Correspondence to: Kenneth Cline, Horticultural Sciences Department, Fifield Hall, University of Florida, Gainesville, FL 32611., kcline{at}ufl.edu (E-mail), (352) 392-4711 x 219 (phone), (352) 392-5653 (fax)
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Abstract |
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Prokaryotes and prokaryote-derived thylakoid membranes of chloroplasts share multiple, evolutionarily conserved pathways for protein export. These include the Sec, signal recognition particle (SRP), and Delta pH/Tat systems. Little is known regarding the thylakoid membrane components involved in these pathways. We isolated a cDNA clone to a novel component of the Delta pH pathway, Tha4, and prepared antibodies against pea Tha4, against maize Hcf106, a protein implicated in Delta pH pathway transport by genetic studies, and against cpSecY, the thylakoid homologue of the bacterial SecY translocon protein. These components were localized to the nonappressed thylakoid membranes. Tha4 and Hcf106 were present in ~10-fold excess over active translocation sites. Antibodies to either Tha4 or Hcf106 inhibited translocation of four known Delta pH pathway substrate proteins, but not of Sec pathway or SRP pathway substrates. This suggests that Tha4 and Hcf106 operate either in series or as subunits of a heteromultimeric complex. cpSecY antibodies inhibited translocation of Sec pathway substrates but not of Delta pH or SRP pathway substrates. These studies provide the first biochemical evidence that Tha4 and Hcf106 are specific components of the Delta pH pathway and provide one line of evidence that cpSecY is used specifically by the Sec pathway.
Key Words: chloroplast protein transport, twin arginine, SecY, Hcf106, Tha4
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Introduction |
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PROTEIN translocation across and into membranes is a fundamental cellular process, responsible for localization of ~50% of the proteins in a eukaryotic cell.
Recently, it has been shown that there are several different export systems present in thylakoid membranes and Escherichia coli plasma membranes (for reviews see
A second pathway, the chloroplast signal recognition particle (SRP) pathway, is responsible for targeting a family of integral thylakoid proteins, the LHCPs (
A third thylakoid pathway is termed the Delta pH pathway because it employs the thylakoidal pH gradient as sole energy source for transport of lumenal proteins (
The Delta pH system was initially thought to be a eukaryotic innovation because of its unique properties (
These observations raise several important questions that are relevant for thylakoids and other prokaryote or prokaryote-derived membranes. First, are Hcf106 and its homologues directly involved in transport on the Delta pH/Tat systems and, if so, what roles do they play in the process? Currently, the only evidence for their involvement in protein transport comes from mutant analysis. Second, do the Delta pH/Tat systems and the cpSRP system employ distinct translocons or do they converge with the Sec-type translocon? Mutant and depletion analyses have addressed this question, but the results are uncertain. With regards to the Delta pH/Tat system, the E. coli Tat substrate trimethylamine N-oxide (TMAO) reductase is exported at normal levels in a conditional SecY mutant strain at the nonpermissive temperature or in a strain depleted of SecE (
Here we have taken a biochemical approach to these questions. Antibodies were raised to the maize Hcf106 protein and a related pea protein, psTha4. These antibodies specifically inhibited transport of Delta pH pathway substrates with the respective maize and pea thylakoid membranes. Similarly, we have isolated a cDNA for pea cpSecY and produced an antibody to its COOH terminus. Antibody to cpSecY exclusively inhibited precursors that employ cpSecA for their transport. This provides direct evidence that cpSecY functions on the thylakoidal Sec pathway. The fact that neither the cpSRP pathway nor especially the Delta pH pathway was affected by antibody to cpSecY argues that the three kinds of thylakoid protein translocation systems are distinct at the level of the translocon.
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Materials and Methods |
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cDNA Cloning of Pea cpSecY and Pea Tha4
The full coding regions of pea cpSecY and pea Tha4 were obtained in two steps. Nearly full-length cDNA clones were isolated by screening a pea lambda ZAP II cDNA library (a generous gift of Dr. Ken Keegstra, Michigan State University, East Lansing, MI) using standard protocols. For cDNA cloning of pea cpSecY, the hybridization probe was a DNA fragment of Arabidopsis cpSecY (accession number AF144684). For cDNA cloning of pea Tha4, the probe was a DNA fragment of maize Hcf106 (accession number AF027808) and Arabidopsis EST clone 182P20T7 (accession number H37534), which was identified by BLAST search as encoding a protein homologous to maize Hcf106. Both of the cDNA clones isolated were nearly complete but lacked initiation methionines. The missing 5' sequences were isolated by 5'-RACE cloning (GIBCO BRL) following the manufacturer's recommendations. Clones containing the full-length coding regions for in vitro transcription and translation were engineered into pGem 4Z (Promega) in the SP6 orientation. A cDNA clone for atHcf106 was isolated by RT-PCR using primers based on genomic sequence (accession number ABO19226) and was cloned into pGem T (Promega) in the SP6 orientation. Sequencing of all clones on both strands was performed by the University of Florida Interdisciplinary Center for Biotechnology Research (ICBR) DNA Sequencing Core Facility.
Expression and Purification of Stroma-exposed Domains of Maize Hcf106 and Pea Tha4
The stroma-exposed domain of maize Hcf106 (hcf106sd) and the stroma-exposed domain of psTha4 (tha4sd) were expressed in E. coli as His-tagged fusion proteins. The coding regions for amino acid residues 90243 of maize Hcf106 and residues 78137 of psTha4 were amplified by PCR with forward primers containing NdeI restriction sites and an in-frame sequence for six histidine residues. A cDNA clone for Hcf106 in pGem 4Z was used as template and the reverse primer was the pUC/M13 forward primer corresponding to the pGem 4Z plasmid. The cDNA clone for psTha4 was used as template and the reverse primer contained a SacI restriction site. The amplified DNA fragments were digested with NdeI and HindIII (maize hcf106sd), or NdeI and SacI (pea tha4sd), and cloned into pETH3c (DE3) and the expression was induced with IPTG. Both recombinant proteins accumulated in the soluble fraction of E. coli cells and were purified by nickel-nitrilotriacetic acid agarose chromatography according to the Novagen protocol. Purified protein was dialyzed against 20 mM Hepes-KOH, pH 8.0, and concentrated with a Centricon YM10.
Production of Antibodies
For cpSecY antibody production, the peptide NH2-CRAEIISQKYKNIELYDFDKY-COOH, equivalent to the COOH terminus of pea cpSecY plus an NH2-terminal cysteine, was synthesized and cross-linked to keyhole limpet hemocyanin by Genosys Biotechnologies. The keyhole limpet hemocyaninlinked peptide was used as antigen. E. coliexpressed maize hcf106sd and pea tha4sd were used an antigens. Antibodies to the proteins were prepared in rabbits by Cocalico Biologicals. IgG was purified from the serum with protein ASepharose as described (
Preparation of Chloroplasts, Lysates, Thylakoids, Nonappressed, and Appressed Thylakoids
Intact pea chloroplasts were isolated from 910-d-old pea (Laxton's Progress 9) seedlings by a combination of differential and Percoll density gradient centrifugation as described (
Preparation of Labeled Precursor Proteins
In vitro transcription plasmids for pLHCP and iOE23 from pea, iOE17 from maize, iOE33 from wheat, pPSI-N from Arabidopsis, and pPSII-T from cotton have been described elsewhere (
Assays for Chloroplast Protein Import and Thylakoid Protein Transport
Import of radiolabeled proteins into chloroplasts was conducted as previously described (
Immunoprecipitation
Radiolabeled cpSecY translation products or chloroplasts repurified after protein import reactions were dissolved in 0.05 M Tris-HCl, pH 6.8, 2% SDS, 4% glycerol, 2% ß-mercaptoethanol, 10 mM EDTA, 0.008% bromophenol blue and were heated at 100°C for 2 min. Samples were then diluted 12-fold in 10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 140 mM NaCl, 1 mM PMSF, 1% Triton X-100. 10 µl of antibody to pea cpSecY or pea Tha4 (as irrelevant antibody) was added and the samples were incubated with shaking for 1.5 h at 4°C. 40 µl of protein ASepharose (packed volume of beads washed in 10 mM Hepes-KOH, pH 8.0) was then added and the samples shaken for an additional 30 min at 4°C. The protein ASepharose/antibody/antigen complexes were pelleted (500 g, 2 min) and washed three times in 10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 140 mM NaCl, 0.2% Triton X-100. The final pellets were resuspended in 40 µl SDS sample buffer, heated 2 min at 100°C, and the supernatant analyzed by SDS-PAGE and fluorography.
Miscellaneous
Chlorophyll concentrations were determined according to
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Results |
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cDNA Cloning of Pea cpSecY and Pea Tha4
Maize Hcf106 was shown to be a component required for the Delta pH pathway in vivo (
Screening for a pea Hcf106 homologue employed a mixed probe consisting of the maize Hcf106 cDNA and an Arabidopsis cDNA obtained from the EST program (Figure 1). A nearly full-length pea cDNA was isolated and extended by 5'-RACE. Similar to the Arabidopsis EST, the pea cDNA encodes a protein that is related to Hcf106 in the transmembrane domain and amphipathic helix, but lacks the extended COOH-terminal acidic domain (Figure 1). Based on sequence comparison, the predicted pea protein is more similar to Tha4, a newly identified maize protein that is related to Hcf106 in sequence and function (Walker, M.B., L.M. Roy, E. Coleman, R. Voelker, and A. Barkan, manuscript submitted for publication), than it is to Hcf106. Accordingly, the pea protein has been designated psTha4. We have now isolated an Arabidopsis cDNA, based on genomic sequence (accession number AB019226), that appears to encode the authentic Hcf106 orthologue (Figure 1).
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The psTha4 cDNA encodes a protein with 137 residues. The amino terminus has characteristics of a chloroplast transit peptide. This was experimentally verified with an in vitro chloroplast import assay. The psTha4 translation product migrated at 19 kD (Figure 2 A, lane 1). Upon incubation with chloroplasts, a faster migrating 16-kD band was produced that copurified with intact chloroplasts and was protected from exogenous protease (Figure 2 A, lanes 2 and 3). This indicates that the precursor was imported into chloroplasts and processed to mature size. The imported psTha4 protein fractionated predominantly with the thylakoid membranes (Figure 2 A, lane 5) and was integrated into the membrane as assessed by resistance to alkaline extraction (Figure 2 A, lane 7). Similar to Hcf106, the hydrophilic domain of imported psTha4 was exposed to the stromal compartment as it was degraded by exogenous protease (Figure 2 A, lane 6).
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A partial pea cpSecY cDNA was isolated and then extended by 5'-RACE (accession number AF144684). The predicted protein is highly homologous to other plant cpSecY proteins, with notable sequence divergence only in the amino-terminal transit peptide and the extreme COOH terminus. Upon in vitro translation, two bands were produced, one at 47 kD (Figure 2 A, lane 1) and one migrating slightly faster. Immunoprecipitation with an antibody to the cpSecY COOH terminus showed that the larger translation product is the full-length precursor (Figure 2 B, lanes 2 and 3). Incubation with chloroplasts produced a major band of processed cpSecY protein at 42 kD and a minor band migrating slightly faster (Figure 2 A, lanes 2 and 3). The imported cpSecY fractionated with the thylakoid membranes (Figure 2 A, lane 5) was resistant to alkaline extraction (Figure 2 A, lane 7), but degraded by protease (Figure 2 A, lane 6) to produce a 20-kD degradation product (data not shown).
Antibodies against Maize Hcf106, Pea Tha4, and Pea cpSecY
The stromal domains of Hcf106 and psTha4 were expressed in E. coli with NH2-terminal His tags for purification. Both proteins accumulated in the soluble fraction of E. coli, presumably in a native conformation. Purified pea tha4sd and maize hcf106sd migrated on SDS-PAGE with molecular masses of ~16 kD and ~35 kD, respectively (Figure 3 A, lanes 1 and 2). Both proteins were used as antigens without further treatment.
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Anti-psTha4 reacted on immunoblots with an ~16-kD pea thylakoid polypeptide (Figure 3 B, lanes 1 and 2). The specificity of the reaction was verified by conducting the antibody incubation in the presence of the tha4sd antigen (Figure 3 B, lanes 3 and 4). Similarly anti-Hcf106 recognized a ~35-kD band in maize thylakoids in an antigen-reversible manner (Figure 3 B, lanes 58). Anti-Hcf106 did not react with pea thylakoids on immunoblots and anti-psTha4 did not react with maize thylakoids (data not shown). However, as will be seen below, anti-Hcf106 binds to a pea Hcf106 orthologue in its native form.
Three cpSecY synthetic peptides were used as antigens in rabbits. These peptides correspond to several different stroma-facing hydrophilic segments based on the topology of E. coli SecY (
Pea cpSecY and psTha4 Are Integral Thylakoid Proteins Located in the Nonappressed Membranes
Antibodies were used to verify the expected properties of endogenous pea cpSecY and psTha4 (Figure 4). As with the imported proteins, endogenous psTha4 and cpSecY were recovered primarily in the thylakoid membrane fraction (data not shown). They were resistant to carbonate extraction, but digested by added thermolysin (Figure 4, lanes 2 and 3). A band corresponding to the 20-kD protease-protected fragment of imported cpSecY was not immunodecorated with anti-cpSecY (data not shown), indicating that the protease protected fragment corresponds to an NH2-terminal or internal fragment. These results verify that cpSecY and psTha4 are integral membrane proteins exposed to the stroma. Thylakoid membranes consist of two structurally and functionally distinct domains, the nonappressed membranes and the appressed membranes. Upon subfractionation of thylakoids with digitonin (see Materials and Methods), cpSecY and psTha4 were recovered with the nonappressed membranes (Figure 4, lane 5) rather than the appressed membranes (Figure 4, lane 4). cpSecA, which is peripherally associated with thylakoids (Figure 4, lanes 1 and 3), was also recovered in the nonappressed membranes (Figure 4, lane 5). Maize Hcf106 was localized in the nonappressed membranes of maize thylakoids (data not shown). These results are consistent with other studies implicating the nonappressed membranes as the site of protein transport/integration (
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Quantification of psTha4 on immunoblots by comparison to a standard dilution series of tha4sd showed that psTha4 is present at ~150 fmol/µg chlorophyll of thylakoid, which corresponds to ~90,000 molecules per chloroplast. A similar quantification showed that Hcf106 is present at ~100,000 molecules per maize chloroplast. This is interesting because our estimate of the number of active Delta pH translocation sites in pea thylakoids is ~8,000 per chloroplast. This was estimated from the Vmax for transport of iOE23 in an intact chloroplast (8,000/chloroplast per minute;
Antibodies to Hcf106 Specifically Inhibit the Delta pH Pathway of Maize Thylakoids
Genetic disruption of the maize Hcf106 gene and genes for homologous proteins in E. coli results in mislocalization of precursor proteins (
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Protein transport on the Delta pH pathway absolutely depends on the thylakoidal pH (
pH was not impaired by the antibody treatment, the assays were conducted in the absence and presence of the ionophores nigericin and valinomycin, which completely dissipate the proton motive force (Figure 5 A, lanes 2, 4, and 6). Transport of OE33 and PC and integration of LHCP are stimulated by the
pH, and translocation levels are reduced 6070% in its absence (
pH-mediated stimulation of Sec pathway transport and LHCP integration was similarly eliminated by ionophores regardless of antibody treatment. Therefore, we conclude that the binding of anti-Hcf106 IgG to maize thylakoids did not compromise the thylakoidal
pH.
As preincubation of anti-Hcf106 IgG with maize thylakoids may induce aggregation of Hcf106 in the plane of thylakoids due to the divalent binding sites of IgG, we prepared monovalent Fab fragments from anti-Hcf106 IgG and tested the Fab fragments with in vitro thylakoid protein transport assays (Figure 5 B). Increasing concentrations of anti-Hcf106 Fab fragments inhibited transport of Delta pH pathway substrates, iOE17 and iOE23, nearly as well as IgGs. Preimmune Fab fragments had no effect on transport, and the inhibition by anti-Hcf106 Fab fragments was suppressed by inclusion of hcf106sd during the antibody incubation step. Anti-Hcf106 Fab fragments had no effect on integration of pLHCP or on transport of iOE33 and pPC. Thus, inhibition of Delta pH pathway transport resulted directly from binding of the antibody, rather than as a secondary effect of aggregation.
Antibodies to psTha4 Specifically Inhibit the Delta pH Pathway of Pea Thylakoids
A similar analysis was conducted with pea thylakoid membranes and antibodies to psTha4 (Figure 6). Preincubation of pea thylakoids with increasing amounts of anti-psTha4 IgG inhibited protein transport of the Delta pH pathway substrates iOE17 and iOE23. Preimmune IgG had no effect on transport, and the inhibition by anti-Tha4 was suppressed by including antigen tha4sd during the preincubation step. Transport of the Sec pathway substrate iOE33 and integration of the SRP pathway substrate pLHCP were unaffected by anti-psTha4 IgG. Assays conducted in the absence and presence of ionophores confirmed that anti-psTha4 IgG binding did not compromise the thylakoidal pH (data not shown). These results indicate that psTha4 also directly functions in protein transport on the Delta pH pathway.
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Anti-Hcf106 or Anti-psTha4 Inhibits Transport of All Known Delta pH Pathway Substrates
E. coli has three genes that are related to Hcf106: tatA, tatB, and tatE. Disruption of each of these genes singly causes partial inhibition of the translocation of overlapping sets of substrates (
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Antibodies to cpSecY Specifically Inhibit Sec Pathway Transport
To explore the function of cpSecY in thylakoid protein transport, the effect of anti-cpSecY IgG on the three pathways was examined (Figure 8). Preincubation of pea thylakoids with increasing amounts of anti-cpSecY inhibited transport of Sec pathway precursors, iOE33 and pPC (Figure 8 A, lanes 15). Inclusion of 20 µM antigen peptide during the antibody preincubation step suppressed the inhibition (Figure 8 A, lanes 6 and 7). No inhibition was observed for control thylakoids preincubated with preimmune IgG (Figure 8 A, lanes 8 and 9). These results demonstrate that cpSecY operates in conjunction with cpSecA for thylakoid Sec pathway transport. Anti-cpSecY IgG had no effect on transport of the Delta pH pathway precursors, iOE23 and iOE17, or on integration of the SRP pathway substrate pLHCP. Even at higher concentrations of cpSecY IgGs (2.0 mg/ml), there was no inhibition of OE23 transport or LHCP integration (data not shown). Of interest is that Fab fragments prepared from anti-cpSecY IgG were ineffective inhibitors of Sec pathway transport (data not shown). This contrasts with the situation of anti-Hcf106 inhibition and suggests either that aggregation of the Sec translocons is required for inhibition or that IgG inhibits Sec pathway transport by steric hindrance.
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Discussion |
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Hcf106 was first identified as a component of the Delta pH transport pathway by genetic studies in maize, wherein mutations in Hcf106 resulted in defective transport of Delta pH pathway substrates in vivo (
We also identified a gene from pea that is related to, but not orthologous to, Hcf106. Our original intention was to isolate the pea Hcf106 orthologue. However, sequence comparisons indicate that the pea protein as well as the protein coded by the Arabidopsis EST are orthologues of maize Tha4 (Figure 1). Our data also support the idea that the Delta pH pathway generally employs at least two Hcf106-like proteins. Genetic studies in maize (
The antibodies against Hcf106 and Tha4 exerted a more complete block in Delta pH transport than would be expected from analyses of mutant plants. In the hcf106 null mutant, Delta pH substrates, OE23 and OE17, accumulate to varying degrees between 10 and 40% (see
There are several possible explanations for the difference between in vivo and in vitro data. One possibility is that in vivo, low transport levels may be compensated by reduced protein turnover and increased protein synthesis. In fact, pulse labeling of hcf106 maize leaves indicates that protein transport is more severely blocked than protein accumulation data suggest (
Another possibility is that Hcf106 and Tha4 are part of the same protein complex. Antibody binding to any of the components of a critical multimeric complex might disable the entire complex, whereas another family member might replace a missing component in vivo. The structural similarity of Tha4 and Hcf106 suggests that they perform highly related functions in the targeting/translocation process. In this regard, it is notable that most bacterial species and chloroplasts from at least three plant species have two Hcf106 homologues, suggesting the possibility that they function in heteromultimeric complexes (
SecY is an indispensable component for the bacterial Sec pathway (
There were at least two reasons to examine the involvement of the cpSecY in other thylakoid pathways. First is that SecYEG/Sec61 is considered to be a general conserved translocation channel (
A similarly important question regards the integration of the SRP substrate LHCP. In E. coli, in vivo and in vitro data indicate that for some precursors, SRP and Sec pathways converge at the translocon level, i.e., the SRP pathway employs SecA and SecY/E for translocation (
If the Sec translocon is not functional for the Delta pH and SRP pathways, it raises intriguing possibilities as to the identity and mode of action of such translocons. For the Delta pH pathway, Hcf106 and Tha4 could conceivably play a role in the translocation step. The topology of these proteins suggests that they serve as receptors for the pathway. However, evidence for that role is currently lacking. On the other hand, both proteins contain conserved acidic and proline residues in their transmembrane domains, suggesting that some of their function is conducted within the bilayer. A TatC homologue is another candidate for the Delta pH pathway translocon. TatC was identified as essential for Tat pathway transport (
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Acknowledgements |
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The authors thank Alice Barkan, Ralph Henry, and Mark Settles for critical review of this manuscript and helpful suggestions, and Drs. Hajime Tokuda and Ken-ichi Nishiyama for advice on immunodetection of cpSecY. The authors also thank Mike McCaffery for excellent technical assistance.
This work was supported in part by National Institutes of Health grant R01 GM46951 and National Science Foundation grant MCB-9419287 to K. Cline.
This manuscript is Florida Agricultural Experiment Station Journal series number R-06936.
Submitted: April 28, 1999; Revised: June 3, 1999; Accepted: June 7, 1999.
1.used in this paper: i, intermediate precursor form; LHCP, light-harvesting chlorophyll a/b protein; m, mature form; OE33, OE23, and OE17, 33-, 23-, and 17-kD subunits of the photosystem II oxygen-evolving complex; p, precursor form; PC, plastocyanin; PSI-N, N subunit of the photosystem I complex; PSII-T, T subunit of the photosystem II complex; SRP, signal recognition particle
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References |
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