coli hydrogenase 1 small subunit (HyaA) by linking it to plastocyanin and assaying the chimeric protein in an in vitro thylakoid transport assay. The chimeric precursor was transported across thylakoids with high efficiency. Transport was characteristic of the Delta pH but not the Sec pathway, i.e. it was eliminated by ionophores that dissipate the Delta pH but occurred in the absence of stromal extract or ATP. This result was confirmed by competition with chemical quantities of a Delta pH pathway precursor. This indicates that the HyaA signal peptide has the necessary elements for efficient and exclusive targeting to the Delta pH pathway and further supports the notion that the alternate targeting pathways in prokaryotes and plant thylakoids are analogous.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

Many thylakoid lumen-resident proteins of plant chloroplasts are synthesized in the cytosol as larger precursors with bipartite amino-terminal extensions called transit peptides (see Ref. 1 for review). The stroma-targeting domain of the transit peptide governs import into the chloroplast stroma; the lumen-targeting domain directs subsequent transport into the thylakoid lumen. Two precursor-specific pathways for protein transport into the thylakoid lumen have been identified by in vitro and genetic studies (1). The thylakoid Sec pathway requires a chloroplast SecA protein (cpSecA) and ATP (1) and appears analogous to the bacterial Sec system. The Delta pH pathway operates independently of ATP and soluble factors, requiring only a thylakoidal pH gradient (1). Targeting specificity for the two pathways is determined primarily by the lumen-targeting domains, which contain motifs of bacterial signal peptides, i.e. an amino-terminal charged N domain, a hydrophobic H domain, and a carboxyl-terminal cleavage C domain (2). Precursors targeted to the Delta pH pathway invariably contain an essential N domain twin arginine that provides access to the Delta pH pathway (Fig. 1) (3). In addition, Delta pH pathway precursors have H and/or C domains that are nonfunctional for Sec pathway transport (4, 5). These latter elements have been termed "Sec-avoidance" elements (4).


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Fig. 1.   Lumen-targeting domains of precursors targeted to the Delta pH pathway and signal peptides of precursors to bacterial redox cofactor-binding proteins. The acidic (A), N-terminal charged (N), hydrophobic (H), and C-terminal cleavage (C) domains are shown for lumen-targeting domains of precursors transported by the Delta pH pathway. These are compared with signal peptides of several bacterial precursor proteins that bind redox cofactors. Bold type R shows a conserved arginine in the N domain; the H domain is underlined. Signal peptides for hydrogenase small subunits of D. vulgaris Hildenborough and E. coli contain two twin arginine motifs. The first twin arginine motif is not required for export (6).

A non-Sec protein export pathway also appears to operate in bacteria. Nivière et al. (6) showed that a chimeric precursor containing the Desulfovibrio vulgaris hydrogenase small subunit signal peptide fused to beta -lactamase was exported efficiently only under anaerobic conditions and this export depended upon a critical N domain twin arginine motif. Export of Pseudomonas stutzeri nitrous oxide reductase also depends upon an N domain RR (7). Recently, it was shown that trimethylamine N-oxide reductase, which bears an N domain twin arginine, is exported by a mechanism independent of SecA, SecY, or SecE, but dependent on the transmembrane Delta µH+ (8). Berks (9) pointed out that many precursors for bacterial proteins that bind redox cofactors share a conserved N domain (S/T)RRXFLK motif (Fig. 1) and suggested that the export system for such proteins may be related to the thylakoid Delta pH pathway. Strong support for such a notion was recently provided by Settles et al. (10). Maize Hcf106 mutant chloroplasts are selectively defective in the Delta pH pathway. The Hcf106 protein has striking homology to several open reading frames from bacterial genomes. In the case of Azotobacter chroococcum, mutation of the Hcf106 homologue results in mislocalization of hydrogenase (10).

Here we show that the signal peptide of Escherichia coli hydrogenase 1 small subunit (HyaA) is functionally equivalent to a lumen-targeting domain for chloroplast Delta pH pathway precursors. This indicates that the HyaA signal peptide has the essential elements that both engage the Delta pH pathway and avoid the Sec pathway.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

Materials-- All reagents, enzymes, and standards were purchased commercially. In vitro transcription plasmids for the stromal intermediate of OE331 (iOE33) from wheat (11), precursors to plastocyanin (pPC) from Arabidopsis and LHCP (12), and the chimeric precursors t23-PC and DT-PC (5) were previously described. For tOE23 expression in E. coli, the coding sequence was amplified from the transcription plasmid with a forward primer that contained an NdeI site encompassing the initiator methionine codon and a reverse primer that also contained a HindIII restriction site. The PCR product was cloned into the NdeI/HindIII sites of pETH3c (12). Expression of tOE23 in E. coli strain BL21 (DE3) and isolation of inclusion bodies were as described (12). The HyaA coding sequence (13) was amplified by PCR with E. coli genomic DNA as template and cloned into pGEM-3z. PCR splicing by overlap extension (SOE) (14) was used to construct a chimeric precursor, Hya-PC, which is an exact fusion between coding sequences for the HyaA signal peptide and the mature domain of Arabidopsis PC. DNA fragments corresponding to the signal peptide and to PC were amplified separately and spliced in a second round of PCR. Forward and reverse primers for the SOE reaction contained restrictions sites for HindIII and SstI sites, respectively, and the SOE product was cloned into the HindIII and SstI sites of pGEM-3z. The sequences of all PCR-cloned constructs were confirmed by Taq DyeDeoxy Terminator cycle sequencing.

Assays for Thylakoid Protein Transport-- Capped RNA for the various precursors was produced in vitro with SP6 polymerase and uncut plasmid. Precursors were translated in a wheat germ system in the presence of [3H]leucine and adjusted to import buffer (50 mM HEPES-KOH, pH 8.0, 0.33 M sorbitol) containing 30 mM unlabeled leucine prior to use (12). Chloroplasts and thylakoids were prepared from pea seedlings as described (15). Transport of radiolabeled proteins into thylakoids was conducted with chloroplast lysates or washed thylakoids in 75-µl assays (12). Precursors and recovered thylakoid membranes were analyzed by SDS-polyacrylamide gel electrophoresis and fluorography. Quantification was accomplished by scintillation counting of radiolabeled proteins extracted from excised gel bands (15).

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

Transport of a Chimeric Precursor Hya-PC into the Thylakoid Lumen-- We initially assayed transport of the HyaA precursor protein into isolated thylakoids. A low level of transport was achieved as determined by expected proteolytic maturation and protection from exogenous protease (data not shown). However, this low level of transport was insufficient for a full examination of the targeting properties. Because the focus here was on the targeting capability of the signal peptide of HyaA, we constructed a chimeric precursor protein (Hya-PC) possessing the HyaA signal peptide fused to the mature domain of Arabidopsis PC. PC was chosen as a passenger protein because it can be transported on the Sec pathway and the Delta pH pathway when linked to appropriate signal peptides (4, 5).

Fig. 2 shows a thylakoid transport assay with Hya-PC. Incubation of Hya-PC with isolated thylakoids produced a smaller product at the location of mature PC (lane 2) that was resistant to thermolysin treatment of the membranes (lane 3). Mature PC was recovered in the lumen subfraction when the recovered thylakoids were sonicated to release the lumenal contents (lane 7). Mature PC was not produced when assays were conducted in the presence of ionophores (lane 4), and the membrane-associated precursor was degraded by thermolysin (lane 5). Fig. 2 also shows control assays for translocation/integration of authentic pPC and the membrane protein LHCP. These assays demonstrate that the HyaA signal peptide directs transport into the thylakoid lumen. Average transport of Hya-PC for three experiments was 32% of the added precursor. This compares very favorably with the 16% of t23-PC transport (Fig. 3, lane 3) for the same three experiments. t23-PC is a fusion protein between the core targeting peptide for the Delta pH substrate OE23 and the mature domain of Arabidopsis PC (5).


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Fig. 2.   Transport of a chimeric precursor Hya-PC into the thylakoid lumen. Transport/integration assays were conducted for 30 min at 25 °C with chloroplast lysate and 5 mM MgATP. Assays were conducted in the light to generate a thylakoidal Delta pH, which was dissipated in assays containing 0.5 µM nigericin and 1 µM valinomycin (lanes 4 and 5). After assay, the thylakoids were recovered by centrifugation, resuspended in import buffer with or without thermolysin, incubated for 40 min on ice, washed, and then resuspended in SDS sample buffer. For thylakoid subfractionation, thermolysin-treated thylakoids were sonicated at 10 watts three times for 10 s. After centrifugation for 30 min at 65,000 rpm with Beckman TLA100.3 rotor, the membrane fraction (lane 6) was resuspended in SDS sample buffer, and lumenal proteins in the supernatant (lane 7) were precipitated with 10% trichloroacetic acid. Lanes were loaded with recovered thylakoids, membrane, or lumen fraction equivalent to 20% of each assay. The radiolabeled precursor (TP) represents 2% of the amount in each assay (lane 1). The precursors used are designated to the left of the fluorogram. The positions of the precursor (p) and mature (m) forms of the proteins are marked. DP designates a characteristic protease degradation product of membrane-inserted LHCP.


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Fig. 3.   Transport requirements suggest that Hya-PC is transported by the Delta pH pathway. Transport of precursors across thylakoid membranes was conducted for 30 min at 25 °C with chloroplast lysate to provide stromal extract (SE) or washed thylakoids. Energy was provided in the form of ATP and/or light to generate a Delta pH. Assay conditions examined the requirement for a Delta pH (lane 4), ATP (lane 5), stromal extract (lane 6), and sensitivity to azide (lane 7). Final concentrations were 5 mM ATP, 10 mM sodium azide, 0.5 µM nigericin (nig), and 1 µM valinomycin (val). Apyrase (0.5 unit per 75 µl) was used to eliminate residual ATP in lysate and translation products for assays conducted in the absence of ATP. These conditions are designated above the panel. Recovered thylakoids were post-treated with thermolysin. Analysis and designations were as in Fig. 2.

Transport Requirements of Hya-PC Are Consistent with Delta pH Pathway Transport-- Assays in Fig. 3 were conducted under a variety of conditions designed to assess energy and stroma requirements that are characteristic for thylakoid translocation pathways. As with t23-PC (a Delta pH pathway substrate), transport of Hya-PC was completely abolished by addition of ionophores that dissipate the thylakoidal Delta pH (lane 4), but was unaffected by the addition of sodium azide (lane 7), a SecA inhibitor (16), or by removal of the stromal extract (lane 6). Depletion of ATP with apyrase in these experiments diminished, but did not abolish, Hya-PC transport (lane 5). A similar reduction of t23-PC in the presence of apyrase also occurred. Such an effect was not previously recognized for t23-PC (5) but appears to be related to the PC mature domain because in parallel assays, tOE23 transport was not reduced by apyrase (data not shown). In contrast, transport of the Sec pathway substrate pPC was only marginally affected by ionophores (lane 4), virtually eliminated by removal of ATP or of stromal extract, the source of ~90% of the cpSecA (lanes 5 and 6), and inhibited by azide (lane 7). These requirements suggest that Hya-PC is transported on the Delta pH pathway rather than the Sec pathway. In addition, Hya-PC transport requirements rule out the participation of two other pathways that are responsible for insertion of membrane proteins (1); i.e. membrane integration by the chloroplast SRP is absolutely dependent on the presence of stroma and NTPs, whereas membrane integration by a spontaneous mechanism occurs even in the absence of a thylakoidal Delta pH.

Competition Assays Verify That Hya-PC Is Targeted to the Delta pH Pathway but Not to the Sec Pathway-- To further clarify the pathway utilized by Hya-PC, competition assays were conducted with bacterially synthesized tOE23 (Fig. 4A). Increasing concentrations of tOE23 progressively competed transport of t23-PC and Hya-PC. At 2 µM tOE23, transport of Hya-PC was reduced to ~5% of that achieved in the absence of competitor. In contrast, transport of Sec pathway substrates pPC and iOE33 was unaffected by tOE23 competitor.


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Fig. 4.   Competition assays verify that Hya-PC is exclusively transported on the Delta pH pathway. A, competition assays were conducted with chloroplast lysates in the presence of 5 mM ATP with increasing concentration of unlabeled tOE23 as described previously (12). The final concentration of urea in each assay was 167 mM. The precursors used are designated to the left of the fluorograms. B, assays were conducted with chloroplast lysates to provide cpSecA (lanes 2, 3, and 8-10) or washed thylakoids (lanes 4-7) with increasing concentration of unlabeled tOE23. For the assay in the absence of ATP (lanes 3 and 9), chloroplast lysates and in vitro translation products were treated with apyrase. The final concentration of urea in each assay was 167 mM. Sodium azide (10 mM final) was added to verify the utilization of cpSecA (lane 10). The precursors used are designated to the left of the fluorogram panels. These conditions and final concentration of tOE23 (µM) are designated above the fluorogram panels.

To determine if the residual Hya-PC transport in the presence of 2 µM tOE23 competitor was mediated by the Sec pathway, the effect of ATP and stromal extract were assessed in the presence of 2 µM tOE23 (Fig. 4B). DT-PC transport served as a positive control for this experiment. DT-PC is a chimeric precursor protein that contains the N domain of the OE23 precursor and the H/C and mature domains of PC. Importantly, DT-PC is transported by both the Sec and the Delta pH pathways (5). In the absence of stromal extract, tOE23 competitor substantially reduced transport of Hya-PC and DT-PC (lanes 4-7). As expected, addition of stromal extract greatly stimulated the DT-PC residual transport (lane 8), and this stimulation was largely eliminated by removal of ATP (lane 9) or inclusion of sodium azide (lane 10). These three effects are characteristic of Sec transport as exemplified by the control assays with iOE33. In contrast to these results, the residual Hya-PC transport was unaffected by the addition of stroma extract, the removal of ATP, or the inclusion of azide. This demonstrates that Hya-PC is not transported by the cpSecA-dependent mechanism even when the transport on the Delta pH pathway is virtually eliminated by competition. Furthermore, these results indicate that the reduction of transport of Hya-PC and t23-PC by apyrase seen in Fig. 3 does not reflect the involvement of the cpSecA translocation ATPase.

Taken together, these results indicate that Hya-PC is exclusively targeted to the Delta pH pathway and does not utilize the Sec pathway (or other thylakoidal pathways). Thus, the signal peptide for HyaA is functionally equivalent to the Delta pH pathway lumen-targeting domain and contains the two essential elements required for exclusive targeting, an N domain twin arginine (3) that provides access to the Delta pH pathway and an element that prevents engagement by the Sec pathway. It was uncertain whether the N domain twin arginine in the HyaA signal peptide would be functional for the Delta pH pathway because it is followed by several amino acids, SFLK, prior to the H domain. For most Delta pH pathway precursors, the twin arginine immediately precedes the first amino acid of the H domain. Furthermore, a chimeric construct that placed an asparagine between the twin arginine motif and the H domain was nonfunctional for Delta pH pathway transport (4). However, the HyaA signal peptide was twice as efficient as the authentic core signal peptide for Delta pH pathway precursor OE23. It is likely, but remains to be demonstrated, that the thylakoid Sec avoidance element in the HyaA signal peptide resides in the H/C domain.

Because of the endosymbiotic origin of chloroplasts from an ancestral cyanobacterium, it was speculated that protein transport into plant thylakoids would be evolutionarily related to prokaryotic transport mechanisms. Work during the past several years has shown that three of the four known mechanisms of thylakoid protein translocation can be ascribed to prokaryote mechanisms. These include a SecA-dependent pathway, an SRP-like pathway for insertion of a membrane protein, and a spontaneous protein insertion mechanism (1). Although characteristics of the thylakoid Delta pH pathway suggest a prokaryote-like mechanism, i.e. a classical signal peptide and initiation via a loop mechanism (17), no analogous system had been identified in bacteria. The recent report of the identity of the Hcf106 protein involved in Delta pH pathway, and the existence of the bacterial homologues (10) now suggests the identity of the bacterial counterpart. Our results further support the notion that the pathway associated with export of redox cofactor-binding proteins in bacteria is related to the Delta pH pathway of chloroplast thylakoids.

    ACKNOWLEDGEMENT

We thank Shan Wu for excellent technical assistance.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant R01 GM46951 and National Science Foundation Grant MCB-9419287 (to K. C.). DNA sequencing was conducted by the University of Florida Interdisciplinary Center for Biotechnology Research (ICBR) DNA Sequencing Core, which is supported by funds supplied by the Division of Sponsored Research and the ICBR at the University of Florida. This paper is Florida Agricultural Experiment Station Journal Series R-06240.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.

Dagger To whom correspondence should be addressed. Tel.: 352-392-4711 (Ext. 219); Fax: 352-392-4711; E-mail: KCC{at}nervm.nerdc.ufl.edu.

1 The abbreviations used are: OE23 and OE33, the 23- and 33-kDa subunits of the photosystem II oxygen evolving complex, respectively; p, i, and t, full-length precursor, intermediate precursor, and truncated precursor form, respectively; PC, plastocyanin; HyaA, E. coli hydrogenase 1 small subunit; DT, dual-targeting; LHCP, the light-harvesting chlorophyll a/b protein; SOE, splicing by overlap extension; PCR, polymerase chain reaction.

    REFERENCES
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Abstract
Introduction
Procedures
Results & Discussion
References

  1. Cline, K., and Henry, R. (1996) Annu. Rev. Cell Dev. Biol. 12, 1-26[CrossRef][Medline] [Order article via Infotrieve]
  2. von Heijne, G., Steppuhn, J., and Herrmann, R. G. (1989) Eur. J. Biochem. 180, 535-545[Abstract]
  3. Chaddock, A. M., Mant, A., Karnauchov, I., Brink, S., Herrmann, R. G., Klösgen, R. B., and Robinson, C. (1995) EMBO J. 14, 2715-2722[Abstract]
  4. Bogsch, E., Brink, S., and Robinson, C. (1997) EMBO J. 16, 3851-3859[Abstract/Free Full Text]
  5. Henry, R., Carrigan, M., McCaffery, M., Ma, X., and Cline, K. (1997) J. Cell Biol. 136, 823-832[Abstract/Free Full Text]
  6. Nivière, V., Wong, S.-L., and Voordouw, G. (1992) J. Gen. Microbiol. 138, 2173-2183[Medline] [Order article via Infotrieve]
  7. Dreusch, A., Bürgisser, D. M., Heizmann, C. W., and Zumft, W. G. (1997) Biochim. Biophys. Acta 1319, 311-318[Medline] [Order article via Infotrieve]
  8. Santini, C.-L, Ize, B., Chanal, A., Müller, M., Giordano, G., and Wu, L.-F. (1998) EMBO J. 17, 101-112[Abstract/Free Full Text]
  9. Berks, B. C. (1996) Mol. Microbiol. 22, 393-404[Medline] [Order article via Infotrieve]
  10. Settles, A. M., Yonetani, A., Baron, A., Bush, D. R., Cline, K., and Martinssen, R. (1997) Science 278, 1467-1470[Abstract/Free Full Text]
  11. Hulford, A., Hazell, L., Mould, R. M., and Robinson, C. (1994) J. Biol. Chem. 269, 3251-3256[Abstract/Free Full Text]
  12. Cline, K., Henry, R., Li, C., and Yuan, J. (1993) EMBO J. 12, 4105-4114[Abstract]
  13. Menon, N. K., Robbins, J., Wendt, J. C., Shanmugam, K. T., and Przybyla, A. E. (1991) J. Bacteriol. 173, 4851-4861[Medline] [Order article via Infotrieve]
  14. Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., and Pease, L. R. (1989) Gene 77, 61-68[CrossRef][Medline] [Order article via Infotrieve]
  15. Cline, K. (1986) J. Biol. Chem. 261, 14804-14810[Abstract/Free Full Text]
  16. Oliver, D. B., Cabelli, R. J., Dolan, K. M., and Jarosik, G. P. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 8227-8231[Abstract]
  17. Fincher, V., McCaffery, M., and Cline, K. (1998) FEBS Lett. 423, 66-70[CrossRef][Medline] [Order article via Infotrieve]


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