INTRODUCTION

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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).

View larger version (24K): [in this window] [in a new window]   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 -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 µ^H+ (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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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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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).

View larger version (46K): [in this window] [in a new window]   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 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.

View larger version (43K): [in this window] [in a new window]   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 pH. Assay conditions examined the requirement for a 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 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 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.

View larger version (33K): [in this window] [in a new window]   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.