Probing the physiological role of the plastid outer-envelope membrane using the oemiR plasmid collection.

Plastids are the site of complex biochemical pathways, most prominently photosynthesis. The organelle evolved through endosymbiosis with a cyanobacterium, which is exemplified by the outer envelope membrane that harbors more than 40 proteins in Arabidopsis. Their evolutionary conservation indicates high significance for plant cell function. While a few proteins are well-studied as part of the protein translocon complex the majority of outer envelope protein functions is unclear. Gaining a deeper functional understanding has been complicated by the lack of observable loss-of-function mutant phenotypes, which is often rooted in functional genetic redundancy. Therefore, we designed outer envelope-specific artificial micro RNAs (oemiRs) capable of downregulating transcripts from several loci simultaneously. We successfully tested oemiR function by performing a proof-of-concept screen for pale and cold-sensitive mutants. An in-depth analysis of pale mutant alleles deficient in the translocon component TOC75 using proteomics provided new insights into putative compensatory import pathways. The cold stress screen not only recapitulated 3 previously known phenotypes of cold-sensitive mutants but also identified 4 mutants of additional oemiR outer envelope loci. Altogether our study revealed a role of the outer envelope to tolerate cold conditions and showcasts the power of the oemiR collection to research the significance of outer envelope proteins.

Chloroplast import of an intermembrane space protein is facilitated by translocon components Toc75 and Tic236.

Chloroplasts are divided into six subcompartments: the outer membrane, intermembrane space, and inner membrane of the envelope, the stroma, the thylakoid membrane, and the thylakoid lumen. Compared with our knowledge of protein import into other subcompartments, extremely little is known about how proteins are imported into the intermembrane space of the envelope. Tic22 was one of the first proteins identified as localizing to the intermembrane space and the only one for which import has been analyzed in some detail. However, conflicting results have been obtained concerning whether the general translocon is used to import Tic22 into the intermembrane space. Taking advantage of available translocon component mutants, we reanalyzed import of Tic22. We reveal reduced in vitro import of Tic22 preprotein (prTic22) into chloroplasts isolated from the Arabidopsis mar1 and tic236 mutants, which are functional knockdown mutants of the outer-membrane channel Toc75 and the intermembrane space linker Tic236, respectively. Import competition experiments also showed that prTic22 import was reduced by excess amounts of a stroma-targeted preprotein. Our results indicate that prTic22 uses at least part of the general translocon for import into the intermembrane space.

Toc75-V/OEP80 is processed during translocation into chloroplasts, and the membrane-embedded form exposes its POTRA domain to the intermembrane space.

The insertion of membrane proteins requires proteinaceous complexes in the cytoplasm, the membrane, and the lumen of organelles. Most of the required complexes have been described, while the components for insertion of ß-barrel-type proteins into the outer membrane of chloroplasts remain unknown. The same holds true for the signals required for the insertion of ß-barrel-type proteins. At present, only the processing of Toc75-III, the ß-barrel-type protein of the central chloroplast translocon with an atypical signal, has been explored in detail. However, it has been debated whether Toc75-V/ outer envelope protein 80 (OEP80), a second protein of the same family, contains a signal and undergoes processing. To substantiate the hypothesis that Toc75-V/OEP80 is processed as well, we reinvestigated the processing in a protoplast-based assay as well as in native membranes. Our results confirm the existence of a cleavable segment. By protease protection and pegylation, we observed intermembrane space localization of the soluble N-terminal domain. Thus, Toc75-V contains a cleavable N-terminal signal and exposes its polypeptide transport-associated domains to the intermembrane space of plastids, where it likely interacts with its substrates.

The POTRA domains of Toc75 exhibit chaperone-like function to facilitate import into chloroplasts.

Protein trafficking across membranes is an essential function in cells; however, the exact mechanism for how this occurs is not well understood. In the endosymbionts, mitochondria and chloroplasts, the vast majority of proteins are synthesized in the cytoplasm as preproteins and then imported into the organelles via specialized machineries. In chloroplasts, protein import is accomplished by the TOC (translocon on the outer chloroplast membrane) and TIC (translocon on the inner chloroplast membrane) machineries in the outer and inner envelope membranes, respectively. TOC mediates initial recognition of preproteins at the outer membrane and includes a core membrane channel, Toc75, and two receptor proteins, Toc33/34 and Toc159, each containing GTPase domains that control preprotein binding and translocation. Toc75 is predicted to have a ß-barrel fold consisting of an N-terminal intermembrane space (IMS) domain and a C-terminal 16-stranded ß-barrel domain. Here we report the crystal structure of the N-terminal IMS domain of Toc75 from Arabidopsis thaliana, revealing three tandem polypeptide transport-associated (POTRA) domains, with POTRA2 containing an additional elongated helix not observed previously in other POTRA domains. Functional studies show an interaction with the preprotein, preSSU, which is mediated through POTRA2-3. POTRA2-3 also was found to have chaperone-like activity in an insulin aggregation assay, which we propose facilitates preprotein import. Our data suggest a model in which the POTRA domains serve as a binding site for the preprotein as it emerges from the Toc75 channel and provide a chaperone-like activity to prevent misfolding or aggregation as the preprotein traverses the intermembrane space.

Toxoplasma gondii Toc75 Functions in Import of Stromal but not Peripheral Apicoplast Proteins.

Apicomplexa are unicellular parasites causing important human and animal diseases, including malaria and toxoplasmosis. Most of these pathogens possess a relict but essential plastid, the apicoplast. The apicoplast was acquired by secondary endosymbiosis between a red alga and a flagellated eukaryotic protist. As a result the apicoplast is surrounded by four membranes. This complex structure necessitates a system of transport signals and translocons allowing nuclear encoded proteins to find their way to specific apicoplast sub-compartments. Previous studies identified translocons traversing two of the four apicoplast membranes. Here we provide functional support for the role of an apicomplexan Toc75 homolog in apicoplast protein transport. We identify two apicomplexan genes encoding Toc75 and Sam50, both members of the Omp85 protein family. We localize the respective proteins to the apicoplast and the mitochondrion of Toxoplasma and Plasmodium. We show that the Toxoplasma Toc75 is essential for parasite growth and that its depletion results in a rapid defect in the import of apicoplast stromal proteins while the import of proteins of the outer compartments is affected only as the secondary consequence of organelle loss. These observations along with the homology to Toc75 suggest a potential role in transport through the second innermost membrane.

Evolution and targeting of Omp85 homologs in the chloroplast outer envelope membrane.

Translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75) is the core component of the chloroplast protein import machinery. It belongs to the Omp85 family whose members exist in various Gram-negative bacteria, mitochondria, and chloroplasts of eukaryotes. Chloroplasts of Viridiplantae contain another Omp85 homolog called outer envelope protein 80 (OEP80), whose exact function is unknown. In addition, the Arabidopsis thaliana genome encodes truncated forms of Toc75 and OEP80. Multiple studies have shown a common origin of the Omp85 homologs of cyanobacteria and chloroplasts but their results about evolutionary relationships among cyanobacterial Omp85 (cyanoOmp85), Toc75, and OEP80 are inconsistent. The bipartite targeting sequence-dependent sorting of Toc75 has been demonstrated but the targeting mechanisms of other chloroplast Omp85 homologs remain largely unexplored. This study was aimed to address these unresolved issues in order to further our understanding of chloroplast evolution. Sequence alignments and recently determined structures of bacterial Omp85 homologs were used to predict structures of chloroplast Omp85 homologs. The results enabled us to identify amino acid residues that may indicate functional divergence of Toc75 from cyanoOmp85 and OEP80. Phylogenetic analyses using Omp85 homologs from various cyanobacteria and chloroplasts provided strong support for the grouping of Toc75 and OEP80 sister to cyanoOmp85. However, this support was diminished when the analysis included Omp85 homologs from other bacteria and mitochondria. Finally, results of import assays using isolated chloroplasts support outer membrane localization of OEP80tr and indicate that OEP80 may carry a cleavable targeting sequence.

Structure and function of POTRA domains of Omp85/TPS superfamily.

The Omp85/TPS (outer-membrane protein of 85 kDa/two-partner secretion) superfamily is a ubiquitous and major class of ß-barrel proteins. This superfamily is restricted to the outer membranes of gram-negative bacteria, mitochondria, and chloroplasts. The common architecture, with an N-terminus consisting of repeats of soluble polypeptide-transport-associated (POTRA) domains and a C-terminal ß-barrel pore is highly conserved. The structures of multiple POTRA domains and one full-length TPS protein have been solved, yet discovering roles of individual POTRA domains has been difficult. This review focuses on similarities and differences between POTRA structures, emphasizing POTRA domains in autotrophic organisms including plants and cyanobacteria. Unique roles, specific for certain POTRA domains, are examined in the context of POTRA location with respect to their attachment to the ß-barrel pore, and their degree of biological dispensability. Finally, because many POTRA domains may have the ability to interact with thousands of partner proteins, possible modes of these interactions are also explored.

Two distinct Omp85 paralogs in the chloroplast outer envelope membrane are essential for embryogenesis in Arabidopsis thaliana.

Homologs of a bacterial beta-barrel protein, Omp85, ubiquitously exist in the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts. Those in non-photosynthetic bacteria and mitochondria are responsible for beta-barrel protein sorting to the outer membranes, and thus are essential for viability of the organisms. There are two distinct Omp85 homologs in chloroplasts of the model plant, Arabidopsis thaliana. One of them, Toc75, functions as the main protein import translocation channel, and was shown to be indispensable from a very early stage of embryogenesis. By contrast, the role of another homolog, OEP80, remains elusive. Recently, we showed that disruption of the OEP80 gene causes embryo abortion in A. thaliana at a stage later than that affected by TOC75 knockout. This indicates that the two chloroplastic Omp85 homologs are both essential for viability of plants from very early stages of development, but may have distinct functions. Defining the functional and evolutionary relationships of Toc75 and OEP80 by further studies should advance our understanding of the importance of plastids during embryogenesis, as well as that of the molecular details of plastid biogenesis.