Insertion of plastidic ß-barrel proteins into the outer envelopes of plastids involves an intermembrane space intermediate formed with Toc75-V/OEP80.

The insertion of organellar membrane proteins with the correct topology requires the following: First, the proteins must contain topogenic signals for translocation across and insertion into the membrane. Second, proteinaceous complexes in the cytoplasm, membrane, and lumen of organelles are required to drive this process. Many complexes required for the intracellular distribution of membrane proteins have been described, but the signals and components required for the insertion of plastidic ß-barrel-type proteins into the outer membrane are largely unknown. The discovery of common principles is difficult, as only a few plastidic ß-barrel proteins exist. Here, we provide evidence that the plastidic outer envelope ß-barrel proteins OEP21, OEP24, and OEP37 from pea (Pisum sativum) and Arabidopsis thaliana contain information defining the topology of the protein. The information required for the translocation of pea proteins across the outer envelope membrane is present within the six N-terminal ß-strands. This process requires the action of translocon of the outer chloroplast (TOC) membrane. After translocation into the intermembrane space, ß-barrel proteins interact with TOC75-V, as exemplified by OEP37 and P39, and are integrated into the membrane. The membrane insertion of plastidic ß-barrel proteins is affected by mutation of the last ß-strand, suggesting that this strand contributes to the insertion signal. These findings shed light on the elements and complexes involved in plastidic ß-barrel protein import.

Membrane Extracts from Plant Tissues.

The comparison of isolated plant cell membranous enclosures can be hampered if their extraction method differs, e.g., in regard to the utilized buffers, the tissue, or the developmental stage of the plant. Thus, for comparable results, different cellular compartments should be isolated synchronously in one procedure. Here, we devise a workflow to isolate different organelles from one tissue, which is applicable to different eudicots such as Medicago x varia and Solanum lycopersicum. We describe this method for the isolation of different organelles from one plant tissue for the example of Arabidopsis thaliana. All compartments are retrieved by utilizing differential centrifugation with organelle-specific parameters.

The signal distinguishing between targeting of outer membrane ß-barrel protein to plastids and mitochondria in plants.

The proteome of the outer membrane of mitochondria and chloroplasts consists of membrane proteins anchored by α-helical or ß-sheet elements. While proteins with α-helical transmembrane domains are present in all cellular membranes, proteins with ß-barrel structure are specific for these two membranes. The organellar ß-barrel proteins are encoded in the nuclear genome and thus, have to be targeted to the outer organellar membrane where they are recognized by surface exposed translocation complexes. In the last years, the signals that ensure proper targeting of these proteins have been investigated as essential base for an understanding of the regulation of cellular protein distribution. However, the organellar ß-barrel proteins are unique as most of them do not contain a typical targeting information in form of an N-terminal cleavable targeting signal. Recently, it was discovered that targeting and surface recognition of mitochondrial ß-barrel proteins in yeast, humans and plants depends on the hydrophobicity of the last ß-hairpin of the ß-barrel. However, we demonstrate that the hydrophobicity is not sufficient for the discrimination of targeting to chloroplasts or mitochondria. By domain swapping between mitochondrial and chloroplast targeted ß-barrel proteins atVDAC1 and psOEP24 we demonstrate that the presence of a hydrophilic amino acid at the C-terminus of the penultimate ß-strand is required for mitochondrial targeting. A mutation of the chloroplast ß-barrel protein psOEP24 which mimics such profile is efficiently targeted to mitochondria. Thus, we present the properties of the signal for mitochondrial targeting of ß-barrel proteins in plants.

The intracellular distribution of the components of the GET system in vascular plants.

The guided entry of tail-anchored proteins (GET) pathway facilitates targeting and insertion of tail-anchored proteins into membranes. In plants, such a protein insertion machinery for the endoplasmic reticulum as well as constituents within mitochondrial and chloroplasts were discovered. Previous phylogenetic analysis revealed that Get3 sequences of Embryophyta form two clades representing cytosolic ("a") and organellar ("bc") GET3 homologs, respectively. Cellular fractionation of Arabidopsis thaliana seedlings and usage of the self-assembly GFP system in protoplasts verified the cytosolic (ATGet3a), plastidic (ATGet3b) and mitochondrial (ATGet3c) localization of the different homologs. The identified plant homologs of Get1 and Get4 in A. thaliana are localized in ER and cytosol, respectively, implicating a degree of conservation of the GET pathway in A. thaliana. Transient expression of Get3 homologs of Solanum lycopersicum, Medicago × varia or Physcomitrella patens with the self-assembly GFP technique in homologous and heterologous systems verified that multiple Get3 homologs with differing subcellular localizations are common in plants. Chloroplast localized Get3 homologs were detected in all tested plant systems. In contrast, mitochondrial localized Get3 homologs were not identified in S. lycopersicum, or P. patens, while we confirmed on the example of A. thaliana proteins that mitochondrial localized Get3 proteins are properly targeted in S. lycopersicum as well.