Chloroplast outer envelope protein P39 in Arabidopsis thaliana belongs to the Omp85 protein family.

Proteins of the Omp85 family chaperone the membrane insertion of ß-barrel-shaped outer membrane proteins in bacteria, mitochondria, and probably chloroplasts and facilitate the transfer of nuclear-encoded cytosolically synthesized preproteins across the outer envelope of chloroplasts. This protein family is characterized by N-terminal polypeptide transport-associated (POTRA) domains and a C-terminal membrane-embedded ß-barrel. We have investigated a recently identified Omp85 family member of Arabidopsis thaliana annotated as P39. We show by in vitro and in vivo experiments that P39 is localized in chloroplasts. The electrophysiological properties of P39 are consistent with those of other Omp85 family members confirming the sequence based assignment of P39 to this family. Bioinformatic analysis showed that P39 lacks any POTRA domain, while a complete 16 stranded ß-barrel including the highly conserved L6 loop is proposed. The electrophysiological properties are most comparable to Toc75-V, which is consistent with the phylogenetic clustering of P39 in the Toc75-V rather than the Toc75-III branch of the Omp85 family tree. Taken together P39 forms a pore with Omp85 family protein characteristics. The bioinformatic comparison of the pore region of Toc75-III, Toc75-V, and P39 shows distinctions of the barrel region most likely related to function. Proteins 2017; 85:1391-1401. © 2014 Wiley Periodicals, Inc.

N-terminal lysines are essential for protein translocation via a modified ERAD system in complex plastids.

Nuclear-encoded pre-proteins being imported into complex plastids of red algal origin have to cross up to five membranes. Thereby, transport across the second outermost or periplastidal membrane (PPM) is facilitated by SELMA (symbiont-specific ERAD-like machinery), an endoplasmic reticulum-associated degradation (ERAD)-derived machinery. Core components of SELMA are enzymes involved in ubiquitination (E1-E3), a Cdc48 ATPase complex and Derlin proteins. These components are present in all investigated organisms with four membrane-bound complex plastids of red algal origin, suggesting a ubiquitin-dependent translocation process of substrates mechanistically similar to the process of retro-translocation in ERAD. Even if, according to the current model, translocation via SELMA does not end up in the classical poly-ubiquitination, transient mono-/oligo-ubiquitination of pre-proteins might be required for the mechanism of translocation. We investigated the import mechanism of SELMA and were able to show that protein transport across the PPM depends on lysines in the N-terminal but not in the C-terminal part of pre-proteins. These lysines are predicted to be targets of ubiquitination during the translocation process. As proteins lacking the N-terminal lysines get stuck in the PPM, a 'frozen intermediate' of the translocation process could be envisioned and initially characterized.

The Omp85-type outer membrane protein p36 of Arabidopsis thaliana evolved by recent gene duplication.

Proteins of the Omp85 family are involved in the insertion of ß-barrel shaped outer membrane proteins in bacteria and mitochondria, and-at least-in the transfer of preproteins across the chloroplast outer envelope. In general these proteins consist of up to five N-terminal "polypeptide transport associated" (POTRA) domains and a C-terminal, membrane embedded ß-barrel domain. In Arabidopsis thaliana two plastidic gene families coding for Omp85-like proteins exist, namely the Toc75-III and the Toc75-V/Oep80 sub-family. The latter is composed of three genes, of which two do not contain POTRA domains. These are annotated as P39 and P36. However, P36 resulted from a very recent gene duplication of P39 and appears to be specific to Arabidopsis thaliana. Furthermore, we show that P39 is specifically expressed in vein tissues, while P36 is expressed at early and late developmental stages. T-DNA insertion in P36 causes a mild phenotype with reduced starch accumulation in chloroplasts of sepals pointing towards a yet to be described plastid function.

Protein targeting and transport as a necessary consequence of increased cellular complexity.

With increasing intracellular complexity, a new cell-biological problem that is the allocation of cytoplasmically synthesized proteins to their final destinations within the cell emerged. A special challenge is thereby the translocation of proteins into or across cellular membranes. The underlying mechanisms are only in parts well understood, but it can be assumed that the course of cellular evolution had a deep impact on the design of the required molecular machines. In this article, we aim to summarize the current knowledge and concepts of the evolutionary development of protein trafficking as a necessary premise and consequence of increased cellular complexity.

Nucleotides and substrates trigger the dynamics of the Toc34 GTPase homodimer involved in chloroplast preprotein translocation.

GTPases are molecular switches that control numerous crucial cellular processes. Unlike bona fide GTPases, which are regulated by intramolecular structural transitions, the less well studied GAD-GTPases are activated by nucleotide-dependent dimerization. A member of this family is the translocase of the outer envelope membrane of chloroplast Toc34 involved in regulation of preprotein import. The GTPase cycle of Toc34 is considered a major circuit of translocation regulation. Contrary to expectations, previous studies yielded only marginal structural changes of dimeric Toc34 in response to different nucleotide loads. Referencing PELDOR and FRET single-molecule and bulk experiments, we describe a nucleotide-dependent transition of the dimer flexibility from a tight GDP- to a flexible GTP-loaded state. Substrate binding induces an opening of the GDP-loaded dimer. Thus, the structural dynamics of bona fide GTPases induced by GTP hydrolysis is replaced by substrate-dependent dimer flexibility, which likely represents a general regulatory mode for dimerizing GTPases.

self-assembling GFP: a versatile tool for plant (membrane) protein analyses.

The investigation of cellular processes on the molecular level is important to understand the functional network within plant cells. self-assembling GFP has evolved to be a versatile tool for (membrane) protein analyses. Based on the autocatalytical reassembling property of the nonfluorescent strands 1-10 and 11, protein distribution and membrane protein topology can be analyzed in vivo. Here, we provide basic protocols to determine membrane protein topology in Arabidopsis thaliana protoplasts.

Defining the core proteome of the chloroplast envelope membranes.

High-throughput protein localization studies require multiple strategies. Mass spectrometric analysis of defined cellular fractions is one of the complementary approaches to a diverse array of cell biological methods. In recent years, the protein content of different cellular (sub-)compartments was approached. Despite of all the efforts made, the analysis of membrane fractions remains difficult, in that the dissection of the proteomes of the envelope membranes of chloroplasts or mitochondria is often not reliable because sample purity is not always warranted. Moreover, proteomic studies are often restricted to single (model) species, and therefore limited in respect to differential individual evolution. In this study we analyzed the chloroplast envelope proteomes of different plant species, namely, the individual proteomes of inner and outer envelope (OE) membrane of Pisum sativum and the mixed envelope proteomes of Arabidopsis thaliana and Medicago sativa. The analysis of all three species yielded 341 identified proteins in total, 247 of them being unique. 39 proteins were genuine envelope proteins found in at least two species. Based on this and previous envelope studies we defined the core envelope proteome of chloroplasts. Comparing the general overlap of the available six independent studies (including ours) revealed only a number of 27 envelope proteins. Depending on the stringency of applied selection criteria we found 231 envelope proteins, while less stringent criteria increases this number to 649 putative envelope proteins. Based on the latter we provide a map of the outer and inner envelope core proteome, which includes many yet uncharacterized proteins predicted to be involved in transport, signaling, and response. Furthermore, a foundation for the functional characterization of yet unidentified functions of the inner and OE for further analyses is provided.

In vivo function of Tic22, a protein import component of the intermembrane space of chloroplasts.

Preprotein import into chloroplasts depends on macromolecular machineries in the outer and inner chloroplast envelope membrane (TOC and TIC). It was suggested that both machineries are interconnected by components of the intermembrane space (IMS). That is, amongst others, Tic22, of which two closely related isoforms exist in Arabidopsis thaliana, namely atTic22-III and atTic22-IV. We investigated the function of Tic22 in vivo by analyzing T-DNA insertion lines of the corresponding genes. While the T-DNA insertion in the individual genes caused only slight defects, a double mutant of both isoforms showed retarded growth, a pale phenotype under high-light conditions, a reduced import rate, and a reduction in the photosynthetic performance of the plants. The latter is supported by changes in the metabolite content of mutant plants when compared to wild-type. Thus, our results support the notion that Tic22 is directly involved in chloroplast preprotein import and might point to a particular importance of Tic22 in chloroplast biogenesis at times of high import rates.

Toc33 and Toc64-III cooperate in precursor protein import into the chloroplasts of Arabidopsis thaliana.

The import of cytosolically synthesized precursor proteins into chloroplasts by the translocon at the outer envelope membrane of chloroplasts (TOC) is crucial for organelle function. The recognition of precursor proteins at the chloroplast surface precedes translocation and involves the membrane-inserted receptor subunits Toc34 and Toc159. A third receptor, Toc64, was discussed to recognize cytosolic complexes guiding precursor proteins to the membrane surface, but this function remains debated. We analysed Arabidopsis thaliana plants carrying a T-DNA insertion in the gene encoding the Toc64 homolog Toc64-III. We observed a light intensity-dependent growth phenotype, which is distinct from the phenotype of ppi1, the previously described mutant of the TOC34 homolog TOC33. Furthermore, chloroplast import of the model precursor proteins pOE33 and pSSU into chloroplasts is reduced in protoplasts isolated from plants with impaired Toc64-III function. This suggests that Toc64-III modulates the translocation efficiency in vivo. A ppi1 and toc64-III double mutant shows a significant increase in the transcript levels of HSP90 and TOC75-III, the latter coding for the pore-forming TOC component. Remarkably, the protein level of Toc75-III is significantly reduced, suggesting that Toc64-III and Toc33 cooperate in the insertion or stabilization of Toc75-III. Accordingly, the results presented support Toc64 as an import-relevant component of the TOC complex.

Chloroplast ß-barrel proteins are assembled into the mitochondrial outer membrane in a process that depends on the TOM and TOB complexes.

Membrane-embedded ß-barrel proteins are found in the outer membranes (OM) of Gram-negative bacteria, mitochondria and chloroplasts. In eukaryotic cells, precursors of these proteins are synthesized in the cytosol and have to be sorted to their corresponding organelle. Currently, the signal that ensures their specific targeting to either mitochondria or chloroplasts is ill-defined. To address this issue, we studied targeting of the chloroplast ß-barrel proteins Oep37 and Oep24. We found that both proteins can be integrated in vitro into isolated plant mitochondria. Furthermore, upon their expression in yeast cells Oep37 and Oep24 were exclusively located in the mitochondrial OM. Oep37 partially complemented the growth phenotype of yeast cells lacking Porin, the general metabolite transporter of this membrane. Similarly to mitochondrial ß-barrel proteins, Oep37 and Oep24 expressed in yeast cells were assembled into the mitochondrial OM in a pathway dependent on the TOM and TOB complexes. Taken together, this study demonstrates that the central mitochondrial components that mediate the import of yeast ß-barrel proteins can deal with precursors of chloroplast ß-barrel proteins. This implies that the mitochondrial import machinery does not recognize signals that are unique to mitochondrial ß-barrel proteins. Our results further suggest that dedicated targeting factors had to evolve in plant cells to prevent mis-sorting of chloroplast ß-barrel proteins to mitochondria.

Specific lipids influence the import capacity of the chloroplast outer envelope precursor protein translocon.

Recent studies demonstrated that lipids influence the assembly and efficiency of membrane-embedded macromolecular complexes. Similarly, lipids have been found to influence chloroplast precursor protein binding to the membrane surface and to be associated with the Translocon of the Outer membrane of Chloroplasts (TOC). We used a system based on chloroplast outer envelope vesicles from Pisum sativum to obtain an initial understanding of the influence of lipids on precursor protein translocation across the outer envelope. The ability of the model precursor proteins p(OE33)titin and pSSU to be recognized and translocated in this simplified system was investigated. We demonstrate that transport across the outer membrane can be observed in the absence of the inner envelope translocon. The translocation, however, was significantly slower than that observed for chloroplasts. Enrichment of outer envelope vesicles with different lipids natively found in chloroplast membranes altered the binding and transport behavior. Further, the results obtained using outer envelope vesicles were consistent with the results observed for the reconstituted isolated TOC complex. Based on both approaches we concluded that the lipids sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylinositol (PI) increased TOC-mediated binding and import for both precursor proteins. In contrast, enrichment in digalactosyldiacylglycerol (DGDG) improved TOC-mediated binding for pSSU, but decreased import for both precursor proteins. Optimal import occurred only in a narrow concentration range of DGDG.

The localization of Tic20 proteins in Arabidopsis thaliana is not restricted to the inner envelope membrane of chloroplasts.

Tic20 is a central, membrane-embedded component of the precursor protein translocon of the inner envelope of chloroplasts (TIC). In Arabidopsis thaliana, four different isoforms of Tic20 exist. They are annotated as atTic20-I, -II, -IV and -V and form two distinct phylogenetic subfamilies in embryophyta. Consistent with atTic20-I being the only essential isoform for chloroplast development, we show that the protein is exclusively targeted to the chloroplasts inner envelope. The same result is observed for atTic20-II. In contrast, atTic20-V is localized in thylakoids and atTic20-IV dually localizes to chloroplasts and mitochondria. These results together with the previously established expression profiles explain the recently described phenotypes of Tic20 knockout plants and point towards a functional diversification of these proteins within the family. For all Tic20 proteins a 4-helix topology is proposed irrespective of the targeted membrane, which in part could be confirmed in vivo by application of a self-assembling GFP-based topology approach. By the same approach we show that the inner envelope localized Tic20 proteins expose their C-termini to the chloroplast stroma. This localization would be consistent with the positive inside rule considering a stromal translocation intermediate as discussed.

Chloroplast Omp85 proteins change orientation during evolution.

The majority of outer membrane proteins (OMPs) from gram-negative bacteria and many of mitochondria and chloroplasts are ß-barrels. Insertion and assembly of these proteins are catalyzed by the Omp85 protein family in a seemingly conserved process. All members of this family exhibit a characteristic N-terminal polypeptide-transport-associated (POTRA) and a C-terminal 16-stranded ß-barrel domain. In plants, two phylogenetically distinct and essential Omp85's exist in the chloroplast outer membrane, namely Toc75-III and Toc75-V. Whereas Toc75-V, similar to the mitochondrial Sam50, is thought to possess the original bacterial function, its homolog, Toc75-III, evolved to the pore-forming unit of the TOC translocon for preprotein import. In all current models of OMP biogenesis and preprotein translocation, a topology of Omp85 with the POTRA domain in the periplasm or intermembrane space is assumed. Using self-assembly GFP-based in vivo experiments and in situ topology studies by electron cryotomography, we show that the POTRA domains of both Toc75-III and Toc75-V are exposed to the cytoplasm. This unexpected finding explains many experimental observations and requires a reevaluation of current models of OMP biogenesis and TOC complex function.

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.

Substrate binding disrupts dimerization and induces nucleotide exchange of the chloroplast GTPase Toc33.

GTPases act as molecular switches to control many cellular processes, including signalling, protein translation and targeting. Switch activity can be regulated by external effector proteins or intrinsic properties, such as dimerization. The recognition and translocation of pre-proteins into chloroplasts [via the TOC/TIC (translocator at the outer envelope membrane of chloroplasts/inner envelope membrane of chloroplasts)] is controlled by two homologous receptor GTPases, Toc33 and Toc159, whose reversible dimerization is proposed to regulate translocation of incoming proteins in a GTP-dependent manner. Toc33 is a homodimerizing GTPase. Functional analysis suggests that homodimerization is a key step in the translocation process, the molecular functions of which, as well as the elements regulating this event, are largely unknown. In the present study, we show that homodimerization reduces the rate of nucleotide exchange, which is consistent with the observed orientation of the monomers in the crystal structure. Pre-protein binding induces a dissociation of the Toc33 homodimer and results in the exchange of GDP for GTP. Thus homodimerization does not serve to activate the GTPase activity as discussed many times previously, but to control the nucleotide-loading state. We discuss this novel regulatory mode and its impact on the current models of protein import into the chloroplast.

Making new out of old: recycling and modification of an ancient protein translocation system during eukaryotic evolution. Mechanistic comparison and phylogenetic analysis of ERAD, SELMA and the peroxisomal importomer.

At first glance the three eukaryotic protein translocation machineries--the ER-associated degradation (ERAD) transport apparatus of the endoplasmic reticulum, the peroxisomal importomer and SELMA, the pre-protein translocator of complex plastids--appear quite different. However, mechanistic comparisons and phylogenetic analyses presented here suggest that all three translocation machineries share a common ancestral origin, which highlights the recycling of pre-existing components as an effective evolutionary driving force. Editor's suggested further reading in BioEssays ERAD ubiquitin ligases Abstract.

Protein-induced modulation of chloroplast membrane morphology.

Organelles are surrounded by membranes with a distinct lipid and protein composition. While it is well established that lipids affect protein functioning and vice versa, it has been only recently suggested that elevated membrane protein concentrations may affect the shape and organization of membranes. We therefore analyzed the effects of high chloroplast envelope protein concentrations on membrane structures using an in vivo approach with protoplasts. Transient expression of outer envelope proteins or protein domains such as CHUP1-TM-GFP, outer envelope protein of 7 kDa-GFP, or outer envelope protein of 24 kDa-GFP at high levels led to the formation of punctate, circular, and tubular membrane protrusions. Expression of inner membrane proteins such as translocase of inner chloroplast membrane 20, isoform II (Tic20-II)-GFP led to membrane protrusions including invaginations. Using increasing amounts of DNA for transfection, we could show that the frequency, size, and intensity of these protrusions increased with protein concentration. The membrane deformations were absent after cycloheximide treatment. Co-expression of CHUP1-TM-Cherry and Tic20-II-GFP led to membrane protrusions of various shapes and sizes including some stromule-like structures, for which several functions have been proposed. Interestingly, some structures seemed to contain both proteins, while others seem to contain one protein exclusively, indicating that outer and inner envelope dynamics might be regulated independently. While it was more difficult to investigate the effects of high expression levels of membrane proteins on mitochondrial membrane shapes using confocal imaging, it was striking that the expression of the outer membrane protein Tom20 led to more elongate mitochondria. We discuss that the effect of protein concentrations on membrane structure is possibly caused by an imbalance in the lipid to protein ratio and may be involved in a signaling pathway regulating membrane biogenesis. Finally, the observed phenomenon provides a valuable experimental approach to investigate the relationship between lipid synthesis and membrane protein expression in future studies.

ERAD components in organisms with complex red plastids suggest recruitment of a preexisting protein transport pathway for the periplastid membrane.

The plastids of cryptophytes, haptophytes, and heterokontophytes (stramenopiles) (together once known as chromists) are surrounded by four membranes, reflecting the origin of these plastids through secondary endosymbiosis. They share this trait with apicomplexans, which are alveolates, the plastids of which have been suggested to stem from the same secondary symbiotic event and therefore form a phylogenetic clade, the chromalveolates. The chromists are quantitatively the most important eukaryotic contributors to primary production in marine ecosystems. The mechanisms of protein import across their four plastid membranes are still poorly understood. Components of an endoplasmic reticulum-associated degradation (ERAD) machinery in cryptophytes, partially encoded by the reduced genome of the secondary symbiont (the nucleomorph), are implicated in protein transport across the second outermost plastid membrane. Here, we show that the haptophyte Emiliania huxleyi, like cryptophytes, stramenopiles, and apicomplexans, possesses a nuclear-encoded symbiont-specific ERAD machinery (SELMA, symbiont-specific ERAD-like machinery) in addition to the host ERAD system, with targeting signals that are able to direct green fluorescent protein or yellow fluorescent protein to the predicted cellular localization in transformed cells of the stramenopile Phaeodactylum tricornutum. Phylogenies of the duplicated ERAD factors reveal that all SELMA components trace back to a red algal origin. In contrast, the host copies of cryptophytes and haptophytes associate with the green lineage to the exclusion of stramenopiles and alveolates. Although all chromalveolates with four membrane-bound plastids possess the SELMA system, this has apparently not arisen in a single endosymbiotic event. Thus, our data do not support the chromalveolate hypothesis.

The physical and functional borders of transit peptide-like sequences in secondary endosymbionts.

BACKGROUND: Plastids rely on protein supply by their host cells. In plastids surrounded by two membranes (primary plastids) targeting of these proteins is facilitated by an N-terminal targeting signal, the transit peptide. In secondary plastids (surrounded by three or four membranes), transit peptide-like regions are an essential part of a bipartite topogenic signal sequence (BTS), and generally found adjacent to a N-terminally located signal peptide of the plastid pre-proteins. As in primary plastids, for which no wealth of functional information about transit peptide features exists, the transit peptide-like regions used for import into secondary ones show some common features only, which are also poorly characterized. RESULTS: We modified the BTS (in the transit peptide-like region) of the plastid precursor fucoxanthin-chlorophyll a/c binding protein D (FcpD) fused to GFP as model substrate for the characterization of pre-protein import into the secondary plastids of diatoms. Thereby we show that (i) pre-protein import is highly charge dependent. Positive net charge is necessary for transport across the plastid envelope, but not across the periplastid membrane. Acidic net charge perturbs pre-protein import within the ER. Moreover, we show that (ii) the mature domain of the pre-protein can provide intrinsic transit peptide functions. CONCLUSIONS: Our results indicate important characteristics of targeting signals of proteins imported into secondary plastids surrounded by four membranes. In addition, we show a self-targeting mechanism, in which the mature protein domain contributes to the transit peptide function. Thus, this phenomenon lowers the demand for pre-sequences evolved during the course of endosymbiosis.

Conserved properties of polypeptide transport-associated (POTRA) domains derived from cyanobacterial Omp85.

Proteins of the Omp85 family are conserved in all kingdoms of life. They mediate protein transport across or protein insertion into membranes and reside in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Omp85 proteins contain a C-terminal transmembrane beta-barrel and a soluble N terminus with a varying number of polypeptide-transport-associated or POTRA domains. Here we investigate Omp85 from the cyanobacterium Anabaena sp. PCC 7120. The crystallographic three-dimensional structure of the N-terminal region shows three POTRA domains, here named P1 to P3 from the N terminus. Molecular dynamics simulations revealed a hinge between P1 and P2 but in contrast show that P2 and P3 are fixed in orientation. The P2-P3 arrangement is identical as seen for the POTRA domains from proteobacterial FhaC, suggesting this orientation is a conserved feature. Furthermore, we define interfaces for protein-protein interaction in P1 and P2. P3 possesses an extended loop unique to cyanobacteria and plantae, which influences pore properties as shown by deletion. It now becomes clear how variations in structure of individual POTRA domains, as well as the different number of POTRA domains with both rigid and flexible connections make the N termini of Omp85 proteins versatile adaptors for a plentitude of functions.