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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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]

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 15N 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. [ABSTRACT FROM AUTHOR]

Histidine-Proline-rich Glycoprotein (HPRG) is a plasma protein of vertebrates and several marine bivalves. Due to its multidomain structure consisting of several regions HPRG can interact with a variety of ligands, however the exact physiological role has not been discovered yet. Past purification approaches out of plasma or serum often led to co-purification of other proteins so that for a profound understanding of the function it is important to obtain a protein of high purity. Recent purification strategies were based upon metale chelate affinity chromatography followed by anion exchange chromatography or size exclusion chromatography, respectively. A large amount of serum albumin, the major plasma protein, also elutes from metale chelate affinity chromatography columns. Separation of rabbit HPRG from rabbit serum albumin could not be achieved via the above named methods by us. We present a method of purification of rabbit serum HPRG by means of metal affinity chromatography and preparative gel electrophoresis, which makes it possible to obtain HPRG practically devoid of impurities as assessed by mass spectrometry analysis. Moreover, we characterize the amount of glycosylation of HPRG and–to the best of our knowledge for the first time–the glycosylation pattern of rabbit HPRG. [ABSTRACT FROM AUTHOR]

Proton gradients are fundamental to chloroplast function. Across thylakoid membranes, the light induced -proton gradient is essential for ATP synthesis. As a result of proton pumping into the thylakoid lumen, an alkaline stromal pH develops, which is required for full activation of pH-dependent Calvin Benson cycle enzymes. This implies that a pH gradient between the cytosol (pH 7) and the stroma (pH 8) is established upon illumination. To maintain this pH gradient chloroplasts actively extrude protons. More than 30 years ago it was already established that these proton fluxes are electrically counterbalanced by Mg2+, K+, or Cl- fluxes, but only recently the first transport systems that regulate the pH gradient were identified. Notably several (Na+,K+)/H+ antiporter systems where identified, that play a role in pH gradient regulation, ion homeostasis, osmoregulation, or coupling of secondary active transport. The established pH gradients are important to drive uptake of essential ions and solutes, but not many transporters involved have been identified to date. In this mini review we summarize the current status in the field and the open questions that need to be addressed in order to understand how pH gradients are maintained, how this is interconnected with other transport processes and what this means for chloroplast function. [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]

Transmitted light and fluorescence microscopy often suffers from the limited Depth of Field (DOF) of microscope objectives. For this reason, plant cells can in most cases not completely be visualized in a single image at suitable magnification. In case of fluorescence signals, this problem can to some extent be circumvented by the use of confocal laser scanning microscopy (CLSM) which is able to remove disturbing light that is out of focus, thus leading to a clear image within the focal plane. Several of such confocal images can then be combined to provide a virtual image pretending a greater DOF. However, this trick is not possible for transmitted bright field microscopy, not even if CLSM operating in the imaging mode is applied. Instead, stacking software packages, which are based on algorithms capable of recognizing those parts of an image that are in focus, might provide an alternative approach to overcome this problem. Here we show that such software packages can be used to assemble mathematically the focal information of a z-stack of arbitrary microscope pictures into a virtual single image. This virtual image shows an enhanced DOF irrespective of whether the original data are epifluorescence or transmitted light microscopy images of plant cells and if they were obtained by conventional microscopy or CLSM. This method thus provides an affordable alternative to confocal image analysis and a meaningful tool in plant cell imaging. Furthermore, this method is even suitable to visualize organelle movement within a single image. [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]

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. [ABSTRACT FROM AUTHOR]