Targeting of proteins to the thylakoids by bipartite presequences: CFoII is imported by a novel, third pathway.

The CFoII subunit of the ATP synthase is an integral component of the thylakoid membrane which is synthesized in the cytosol with a bipartite, lumen-targeting presequence similar in structural terms to those of imported lumenal proteins such as plastocyanin. This presequence is shown to possess a terminal cleavage site for the thylakoidal processing peptidase, but no intermediate site for the stromal processing peptidase. The integration of CFoII into the thylakoid membrane of Pisum sativum has been analysed using in vitro assays for the import of proteins into intact chloroplasts or isolated thylakoids. Efficient integration into thylakoids is observed in the light and dark, and the integration process does not require the presence of either stromal extracts or nucleoside triphosphates. The uncoupler nigericin inhibits integration only very slightly, indicating that the thylakoidal delta pH does not play a significant role in the integration mechanism. In each of these respects, the requirements for CFoII integration differ notably from those determined for integration of the light-harvesting chlorophyll-binding protein of photosystem II. The integration mechanism also differs significantly from the two mechanisms involved in the translocation of lumenal proteins across the thylakoid membrane, since one of these processes requires the presence of stromal protein factors and ATP, and the other mechanism is dependent on the thylakoidal delta pH. This conclusion is reinforced by the finding that saturation of the translocation system for the precursor to the lumenal 23 kDa oxygen-evolving complex protein does not affect integration of CFoII into thylakoids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein-specific energy requirements for protein transport across or into thylakoid membranes. Two lumenal proteins are transported in the absence of ATP.

Cytosolically synthesized thylakoid proteins must be translocated across the chloroplast envelope membranes, traverse the stroma, and then be translocated into or across the thylakoid membrane. Protein transport across the envelope requires ATP hydrolysis but not electrical or proton gradients. The energy requirements for the thylakoid translocation step were studied here for the light-harvesting chlorophyll a/b protein (LHCP), an integral membrane protein, and for several thylakoid lumen-resident proteins: plastocyanin and OE33, OE23, and OE17 (the 33-, 23-, and 17-kDa subunits of the oxygen-evolving complex, respectively). Dissipation of the thylakoid protonmotive force during an in organello protein import assay partially inhibited the thylakoid localization of LHCP and OE33, totally inhibited localization of OE23 and OE17, and had no effect on localization of plastocyanin. We used reconstitution assays for LHCP insertion and for OE23 and OE17 transport into isolated thylakoids to investigate the energy requirements in detail. The results indicated that LHCP insertion absolutely requires ATP hydrolysis and is enhanced by a transthylakoid delta pH and that transport of OE23 and OE17 is absolutely dependent upon a delta pH. Surprisingly, OE23 and OE17 transport occurred maximally in the complete absence of ATP. These results establish the thylakoid membrane as the only membrane system in which a delta pH can provide all of the energy required to translocate proteins across the bilayer. They also demonstrate that the energy requirements for integration into or translocation across the thylakoid membranes are protein-specific.     

The thylakoid delta pH/delta psi are not required for the initial stages of Tat-dependent protein transport in tobacco protoplasts

The twin-arginine translocation (Tat) system transports folded proteins across the chloroplast thylakoid membrane and bacterial plasma membrane. In vitro import assays have pointed to a key role for the thylakoid delta pH in the initial assembly of the full translocon from two subcomplexes; more generally, the delta pH is believed to provide the overall driving force for translocation. Here, we have studied the role of the delta pH in vivo by analyzing the translocation of Tat substrates in transfected tobacco protoplasts. We show that the complete maturation of the precursor of the 23-kDa lumenal protein (pre-23K) and of a fusion of the 23K presequence linked to green fluorescent protein (pre-GFP) are unaffected by dissipation of the delta pH. High level expression of Tat substrates in protoplasts has recently been shown to result in "translocation reversal" in that a large proportion of a given substrate is partially translocated across the thylakoid membrane, processed to the mature size, and returned to the stroma. However, the efficiency of translocation of pre-23K is undiminished in the absence of the delta pH and/or delta psi, and the rate and extent of maturation of both pre-23K and pre-GFP by the lumen-facing processing peptidase is similarly unaffected. These data demonstrate that the proton motive force is not required for the functional assembly of the Tat translocon and the initial stages of translocation in higher plant chloroplasts in vivo. We conclude that unknown factors play an influential role in both the mechanism and energetics of this system under in vivo conditions.     

Import of the barley PSI-F subunit into the thylakoid lumen of isolated chloroplasts

A full-length cDNA clone encoding the PSI-F subunit of barley photosystem I has been isolated and sequenced. The open reading frame encodes a precursor polypeptide with a deduced molecular mass of 24837 Da. The barley PSI-F precursor contains a bipartite presequence with characteristics similar to the presequences of proteins destined to the thylakoid lumen. In vitro import studies demonstrate that an in vitro synthesized precursor is transported across the chloroplast envelope and directed to the thylakoid membrane, where it accumulates in a protease-resistant form. Incubation of the precursor with a chloroplast stromal extract results in processing to a form intermediate in size between the precursor and mature forms. Hydrophobicity analysis of the barley PSI-F protein reveals a hydrophobic region predicted to be a membrane spanning -helix. The hydrophobic nature of PSI-F combined with a bipartite presequence is unusual. We postulate that the second domain in the bipartite presequence of the PSI-F precursor proteins is required to ensure the proper orientation of PSI-F in the thylakoid membrane. The expression of the PsaF gene is light-induced similar to other barley photosystem I genes.Abbreviations 16K 23K and 33K proteins, the 16 kDa, 23 kDa and 33 kDa subunits of the photosystem II oxygen-evolving complex - PSI-N and PSI-F photosystem I subunit N and F - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

One signal is enough: Stepwise transport of two distinct passenger proteins by the Tat pathway across the thylakoid membrane

The twin-arginine translocation (Tat) pathway, one of four protein transport pathways operating at the thylakoid membrane of chloroplasts, shows remarkable substrate flexibility. Here, we have analyzed the thylakoid transport of chimeric tandem substrates that are composed of two different passenger proteins fused to a single Tat transport signal. The chimera 23/23-EGFP in which the reporter protein EGFP is connected to the C-terminus of the OEC23 precursor shows that a single Tat transport signal is sufficient to mediate transport of two distinct passenger proteins in a row. Replacing the transit peptide of OEC23 in 23/23-EGFP by its homolog from OEC16 yields the chimera 16/23-EGFP, which can likewise be fully translocated by the Tat pathway across the thylakoid membrane. However, transport of 16/23-EGFP is retarded at specific steps in the transport process leading to the temporary and consecutive accumulation of three translocation intermediates with distinct membrane topology. They are associated with two oligomeric membrane complexes presumably representing TatBC-receptor complexes. The composition of the translocation intermediates as determined by immunoprecipitation experiments suggests that the two passenger proteins are translocated in a stepwise manner across the membrane.     

A stromal pool of TatA promotes Tat-dependent protein transport across the thylakoid membrane

In chloroplasts and bacteria, the Tat (twin-arginine translocation) system is engaged in transporting folded passenger proteins across the thylakoid and cytoplasmic membranes, respectively. To date, three membrane proteins (TatA, TatB, and TatC) have been identified to be essential for Tat-dependent protein translocation in the plant system, whereas soluble factors seem not to be required. In contrast, in the bacterial system, several cytosolic chaperones were described to be involved in Tat transport processes. Therefore, we have examined whether stromal or peripherally associated membrane proteins also play a role in Tat transport across the thylakoid membrane. Analyzing both authentic precursors as well as the chimeric 16/23 protein, which allows us to study each step of the translocation process individually, we demonstrate that a soluble form of TatA is present in the chloroplast stroma, which significantly improves the efficiency of Tat-dependent protein transport. Furthermore, this soluble TatA is able to reconstitute the Tat transport properties of thylakoid membranes that are transport-incompetent due to extraction with solutions of chaotropic salts.     

Two distinct translocation intermediates can be distinguished during protein transport by the TAT (Deltaph) pathway across the thylakoid membrane.

During thylakoid transport of the chimeric precursor protein 16/23 which takes place by the twin arginine translocation (TAT) (Deltaph)-dependent pathway, two distinct translocation intermediates can be identified which represent successive steps in the translocation process. Both intermediates are partially inserted into the thylakoid membrane and can be distinguished by specific degradation fragments occurring after thermolysin treatment of the thylakoids. While the formation of the early translocation intermediate does not depend on a functional TAT translocation machinery, the appearance of the late intermediate is strictly coupled to the Deltaph-dependent transport of the 16/23 chimera. Accordingly, this translocation intermediate is found associated with two distinct complexes in the thylakoid membrane having apparent molecular masses of approximately 560 and 620 kDa, respectively.     

Full-length plastocyanin precursor is translocated across isolated thylakoid membranes

In higher plants, the chloroplastic protein plastocyanin is synthesized as a transit peptide-containing precursor by cytosolic ribosomes and posttranslationally transported to the thylakoid lumen. En route to the lumen, a plastocyanin precursor is first imported into chloroplasts and then further directed across the thylakoid membrane by a second distinct transport event. A partially processed form of plastocyanin is observed in the stroma during import experiments using intact chloroplasts and has been proposed to be the translocation substrate for the second step (Smeekens, S., Bauerle, C., Hageman, J., Keegstra, K., and Weisbeek, P. (1986) Cell 46, 365-375). To further characterize this second step, we have reconstituted thylakoid transport in a system containing in vitro-synthesized precursor proteins and isolated thylakoid membranes. This system was specific for lumenal proteins since stromal proteins lacking the appropriate targeting information did not accumulate in the thylakoid lumen. Plastocyanin precursor was taken up by isolated thylakoids, proteolytically processed to mature size, and converted to holo form. Translocation was temperature-dependent and was stimulated by millimolar levels of ATP but did not strictly require the addition of stromal factors. We have examined the substrate requirements of thylakoid translocation by testing the ability of different processed forms of plastocyanin to transport in the in vitro system. Interestingly, only the full-length plastocyanin precursor, not the partially processed intermediate form, was competent for transport in this in vitro system.     

Targeting of proteins to the thylakoid lumen by the bipartite transit peptide of the 33 kd oxygen-evolving protein.

Various chimeric precursors and deletions of the 33 kd oxygen-evolving protein (OEE1) were constructed to study the mechanism by which chloroplast proteins are imported and targeted to the thylakoid lumen. The native OEE1 precursor was imported into isolated chloroplasts, processed and localized in the thylakoid lumen. Replacement of the OEE1 transit peptide with the transit peptide of the small subunit of ribulose-1,5-bisphosphate carboxylase, a stromal protein, resulted in redirection of mature OEE1 into the stromal compartment of the chloroplast. Utilizing chimeric transit peptides and block deletions we demonstrated that the 85 residue OEE1 transit peptide contains separate signal domains for importing and targeting the thylakoid lumen. The importing domain, which mediates translocation across the two membranes of the chloroplast envelope, is present in the N-terminal 58 amino acids. The thylakoid lumen targeting domain, which mediates translocation across the thylakoid membrane, is located within the C-terminal 27 residues of the OEE1 transit peptide. Chimeric precursors were constructed and used in in vitro import experiments to demonstrate that the OEE1 transit peptide is capable of importing and targeting foreign proteins to the thylakoid lumen.     

Azide-sensitive thylakoid membrane insertion of chimeric cytochrome f polypeptides imported by isolated pea chloroplasts

Post-translational integration of cytochrome f into thylakoid membranes was observed after import by isolated pea chloroplasts of a chimeric protein consisting of the presequence of the small subunit of ribulose 1,5-bisphosphate carboxylase fused to the cytochrome f precursor. Import of a similar chimeric protein lacking the C-terminal 33 amino acid residues resulted in a soluble cytochrome f protein in the thylakoid lumen, indicating that the C-terminal region contains a stop-transfer sequence for membrane integration. Azide inhibited the insertion of cytochrome f into the thylakoid membrane, whereas the ionophores nigericin and valinomycin had little effect on membrane insertion. The precursor of the 33 kDa protein, but not the 23 kDa protein, of the photosystem II oxygen-evolving complex inhibited the thylakoid insertion of cytochrome f , suggesting competition for a component of the transport pathway. These experiments suggest that the post-translational insertion of cytochrome f into the thylakoid membrane uses a SecA-dependent pathway.     

Large-scale translocation reversal within the thylakoid Tat system in vivo

In vitro import assays have shown that the thylakoid twin-arginine translocase (Tat) system transports folded proteins in a unidirectional manner. Here, we expressed a natural substrate, pre-23K, and a 23K presequence-green fluorescent protein (GFP) chimera in vivo in tobacco protoplasts. Both are imported into chloroplasts, targeted to the thylakoids, and processed to the mature size by the lumen-facing processing peptidase. However, the vast majority of mature GFP and about half of the 23K are then returned to the stroma. Mutations in the twin-arginine motif block thylakoid targeting and maturation, confirming an involvement of the Tat apparatus. Mutation of the processing site yields membrane-associated intermediate-size protein in vivo, indicating a delayed reversal of translocation to the stroma and suggesting a longer lived interaction with the Tat machinery. We conclude that, in vivo, the Tat system can reject substrates at a late stage in translocation and on a very large scale, indicating the influence of factors that are absent in reconstitution assays.     

A proton gradient is required for the transport of two lumenal oxygen-evolving proteins across the thylakoid membrane.

The 33- and 23-kDa proteins of the photosynthetic oxygen-evolving complex are synthesized in the cytosol as larger precursors and transported into the thylakoid lumen via stromal intermediate forms. We have investigated the energetics of protein transport across the thylakoid membrane using import assays that utilize either intact chloroplasts or isolated thylakoids. We have found that the light-driven import of the 23-kDa protein into isolated thylakoids is almost completely inhibited by electron transport inhibitors or by the ionophore nigericin but not by valinomycin. These compounds have similar effects in chloroplast import assays: precursors of both the 33- and 23-kDa proteins are imported and processed to intermediate forms in the stroma, but transport into the thylakoid lumen is blocked when electron transport is inhibited or nigericin is present. These results indicate that the transport of these proteins across the thylakoid membrane requires a protonmotive force and that the dominant component in this respect is the proton gradient and not the electrical potential.     

Precursors of one integral and five lumenal thylakoid proteins are imported by isolated pea and barley thylakoids: optimisation of in vitro assays

In vitro assays for the import of proteins by isolated pea thylakoids have been refined and optimised with respect to (a) the method of thylakoid preparation, (b) the concentration of thylakoids in the import assay, and (c) the pH and temperature of the import assay. As a result, the 23 kDa and 16 kDa proteins of the photosynthetic oxygen-evolving complex are imported with efficiencies approaching 100%; import of the third oxygen-evolving complex protein is also observed, albeit with lower efficiencies. We have also demonstrated import of three further thylakoid proteins: plastocyanin, the CF_oII subunit of the ATP synthase, and the photosystem I subunit, PSI-N, using this import assay. Import of plastocyanin, PSI-N and the 33 kDa oxygen-evolving complex protein subunit requires the presence of stromal extract whereas the other three proteins are efficiently imported in the absence of added soluble proteins. Import into isolated barley thylakoids was achieved under identical assay conditions, although with somewhat lower efficiency than into pea thylakoids.     

Sequential translocation of an artificial precursor protein across the two mitochondrial membranes.

We have constructed a chimeric mitochondrial precursor protein consisting of a mutant bovine pancreatic trypsin inhibitor coupled to the C terminus of a purified artificial precursor protein. This construct fails to complete its import into isolated mitochondria and becomes stuck across sites of close contact between the two mitochondrial membranes. When the mitochondria are then depleted of ATP and the intramolecular disulfide bridges of the trypsin inhibitor are cleaved by dithiothreitol, the trypsin inhibitor moiety is transported across the outer membrane into the intermembrane space. This translocation intermediate can be chased across the inner membrane by restoring the ATP levels in the matrix. These results show that translocation of pancreatic trypsin inhibitor across a biological membrane is prevented by its intramolecular disulfide bridges, that import into the matrix involves two distinct translocation system operating in tandem, and that ATP is required for protein translocation across the inner but not the outer membrane.     

The role of the transit peptide in the routing of precursors toward different chloroplast compartments

The role of the transit peptide in the routing of imported proteins inside the chloroplast was investigated with chimeric proteins in which the transit peptides for the nuclear-encoded ferredoxin and plastocyanin precursors were exchanged. Import and localization experiments with a reconstituted chloroplast system show that the ferredoxin transit peptide directs mature plastocyanin away from its correct location, the thylakoid lumen, to the stroma. With the plastocyanin transit peptide-mature ferredoxin chimera, a processing intermediate is arrested on its way to the lumen. We propose a two domain hypothesis for the plastocyanin transit peptide: the first domain functions in the chloroplast import process, whereas the second is responsible for transport across the thylakoid membrane. Thus, the transit peptide not only targets proteins to the chloroplast, but also is a major determinant in their subsequent localization within the organelle.     

Genetic Interactions between a Pep7 Mutation and the Pep12 and Vps45 Genes: Evidence for a Novel Snare Component in Transport between the Saccharomyces Cerevisiae Golgi Complex and Endosome

A nuclear mutant of maize, tha1, which exhibited defects in the translocation of proteins across the thylakoid membrane, was described previously. A transposon insertion at the tha1 locus facilitated the cloning of portions of the tha1 gene. Strong sequence similarity with secA genes from bacteria, pea and spinach indicates that tha1 encodes a SecA homologue (cp-SecA). The tha1-ref allele is either null or nearly so, in that tha1 mRNA is undetectable in mutant leaves and cp-SecA accumulation is reduced >=40-fold. These results, in conjunction with the mutant phenotype described previously, demonstrate that cp-SecA functions in vivo to facilitate the translocation of OEC33, PSI-F and plastocyanin but does not function in the translocation of OEC23 and OEC16. Our results confirm predictions for cp-SecA function made from the results of in vitro experiments and establish several new functions for cp-SecA, including roles in the targeting of a chloroplast-encoded protein, cytochrome f, and in protein targeting in the etioplast, a nonphotosynthetic plastid type. Our finding that the accumulation of properly targeted plastocyanin and cytochrome f in tha1-ref thylakoid membranes is reduced only a few-fold despite the near or complete absence of cp-SecA suggests that cp-SecA facilitates but is not essential in vivo for their translocation across the membrane.     

Transport of proteins into chloroplasts. Import and maturation of precursors to the 33-, 23-, and 16-kDa proteins of the photosynthetic oxygen-evolving complex

The 33-, 23-, and 16-kDa proteins of the photosynthetic oxygen-evolving complex are synthesized as precursors in the cytoplasm and transported into the thylakoid lumen of higher plant chloroplasts. In this report we have analyzed the import and maturation of these precursors, using reconstituted protein import assays and partially purified preparations of the processing peptidases involved. Precursors of the 33- and 23-kDa proteins from Spinacia and Triticum aestivum are processed by a stromal peptidase to intermediate forms; polypeptides of similar size are observed during the transport of these precursors and possibly that of the 16-kDa protein, into isolated chloroplasts. Complete maturation of the 33- and 23-kDa proteins is carried out by a thylakoidal peptidase shown previously to be involved in plastocyanin biogenesis. The data support an import mechanism involving successive cleavages by the stromal and thylakoidal processing peptidases.     

Isolated plant mitochondria import chloroplast precursor proteins in vitro with the same efficiency as chloroplasts.

Most chloroplast and mitochondrial proteins are synthesized with N-terminal presequences that direct their import into the appropriate organelle. In this report we have analyzed the specificity of standard in vitro assays for import into isolated pea chloroplasts and mitochondria. We find that chloroplast protein import is highly specific because mitochondrial proteins are not imported to any detectable levels. Surprisingly, however, pea mitochondria import a range of chloroplast protein precursors with the same efficiency as chloroplasts, including those of plastocyanin, the 33-kDa photosystem II protein, Hcf136, and coproporphyrinogen III oxidase. These import reactions are dependent on the Deltaphi across the inner mitochondrial membrane, and furthermore, marker enzyme assays and Western blotting studies exclude any import by contaminating chloroplasts in the preparation. The pea mitochondria specifically recognize information in the chloroplast-targeting presequences, because they also import a fusion comprising the presequence of coproporphyrinogen III oxidase linked to green fluorescent protein. However, the same construct is targeted exclusively into chloroplasts in vivo indicating that the in vitro mitochondrial import reactions are unphysiological, possibly because essential specificity factors are absent in these assays. Finally, we show that disruption of potential amphipathic helices in one presequence does not block import into pea mitochondria, indicating that other features are recognized.     

A folded protein can be transported across the chloroplast envelope and thylakoid membranes.

Many thylakoid lumenal proteins are nuclear encoded, cytosolically synthesized, and reach their functional location after posttranslational targeting across two chloroplast envelope membranes and the thylakoid membrane via proteinaceous transport systems. To study whether these transmembrane transport machineries can translocate folded structures, we overexpressed the 17-kDa subunit of the oxygen-evolving complex of photosystem II (prOE17) that had been modified to contain a unique C-terminal cysteine. This allowed us to chemically link a terminal 6.5-kDa bovine pancreatic trypsin inhibitor (BPTI) moiety to prOE17 to create the chimeric protein prOE17-BPTI. Redox reagents and an irreversible sulfhydryl-specific cross-linker, bis-maleimidohexane, were used to manipulate the structure of BPTI. Import of prOE17-BPTI into isolated chloroplasts and thylakoids demonstrates that the small tightly folded BPTI domain is carried across both the chloroplast envelopes and the delta pH-dependent transmembrane transporter of the thylakoid membrane when linked to the correctly targeted OE17 precursor. Transport proceeded even when the BPTI moiety was internally cross-linked into a protease-resistant form. These data indicate that unfolding is not a ubiquitous requirement for protein translocation and that at least some domains of targeted proteins can maintain a nonlinear structure during their translocation into and within chloroplasts.