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The twin-arginine translocation (Tat(1)) pathway is unique with respect to its property to translocate proteins in a fully folded conformation across ion-tight membranes. In chloroplasts and Gram-negative bacteria, Tat translocase consists of the integral subunits TatB and TatC, which are assumed to constitute the membrane receptor, and TatA, a bitopic membrane protein being responsible in a yet unknown manner for the membrane translocation step. Antibody inhibition of intrinsic thylakoidal TatA activity and recovery of transport by heterologously expressed, purified TatA allowed to exactly quantify the amount of TatA required to catalyse membrane transport of the model Tat substrate 16/23. We can show that TatA concentrations in the 100nM range are sufficient to efficiently catalyse membrane transport of the protein, which corresponds well to the amount of TatA identified in thylakoids. Furthermore, TatA shows cooperativity in its catalytic activity suggesting that Tat translocase operates as an allosteric enzyme complex., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The initial step in twin arginine translocation (Tat)-dependent thylakoid transport of the 16/23 chimera is the interaction of the protein with the lipid bilayer. It results in the formation of the early translocation intermediate Ti-1, which is represented by a protease-protected fragment of 14 kDa. Cys-scanning mutagenesis in combination with in thylakoido and liposome insertion assays was used to precisely map this membrane-interacting and protease-protected fragment within the 16/23 chimera. The fragment comprises 124 residues, which are provided both by the transit peptide (31 residues) and the mature protein (93 residues), demonstrating that the passenger protein directly participates in membrane binding. The implications of this finding on the mechanism of Tat-dependent protein transport are discussed.

In bacteria and chloroplasts, the Tat (twin arginine translocation) system is capable of translocating folded passenger proteins across the cytoplasmic and thylakoidal membranes, respectively. Transport depends on signal peptides that are characterized by a twin pair of arginine residues. The signal peptides are generally removed after transport by specific processing peptidases, namely the leader peptidase and the thylakoidal processing peptidase. To gain insight into the prerequisites for such signal peptide removal, we mutagenized the vicinity of thylakoidal processing peptidase cleavage sites in several thylakoidal Tat substrates. Analysis of these mutants in thylakoid transport experiments showed that the amino acid composition of both the C-terminal segment of the signal peptide and the N-terminal part of the mature protein plays an important role in the maturation process. Efficient removal of the signal peptide requires the presence of charged or polar residues within at least one of those regions, whereas increased hydrophobicity impairs the process. The relative extent of this effect varies to some degree depending on the nature of the precursor protein. Unprocessed transport intermediates with fully translocated passenger proteins are found in membrane complexes of high molecular mass, which presumably represent Tat complexes, as well as free in the lipid bilayer. This seems to indicate that the Tat substrates can be laterally released from the complexes prior to processing and that membrane transport and terminal processing of Tat substrates are independent processes.

In the thylakoid membrane of chloroplasts as well as in the cytoplasmic membrane of bacteria, the DeltapH/Tat-dependent protein transport pathway is responsible for the translocation of folded proteins. Using the chimeric 16/23 protein as model substrate in thylakoid transport experiments, we dissected the transport process into several distinct steps that are characterized by specific integral translocation intermediates. Formation of the early translocation intermediate Ti-1, which still exposes the N and the C terminus to the stroma, is observed with thylakoids pretreated with (i) solutions of chaotropic salts or alkaline pH, (ii) protease, or (iii) antibodies raised against TatA, TatB, or TatC. Membrane insertion takes place even into liposomes, demonstrating that proteinaceous components are not required. This suggests that Tat-dependent transport may be initiated by the unassisted insertion of the substrate into the lipid bilayer, and that interaction with the Tat translocase takes place only in later stages of the process.

Dual targeting of a nuclearly encoded protein into two different cell organelles is an exceptional event in eukaryotic cells. Yet, the frequency of such dual targeting is remarkably high in case of mitochondria and chloroplasts, the two endosymbiotic organelles of plant cells. In most instances, it is mediated by “ambiguous” transit peptides, which recognize both organelles as the target. A number of different approaches including in silico, in organello as well as both transient and stable in vivo assays are established to determine the targeting specificity of such transit peptides. In this review, we will describe and compare these approaches and discuss the potential role of this unusual targeting process. Furthermore, we will present a hypothetical scenario how dual targeting might have arisen during evolution. [ABSTRACT FROM AUTHOR]

Plant cells are unique as they carry two organelles of endosymbiotic origin, namely mitochondria and chloroplasts (plastids) which have specific but partially overlapping functions, e. g., in energy and redox metabolism. Despite housing residual genomes of limited coding capacity, most of their proteins are encoded in the nucleus, synthesized by cytosolic ribosomes and need to be transported "back" into the respective target organelle. While transport is in most instances strictly monospecific, a group of proteins carries "ambiguous" transit peptides mediating transport into both, mitochondria and plastids. However, such dual targeting is often disputed due to variability in the results obtained from different experimental approaches. We have therefore compared and evaluated the most common methods established to study protein targeting into organelles within intact plant cells. All methods are based on fluorescent protein technology and live cell imaging. For our studies, we have selected four candidate proteins with proven dual targeting properties and analyzed their subcellular localization in vivo utilizing four different methods (particle bombardment, protoplast transformation, Agrobacterium infiltration, and transgenic plants). Though using identical expression constructs in all instances, a given candidate protein does not always show the same targeting specificity in all approaches, demonstrating that the choice of method is important, and depends very much on the question to be addressed. [ABSTRACT FROM AUTHOR]

Plant cells harbor two types of endosymbiotic organelle: mitochondria and chloroplasts. As a consequence of endosymbiotic gene transfer, the majority of their proteins are encoded in the nucleus and post-translationally 're'-imported into the respective target organelle. The corresponding transport signals are usually selective for a single organelle, but several proteins are transported into both the mitochondria and chloroplasts., To estimate the number of proteins with such dual targeting properties in Arabidopsis, we classified the proteins encoded by nuclear genes of endosymbiotic origin according to the respective targeting specificity of their N-terminal transport signals as predicted by the TargetP software package. Selected examples of the resulting protein classes were subsequently analyzed by transient transformation assays as well as by in organello protein transport experiments., It was found that most proteins with high prediction values for both organelles show dual targeting with both experimental approaches. Unexpectedly, however, dual targeting was even found among those proteins that are predicted to be localized solely in one of the two endosymbiotic organelles. In total, among the 16 candidate proteins analyzed, we identified 10 proteins with dual targeting properties., This unexpectedly high proportion suggests that such transport properties are much more abundant than anticipated. [ABSTRACT FROM AUTHOR]

As a result of the endosymbiotic gene transfer, the majority of proteins of mitochondria and chloroplasts are encoded in the nucleus and synthesized in the cytosol as precursor proteins carrying N-terminal transport signals for the ‘re-import’ into the respective target organelle. Most of these transport signals are monospecific, although some of them have dual targeting properties, that is, they are recognized both by mitochondria and by chloroplasts as target organelles. We have identified alpha-MPP2, one of the two isoforms of the substrate binding subunit of mitochondrial processing peptidase of Arabidopsis thaliana, as a novel member of this class of nuclear-encoded organelle proteins. As demonstrated by in organello transport experiments with isolated organelles and by in vivo localization studies employing fluorescent chimeric reporter proteins, the N-terminal region of the alpha-MPP2 precursor comprises transport signals for the import into mitochondria as well as into chloroplasts. Both signals are found within the N-terminal 79 residues of the precursor protein, where they occupy partly separated and partly overlapping regions. Deletion mapping combined with in organello and in vivo protein transport studies demonstrate an unusual architecture of this transport signal, suggesting a composition of three functionally separated domains. [ABSTRACT FROM PUBLISHER]

As a result of the endosymbiotic gene transfer, the majority of proteins of mitochondria and chloroplasts is encoded in the nucleus and synthesized in the cytosol as precursor molecules carrying N-terminal transit peptides for the transport into the respective target organelle. In most instances, transport takes place into either mitochondria or chloroplasts, although a few examples of dual targeting into both organelles have been described. Here, we show by a combination of three different experimental strategies that also cytochrome c1 of potato, a component of the respiratory electron transport chain, is imported not only into mitochondria, but also into plastids. In organello import experiments with isolated mitochondria and chloroplasts, which were analyzed in both single and mixed organelle assays, demonstrate that the processing products accumulating after import within the two endosymbiotic organelles are different in size. Dual targeting of cytochrome c1 is observed also in vivo, after biolistic transformation of leaf epidermal cells with suitable reporter constructions. Finally, Western analyses employing cytochrome c1-specific antiserum provide evidence that the protein accumulates in significant amounts in mitochondria and chloroplasts of both pea and spinach. The possible consequences of our findings on the relevance of the dual targeting phenomenon are discussed. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM AUTHOR]

The CtpA protein in the cyanobacterium Synechocystis 6803 is a C-terminal processing protease that is essential for the assembly of the manganese cluster of the photosystem II complex. When fused to different chloroplast-targeting transit peptides, CtpA can be imported into isolated spinach chloroplasts and is subsequently translocated into the thylakoid lumen. Thylakoid transport is mediated by the cyanobacterial signal peptide which demonstrates that the protein transport machinery in thylakoid membranes is functionally conserved between chloroplasts and cyanobacteria. Transport of CtpA across spinach thylakoid membranes is affected by both nigericin and sodium azide indicating that the Seca protein and a transthylakoidal proton gradient are involved in this process. Saturation of the Sec-dependent thylakoid transport route by high concentrations of the precursor of the 33-kDa subunit of the oxygen-evolving system leads to a strongly reduced rate of thylakoid translocation of CtpA which demonstrates transport by the Sec pathway. However, thylakoid transport of CtpA is affected also by excess amounts of the 23-kDa subunit of the oxygen-evolving system, though to a lesser extent. This suggests that the cyanobacterial protein is capable of also interacing with components of the ΔpH-dependent route and that transport of a protein across the thylakoid membrane may not always be restricted to a single pathway. [ABSTRACT FROM AUTHOR]

Abstract: We have studied the membrane transport of the chimeric precursor protein 16/33, which is composed of the Tat1-specific transport signal of OEC16 and the Sec passenger protein OEC33, both subunits of the oxygen-evolving system associated with photosystem II. Protein transport experiments performed with isolated pea thylakoids show that the 16/33 chimera is transported in a strictly Tat-dependent manner into the thylakoid vesicles yielding mature OEC33 (mOEC33) in two different topologies. One fraction accumulates in the thylakoid lumen and is thus resistant to externally added protease. A second fraction is arrested during transport in an N-in/C-out topology within the membrane. Chase experiments demonstrate that this membrane-arrested mOEC33 moiety does not represent a translocation intermediate but instead an alternative end product of the transport process. Transport arrest of mOEC33, which is embedded in the membrane with a mildly hydrophobic protein segment, requires more than 26 additional and predominantly hydrophilic residues C-terminal of the membrane-embedded segment. Furthermore, it is stimulated by mutations which potentially affect the conformation of mOEC33 suggesting that at least partial folding of the passenger protein is required for complete membrane translocation. [Copyright &y& Elsevier]

Abstract: 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. [ABSTRACT FROM AUTHOR]