The delta pH‐driven and Sec‐related thylakoidal protein translocases recognise distinct types of thylakoid transfer signal, yet all transfer signals resemble bacterial signal peptides in structural terms. Comparison of known transfer signals reveals a single concrete difference: signals for the delta pH‐dependent system contain a common twin‐arginine motif immediately before the hydrophobic region. We show that this motif is critical for the delta pH‐driven translocation process; substitution of the arg‐arg by gln‐gln or even arg‐lys totally blocks translocation across the thylakoid membrane, and replacement by lys‐arg reduces the rate of translocation by > 100‐fold. The targeting information in this type of signal thus differs fundamentally from that of bacterial signal peptides, where the required positive charge can be supplied by any basic amino acid. Insertion of a twin‐arg motif into a Sec‐dependent substrate does not alter the pathway followed but reduces translocation efficiency, suggesting that the motif may also repel the Sec‐type system. Other information must help to specify the choice of translocation mechanism, but this information is unlikely to reside in the hydrophobic region because substitution by a hydrophobic section from an integral membrane protein does not affect the translocation pathway.

Mutant plastocyanins with Leu at position 10, 90 or 83 (Gly, Ala and Tyr respectively in wildtype) were constructed by site‐specific mutagenesis of the spinach gene, and expressed in transgenic potato plants under the control of the authentic plastocyanin promoter, as well as in Escherichia coli as truncated precursor intermediates carrying the C‐terminal 22 amino acid residues of the transit peptide, i.e. the thylakoid‐targeting domain that acts as a bacterial export signal. The identity of the purified plastocyanins was verified by matrix‐assisted laser desorption/ionization mass spectrometry. The formation of a complex between authentic or mutant spinach plastocyanin and isolated photosystem I and the electron transfer has been studied from the biphasic reduction kinetics of P700+ after excitation with laser flashes. The formation of the complex was abolished by the bulky hydrophobic group of Leu at the respective position of G10 or A90 which are part of the conserved flat hydrophobic surface around the copper ligand H87. The rate of electron transfer decreased by both mutations to < 20% of that found with wildtype plastocyanin. We conclude that the conserved flat surface of plastocyanin represents one of two crucial structural elements for both the docking at photosystem I and the efficient electron transfer via H87 to P700+. The Y83L mutant exhibited faster electron transfer to P700+ than did authentic plastocyanin. This proves that Y83 is not involved in electron transfer to P700 and suggests that electron transfer from cytochrome f and to P700 follows different routes in the plastocyanin molecule. Plastocyanin (Y83L) expressed in either E. coli or potato exhibited different isoelectric points and binding constants to photosystem I indicative of differences in the folding of the protein. The structure of the binding site at photosystem I and the mechanism of electron transfer are discussed.

The waxy (Wx) locus of Zea mays was cloned from strains carrying the wild‐type and wxm‐8 mutant alleles. The receptor component of the Suppressor‐Mutator (Spm) controlling element system in the wxm‐8 allele was shown to be a 2 kb long insertion within the transcribed region of the Wx gene. The insertion, termed Spm‐I8, is excised during somatic reversion events induced by the autonomous controlling element Enhancer (En), which is an equivalent to Spm. Integration of Spm‐I8 into the Wx gene generates a 3‐bp target site duplication. Spm‐I8 has a 13 bp long inverted repeat at its termini. The ends of the element can be further folded to build a large double‐stranded structure consisting of five perfectly matching double‐stranded regions of 9–13 bp in length, interrupted by single‐stranded loops. A comparison of the wild‐type and wxm‐8 alleles revealed two additional insertions 6 (insert‐1) and 0.25 (insert‐2) kb in length. No En‐induced excision of insert‐1 and insert‐2 could be detected so far. There is remarkable structure and sequence homology between Spm‐I8 and the transposable elements Tam1 and Tam2 of Antirrhinum majus at their termini, reflecting a possible evolutionary and/or functional relationship between transposons in different plant species.

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Stromules are stroma-filled tubules extending from plastids whose rapid extension toward or retraction from other plastids has suggested a role in interplastidic communication and exchange of metabolites. Several studies point to sporadic dilations, kinks, and branches occurring along stromule length but have not elucidated the underlying basis for these occurrences. Similarly, although specific details on interacting partners have been missing, a consensus viewpoint suggests that stromules increase the interactive surface of a plastid with its cytoplasmic surroundings. Here, using live imaging, we show that the behavior of dynamic, pleomorphic stromules strongly coincides with that of cortical endoplasmic reticulum (ER) tubules. Covisualization of fluorescent protein-highlighted stromules and the ER in diverse cell types clearly suggests correlative dynamics of the two membrane-bound compartments. The extension and retraction, as well as directional changes in stromule branches occur in tandem with the behavior of neighboring ER tubules. Three-dimensional and four-dimensional volume rendering reveals that stromules that extend into cortical regions occupy channels between ER tubules possibly through multiple membrane contact sites. Our observations clearly depict coincidental stromule-ER behavior and suggest that either the neighboring ER tubules shape stromules directly or the behavior of both ER and stromules is simultaneously dictated by a shared cytoskeleton-based mechanism. These new observations strongly implicate the ER membrane in interactions with stromules and suggest that their interacting surfaces might serve as major conduits for bidirectional exchange of ions, lipids, and metabolites between the two organelles.

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)

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Both in prokaryotic organisms and in chloroplasts, a specialized protein transport pathway exists which is capable of translocating proteins in a fully folded conformation. Transport is mediated in both instances by signal peptides harbouring a twin-arginine consensus motif (twin-arginine translocation (Tat) pathway). The Tat translocase comprises the three functionally different membrane proteins TatA, TatB, and TatC. While TatB and TatC are involved in the specific recognition of the substrate, TatA might be the major pore-forming component. Current evidence suggests that a functional Tat translocase is assembled from separate TatBC and TatA assemblies only on demand, i.e., in the presence of transport substrate and a transmembrane H+-motive force.