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The recognition of stromules as sporadically extended stroma filled tubules from all kinds of plastids constitutes one of the major insights that resulted from the direct application of green fluorescent protein aided imaging of living plant cells. Observations of dynamic green fluorescent stromules strongly suggested that plastids frequently interact with each other while photo-bleaching of interconnected plastids indicated that proteins can move within the stroma filled tubules. These observations readily fit into the prevailing concept of the endosymbiogenic origins of plastids and provided stromules the status of conduits for inter-plastid communication and macromolecule transfer. However, experimental evidence obtained recently through the use of photoconvertible protein labeled stromules strongly supports plastid independence rather than their interconnectivity. Additional information on stress conditions inducing stromules and observations on their alignment with other organelles suggests that the major role of stromules is to increase the interactive surface of a plastid with the rest of the cytoplasm.

Glucose‐fructose oxidoreductase (GFOR), a periplasmic protein of Zymomonas mobilis, is synthesized as a precursor polypeptide with a twin‐R signal peptide for Sec‐independent protein export in bacteria. In higher plant chloroplasts, twin‐R signal peptides are specific targeting signals for the Sec‐independent ΔpH pathway of the thylakoid membrane system. In agreement with the assumed common phylogenetic origin of the two protein transport mechanisms, GFOR can be efficiently translocated by the ΔpH‐dependent pathway when analyzed with isolated thylakoid membranes. Transport is sensitive to the ionophore nigericin and competes with specific substrates for the ΔpH‐dependent transport route. In contrast, neither sodium azide nor enzymatic destruction of the nucleoside triphosphates in the assays affects thylakoid transport of GFOR indicating that the Sec apparatus is not involved in this process. Mutagenesis of the twin‐R motif in the GFOR signal peptide prevents membrane translocation of the protein emphasizing the importance of these residues for the transport process.

During thylakoid transport of the chimeric precursor protein 16/23 which takes place by the twin arginine translocation (TAT) (Δph)‐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 Δph‐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.

The topology of the Rieske protein of the cytochrome b6/fcomplex in thylakoids from spinach chloroplasts was examined by protease protection experiments as well as polypeptide extraction assays using solutions of chaotropic salts or alkaline pH. While neither thermolysin nor trypsin cleave any of the Rieske protein when added to the stromal side of the thylakoid membrane, proteinase K is capable of removing approximately four residues from its NH2‐terminus. The protein is resistant to membrane extraction by 0.1 M Na2CO3or 2 M NaBr but is quantitatively released by 0.1 M NaOH. Treatment of thylakoids with 2 M NaSCN leads to extraction of variable amounts of the protein, depending on the presence or absence of sucrose in the medium which apparently stabilizes the cytochrome complex. From these results we conclude that the Rieske protein is an integral component of the cytochrome complex which spans the thylakoid membrane with a single hydrophobic segment and is anchored predominantly by electrostatic interactions.

Preproteins targeted to the Sec‐independent protein transport systems of plant thylakoids and of bacteria both have unusual transfer peptides bearing a consensus twin‐arginine motif. Possible mechanistic similarity between the two Sec‐independent transport pathways was investigated by assessing the ability of bacterial twin‐arginine transfer peptides to direct thylakoid import. High efficiency import was observed. This process was demonstrated to occur specifically via the Sec‐independent ΔpH pathway and to depend on an intact twin‐arginine motif on the transfer peptide. These results provide strong evidence for the operation of mechanistically related Sec‐independent protein transport pathways in chloroplasts and bacteria.

The spinach triose phosphate/phosphate translocator and the 37-kDa protein are both integral components of the chloroplast inner envelope membrane. They are synthesized in the cytosol with N-terminal extensions, the transit peptides, that are different in structural terms from those of imported stromal or thylakoid proteins. In order to determine if these N-terminal extensions are essential for the correct localization to the envelope membrane, they were linked to the mature parts of thylakoid membrane proteins, the light-harvesting chlorophyll a/bbinding protein and the CF0II-subunit of the thylakoid ATP synthase, respectively. In addition, the transit peptide of the CF0II-subunit that contains signals for the transport across both the envelope and the thylakoid membrane was fused to the mature parts of both envelope membrane proteins. The chimeric proteins were imported into isolated spinach chloroplasts, and the intraorganellar routing of the proteins was analyzed. The results obtained show that the N-terminal extensions of both envelope membrane proteins possess a stroma-targeting function only and that the information for the integration into the envelope membrane is contained in the mature parts of the proteins. At least part of the integration signal is provided by hydrophobic domains in the mature sequences since the removal of such a hydrophobic segment from the 37-kDa protein leads to missorting of the protein to the stroma and the thylakoid membrane.

The transit peptide of the waxy protein of maize which in the maize plant targets this protein only into amyloplasts was used for in vitro protein transport experiments with isolated amyloplasts from maize and chloroplasts from maize, pea and potato. In the presence of both intact and disrupted amyloplasts an artificial preprotein (TP30), consisting of the waxy transit peptide plus the first 34 amino acids of the mature waxy protein fused in-frame to the ß-glucuronidase of Escherichia coli, is processed to the size expected when the transit peptide is cleaved off. The chloroplasts studied show in vitro import and correct processing of both TP30 and the authentic waxy protein, but not of the ß-glucuronidase without the waxy transit peptide. The in vitro import of TP30 into chloroplasts is almost as efficient as that of the precursor of the small subunit of pea ribulose-1,5-bisphosphate carboxylase, a nuclear-encoded chloroplast protein, whereas the waxy protein accumulates to a lesser extent in the chloroplasts. Since the amino-terminal transit peptides of TP30 and the waxy precursor are the same, this difference must be due to the mature part of the waxy protein. One possible explanation is the observed instability of the waxy protein in the presence of chloroplasts.

A Sec-type system is responsible for the translocation of a subset of proteins across the thylakoid membrane in higher plant chloroplasts. Previous studies have suggested that the thylakoidal ΔpH plays a minor role in this translocation mechanism, but we show here that it can be essential for the translocation process, depending on the identity of the passenger protein and the concentration of ATP. Studies using chimeric proteins show that, whereas the presequence dictates the translocation pathway, the ΔpH requirement is dictated exclusively by the passenger protein; some passenger proteins are virtually ΔpH-independent whereas others are absolutely dependent. ΔpH requirement is not related to charge characteristics of the passenger proteins, ruling out an electrophoretic effect. Analysis of the 33-kDa photosystem II protein reveals an inverse relationship between ΔpH requirement and ATP concentration; import into isolated thylakoids is inhibited 14-fold by nigericin at moderate ATP concentrations, and totally inhibited when the ATP concentration is reduced to 2 μM. The results indicate that the roles of the ΔpH and ATP overlap and suggest that the ΔpH may be obligatory when the passenger protein is abnormally difficult to translocate, possibly due to the folding of the polypeptide chain. We compare the energetics of this system with those of prokaryotic systems from which the chloroplast system is believed to have evolved.

Thylakoid membranes of chloroplasts in higher plants harbor different pathways for the translocation of proteins. One of these routes is related to the prokaryotic Sec pathway, which mediates the secretion of particular proteins into the periplasmic space and involves the SecA protein as an essential component. We have isolated a full size cDNA of 3739 nucleotides encoding the SecA homologue from spinach. It contains an open reading frame of 1036 codons corresponding to a polypeptide with a calculated mass of 117 kDa. The deduced amino acid sequence shows between 43 and 49% identity to SecA proteins from bacteria and lower algae and 62% identity to SecA of the cyanobacterium Synechococcussp. PCC7942. Compared with the Escherichia coliprotein, spinach SecA carries an amino-terminal extension of approximately 80 residues. In organelloexperiments performed with the protein made in vitroby transcription of the cDNA and cell-free translation of the resulting RNA showed that this extension comprises a transit peptide that mediates the import of the protein into the chloroplast. The processed product of approximately 107 kDa accumulates predominantly in the stroma and to a lower extent associates with the thylakoid membrane. Comparably to E. coli,in which SecA activity can be inhibited by sodium azide, thylakoid translocation of a subset of lumenal proteins is sensitive to sodium azide in pea but not in spinach chloroplasts, suggesting that the latter contain an azide-resistant SecA variant.

The structure of the wild-type waxy (wx+) locus was determined by sequence analysis of both a genomic and an almost full-size cDNA clone. The coding region comprises 3,718 bp and is composed of 14 exons and 13 small introns. The exons and the promoter region are G/C rich (60%–80%). All three waxy transcripts analysed so far reveal different polyadenylation sites and corresponding polyadenylation signals. The smallest of these mRNAs has a size of 2,263 nucleotides. Northern blot analysis suggests that the tissue-specific expression of the locus is due to transcriptional control. The insertion sites of all transposable element induced waxy mutations analysed have been mapped precisely within the locus. N-terminal sequencing of the mature wx+ protein leads to the identification of a maize amyloplast-specific transit peptide of 72 aminoacid residues.

The transit peptide of the maize waxy protein (a nuclear-encoded amyloplast protein of the maize endosperm) was studied with respect to its role in subcellular protein targeting in transgenic potato plants. TP30, a chimeric precursor protein consisting of the waxy transit peptide and an additional 34 amino acids of the mature waxy protein fused to the ß-glucuronidase of Escherichia coli, was expressed in potato plants under the control of the 35S promoter of cauliflower mosaic virus. This fusion protein is imported not only into amyloplasts, the natural target organelles in the maize plant, but also into chloroplasts. In contrast, Gus, the ß-glucuronidase alone, which was also expressed in parallel experiments in transgenic potato plants is always found in the cytosol of the plant cells. As a consequence of the different subcellular locations of TP30 and Gus, we observed differences in the expression rates of the respective proteins in leaf cells, resulting in higher steady state levels of TP30 compared to Gus. In tuber cells, no correlation between intracellular location and expression of the proteins was found.

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 CFoII 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.

Transit peptides are N-terminal extensions that serve to route nuclearencoded proteins into chloroplasts. We have collected amino acid sequences for approximately 260 transit peptides, and have constructed a database that presently runs under FileMaker Plus on Macintosh personal computers. In addition, the collection is available as an ASCII-file, either in a tab-delimited format suitable for import into other database programs, or in a format with line-identifiers similar to the widely used Swiss Prot database. We hope that the plant molecular biology community will help us ensure that the collection is kept up to date by submitting new sequences directly to us.

As a consequence of the endosymbiotic gene transfer, most mitochondrial and chloroplastic proteins are nuclear encoded and synthesized in the cytosol as precursor proteins with transit peptides mediating transport to their subcellular destination. It is often assumed that these transit peptides are strictly monospecific for a single organelle. But in recent years more and more proteins have been identified which carry transit peptides that are capable of mediating transport into both mitochondria and chloroplasts. In a recent study we showed with a combination of in silico, in organello, and in vivo approaches that the frequency of such proteins is apparently much higher than usually anticipated.1Here we demonstrate with in organello competition experiments that the import of 2 of these dually targeted proteins (GrpE and EF-Tu) takes place by the same import pathways that are used by organelle proteins with “typical” monospecific targeting properties.