Targeting specificity of nuclear-encoded organelle proteins with a self-assembling split-fluorescent protein toolkit.

A large number of nuclear-encoded proteins are targeted to the organelles of endosymbiotic origin, namely mitochondria and plastids. To determine the targeting specificity of these proteins, fluorescent protein tagging is a popular approach. However, ectopic expression of fluorescent protein fusions commonly results in considerable background signals and often suffers from the large size and robust folding of the reporter protein, which may perturb membrane transport. Among the alternative approaches that have been developed in recent years, the self-assembling split-fluorescent protein (sasplit-FP) technology appears particularly promising to analyze protein targeting specificity in vivo Here, we improved the sensitivity of this technology and systematically evaluated its utilization to determine protein targeting to plastids and mitochondria. Furthermore, to facilitate high-throughput screening of candidate proteins we developed a Golden Gate-based vector toolkit (PlaMinGo). As a result of these improvements, dual targeting could be detected for a number of proteins that had earlier been characterized as being targeted to a single organelle only. These results were independently confirmed with a plant phenotype complementation approach based on the immutans mutant.This article has an associated First Person interview with the first author of the paper.

Rather rule than exception? How to evaluate the relevance of dual protein targeting to mitochondria and chloroplasts.

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.

Tat transport of a Sec passenger leads to both completely translocated as well as membrane-arrested passenger proteins.

We have studied the membrane transport of the chimeric precursor protein 16/33, which is composed of the Tat(1)-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.

Enough is enough: TatA demand during Tat-dependent protein transport.

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.

The exception proves the rule? Dual targeting of nuclear-encoded proteins into endosymbiotic organelles.

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.

Fluorescent Protein-Based Approaches for Subcellular Protein Localization in Plants.

Fluorescent proteins (FPs) remarkably advanced the study of cellular biology of plants. The most common application is their use as reporter proteins to determine the subcellular localization of a protein of interest (POI) by endogenous expression of a suitable FP-POI fusion construct in plant cells. In this chapter we describe three approaches, namely, particle bombardment, protoplast transformation, and Agrobacterium infiltration, to transiently express such fusion constructs in plant cells of different species. These approaches are versatile and can be utilized for diverse fluorescent protein-based applications.

Preparation of physiologically active inside-out vesicles from plant inner mitochondrial membranes.

For many metabolites, the major barrier between cytosol and mitochondrial matrix is the inner membrane of mitochondria, the site of the respiratory electron transport chain. In consequence, it houses numerous transporters which facilitate the controlled exchange of metabolites, ions, and even proteins between these cellular compartments. While their import into the organelle can be studied with isolated mitochondria or mitoplasts, the analysis of their export from the matrix into the intermembrane space or even the cytosol demands for more sophisticated approaches. Among those, inside-out inner membrane vesicles are particularly useful, since they allow the direct presentation of the potential export substrates to the membrane without prior import into the organelle. Here we present a protocol for the isolation of such inside-out vesicles of the inner membrane of plant mitochondria based on repeated freeze/thaw-cycles of freshly prepared mitoplasts. Electron microscopy and Western analysis could show that the majority of the vesicles have single envelope membranes in an inside-out topology. The vesicles are furthermore physiologically active, as demonstrated by assays measuring the enzymatic activities of Complex I (NADH dehydrogenase), Complex V (ATP synthase) and the mitochondrial processing peptidase (MPP) associated with Complex III. Hence, the method presented here provides a good basis for further studies of the inner mitochondrial membrane and mitochondrial export processes.

A high-definition native polyacrylamide gel electrophoresis system for the analysis of membrane complexes.

Native polyacrylamide gel electrophoresis (PAGE) is an important technique for the analysis of membrane protein complexes. A major breakthrough was the development of blue native (BN-) and high resolution clear native (hrCN-) PAGE techniques. Although these techniques are very powerful, they could not be applied to all systems with the same resolution. We have developed an alternative protocol for the analysis of membrane protein complexes of plant chloroplasts and cyanobacteria, which we termed histidine- and deoxycholate-based native (HDN-) PAGE. We compared the capacity of HDN-, BN- and hrCN-PAGE to resolve the well-studied respiratory chain complexes in mitochondria of bovine heart muscle and Yarrowia lipolytica, as well as thylakoid localized complexes of Medicago sativa, Pisum sativum and Anabaena sp. PCC7120. Moreover, we determined the assembly/composition of the Anabaena sp. PCC7120 thylakoids and envelope membranes by HDN-PAGE. The analysis of isolated chloroplast envelope complexes by HDN-PAGE permitted us to resolve complexes such as the translocon of the outer envelope migrating at approximately 700 kDa or of the inner envelope of about 230 and 400 kDa with high resolution. By immunodecoration and mass spectrometry of these complexes we present new insights into the assembly/composition of these translocation machineries. The HDN-PAGE technique thus provides an important tool for future analyses of membrane complexes such as protein translocons.

Interaction of a Tat substrate and a Tat signal peptide with thylakoid lipids at the air-water interface.

The Tat machinery enables folded proteins to be translocated across biological membranes. In vitro studies have shown that Tat substrates can interact with membranes prior to translocation. In this study we investigated the initial states of this interaction with thylakoid lipid monolayers at the air-water interface by using monolayer techniques combined with infrared reflection-absorption spectroscopy (IRRAS). We used enhanced green fluorescent protein (EGFP) as a model substrate and the signal peptide SP16 from the 16 kDa protein of the spinach oxygen-evolving complex (OEC16). We found that the signal peptide is essential for the interaction of the model substrate with lipid monolayers. IRRA spectroscopy showed an increased amount of α-helical secondary structure elements for the chimeric model substrate i16/EGFP (SP16 fused to EGFP) compared with EGFP; this can be attributed to the signal peptide.

Plastid stromule branching coincides with contiguous endoplasmic reticulum dynamics.

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.

Induction of stromule formation by extracellular sucrose and glucose in epidermal leaf tissue of Arabidopsis thaliana.

BACKGROUND: Stromules are dynamic tubular structures emerging from the surface of plastids that are filled with stroma. Despite considerable progress in understanding the importance of certain cytoskeleton elements and motor proteins for stromule maintenance, their function within the plant cell is still unknown. It has been suggested that stromules facilitate the exchange of metabolites and/or signals between plastids and other cell compartments by increasing the cytosolically exposed plastid surface area but experimental evidence for the involvement of stromules in metabolic processes is not available. The frequent occurrence of stromules in both sink tissues and heterotrophic cell cultures suggests that the presence of carbohydrates in the extracellular space is a possible trigger of stromule formation. We have examined this hypothesis with induction experiments using the upper epidermis from rosette leaves of Arabidopsis thaliana as a model system. RESULTS: We found that the stromule frequency rises significantly if either sucrose or glucose is applied to the apoplast by vacuum infiltration. In contrast, neither fructose nor sorbitol or mannitol are capable of inducing stromule formation which rules out the hypothesis that stromule induction is merely the result of changes in the osmotic conditions. Stromule formation depends on translational activity in the cytosol, whereas protein synthesis within the plastids is not required. Lastly, stromule induction is not restricted to the plastids of the upper epidermis but is similarly observed also with chloroplasts of the palisade parenchyma. CONCLUSIONS: The establishment of an experimental system allowing the reproducible induction of stromules by vacuum infiltration of leaf tissue provides a suitable tool for the systematic analysis of conditions and requirements leading to the formation of these dynamic organelle structures. The applicability of the approach is demonstrated here by analyzing the influence of apoplastic sugar solutions on stromule formation. We found that only a subset of sugars generated in the primary metabolism of plants induce stromule formation, which is furthermore dependent on cytosolic translational activity. This suggests regulation of stromule formation by sugar sensing mechanisms and a possible role of stromules in carbohydrate metabolism and metabolite exchange.

Soluble TatA forms oligomers that interact with membranes: Structure and insertion studies of a versatile protein transporter.

The twin-arginine translocase (Tat) mediates the transport of already-folded proteins across membranes in bacteria, plants and archaea. TatA is a small, dynamic subunit of the Tat-system that is believed to be the active component during target protein translocation. TatA is foremost characterized as a bitopic membrane protein, but has also been found to partition into a soluble, oligomeric structure of yet unknown function. To elucidate the interplay between the membrane-bound and soluble forms we have investigated the oligomers formed by Arabidopsis thaliana TatA. We used several biophysical techniques to study the oligomeric structure in solution, the conversion that takes place upon interaction with membrane models of different compositions, and the effect on bilayer integrity upon insertion. Our results demonstrate that in solution TatA oligomerizes into large objects with a high degree of ordered structure. Upon interaction with lipids, conformational changes take place and TatA disintegrates into lower order oligomers. The insertion of TatA into lipid bilayers causes a temporary leakage of small molecules across the bilayer. The disruptive effect on the membrane is dependent on the liposome's negative surface charge density, with more leakage observed for purely zwitterionic bilayers. Overall, our findings indicate that A. thaliana TatA forms oligomers in solution that insert into bilayers, a process that involves reorganization of the protein oligomer.

Tat subunit stoichiometry in Arabidopsis thaliana challenges the proposed function of TatA as the translocation pore.

The twin arginine translocation (Tat) machinery which is capable of transporting folded proteins across lipid bilayers operates in the thylakoid membrane of plant chloroplasts as well as in the cytoplasmic membrane of bacteria. It is composed of three integral membrane proteins (TatA, TatB, and TatC) which form heteromeric complexes of high molecular weight that accomplish binding and transport of substrates carrying Tat pathway-specific signal peptides. Western analyses using affinity purified antibodies showed in both, juvenile and adult tissue from Arabidopsis thaliana, an approximately equimolar ratio of the TatB and TatC components, whereas TatA was detectable only in minor amounts. Upon Blue Native-PAGE, TatB and TatC were found in four heteromeric TatB/C complexes possessing molecular weights of approximately 310, 370, 560 and 620 kDa, respectively, while TatA was detected only in a molecular weight range below 200 kDa. The implications of these findings on the currently existing models explaining the mechanism of Tat transport are discussed.

Diversity in degrees of freedom of mitochondrial transit peptides.

Most mitochondrial proteins are synthesized in the cytosol of eukaryotic cells as precursor proteins carrying N-terminal extensions called transit peptides or presequences, which mediate their specific transport into mitochondria. However, plant cells possess a second potential target organelle for such transit peptides, the chloroplast. It can therefore be assumed that mitochondrial transit peptides in plants are exposed to an increased demand of specificity, which in turn leads to reduced degrees of freedom in these transit peptides compared with those of nonplant organisms. Our study investigates this hypothesis using fractal dimension. Statistical analysis of sequence data shows that the fractal dimension of mitochondrial transit peptides in plants is indeed significantly lower than that from nonplant organisms.

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.

Twin arginine translocation (Tat)-dependent protein transport: the passenger protein participates in the initial membrane binding step.

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.

Functional reconstitution of TatB into the thylakoidal Tat translocase.

We have established an experimental system for the functional analysis of thylakoidal TatB, a component of the membrane-integral TatBC receptor complex of the thylakoidal Twin-arginine protein transport (Tat) machinery. For this purpose, the intrinsic TatB activity of isolated pea thylakoids was inhibited by affinity-purified antibodies and substituted by supplementing the assays with TatB protein either obtained by in vitro translation or purified after heterologous expression in E. coli. Tat transport activity of such reconstituted thylakoids, which was analysed with the authentic Tat substrate pOEC16, reached routinely 20-25% of the activity of mock-treated thylakoid vesicles analysed in parallel. In contrast, supplementation of the assays with the purified antigen comprising all but the N-terminal transmembrane helix of thylakoidal TatB did not result in Tat transport reconstitution which confirms that transport relies strictly on the activity of the TatB protein added and is not due to restoration of the intrinsic TatB activity by antibody release. Unexpectedly, even a mutated TatB protein (TatB,E10C) assumed to be incapable of assembling into the TatBC receptor complex showed low but considerable transport reconstitution underlining the sensitivity of the approach and its suitability for further functional analyses of protein variants. Finally, quantification of TatB demand suggests that TatA and TatB are required in approximately equimolar amounts to achieve Tat-dependent thylakoid transport.

Dual targeting of TatA points to a chloroplast-like Tat pathway in plant mitochondria.

The biogenesis of membrane-bound electron transport chains requires membrane translocation pathways for folded proteins carrying complex cofactors, like the Rieske Fe/S proteins. Two independent systems were developed during evolution, namely the Twin-arginine translocation (Tat) pathway, which is present in bacteria and chloroplasts, and the Bcs1 pathway found in mitochondria of yeast and mammals. Mitochondria of plants carry a Tat-like pathway which was hypothesized to operate with only two subunits, a TatB-like protein and a TatC homolog (OrfX), but lacking TatA. Here we show that the nuclearly encoded TatA from pea has dual targeting properties, i.e., it can be imported into both, chloroplasts and mitochondria. Dual targeting of TatA was observed with in organello experiments employing chloroplasts and mitochondria isolated from pea as well as after transient expression of suitable reporter constructs in leaf tissue from pea and Nicotiana benthamiana. The extent of transport of these constructs into mitochondria of transiently transformed leaf cells was relatively low, causing a demand for highly sensitive methods to be detected, like the sasplitGFP approach. Yet, the dual import of TatA into mitochondria and chloroplasts observed here points to a common mechanism of Tat transport for folded proteins within both endosymbiotic organelles in plants.

Dual or Not Dual?-Comparative Analysis of Fluorescence Microscopy-Based Approaches to Study Organelle Targeting Specificity of Nuclear-Encoded Plant Proteins.

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.

Structure and dynamics of plant TatA in micelles and lipid bilayers studied by solution NMR.

The twin-arginine translocase (Tat) transports folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. In Gram-negative bacteria and chloroplasts, the translocon consists of three subunits, TatA, TatB, and TatC, of which TatA is responsible for the actual membrane translocation of the substrate. Herein we report on the structure, dynamics, and lipid interactions of a fully functional C-terminally truncated 'core TatA' from Arabidopsis thaliana using solution-state NMR. Our results show that TatA consists of a short N-terminal transmembrane helix (TMH), a short connecting linker (hinge) and a long region with propensity to form an amphiphilic helix (APH). The dynamics of TatA were characterized using 15 N relaxation NMR in combination with model-free analysis. The TMH has order parameters characteristic of a well-structured helix, the hinge is somewhat less rigid, while the APH has lower order parameters indicating structural flexibility. The TMH is short with a surprisingly low protection from solvent, and only the first part of the APH is protected to some extent. In order to uncover possible differences in TatA's structure and dynamics in detergent compared to in a lipid bilayer, fast-tumbling bicelles and large unilamellar vesicles were used. Results indicate that the helicity of TatA increases in both the TMH and APH in the presence of lipids, and that the N-terminal part of the TMH is significantly more rigid. The results indicate that plant TatA has a significant structural plasticity and a capability to adapt to local environments.