Prediction of β-barrel membrane proteins by searching for restricted domains

BACKGROUND: The identification of beta-barrel membrane proteins out of a genomic/proteomic background is one of the rapidly developing fields in bioinformatics. Our main goal is the prediction of such proteins in genome/proteome wide analyses. RESULTS: For the prediction of beta-barrel membrane proteins within prokaryotic proteomes a set of parameters was developed. We have focused on a procedure with a low false positive rate beside a procedure with lowest false prediction rate to obtain a high certainty for the predicted sequences. We demonstrate that the discrimination between beta-barrel membrane proteins and other proteins is improved by analyzing a length limited region. The developed set of parameters is applied to the proteome of E. coli and the results are compared to four other described procedures. CONCLUSION: Analyzing the beta-barrel membrane proteins revealed the presence of a defined membrane inserted beta-barrel region. This information can now be used to refine other prediction programs as well. So far, all tested programs fail to predict outer membrane proteins in the proteome of the prokaryote E. coli with high reliability. However, the reliability of the prediction is improved significantly by a combinatory approach of several programs. The consequences and usability of the developed scores are discussed

Phosphorylation regulates the assembly of chloroplast import machinery

Chloroplast function depends on the translocation of cytosolically synthesized precursor proteins into the organelle. The recognition and transfer of most precursor proteins across the outer membrane depend on a membrane inserted complex. Two receptor components of this complex, Toc34 and Toc159, are GTPases, which can be phosphorylated by kinases present in the hosting membrane. However, the physiological function of phosphorylation is not yet understood in detail. It is demonstrated that both receptors are phosphorylated within their G-domains. In vitro, the phosphorylation of Toc34 disrupts both homo- and heterodimerization of the G-domains as determined using a phospho-mimicking mutant. In endogenous membranes this mutation or phosphorylation of the wild-type receptor disturbs the association of Toc34, but not of Toc159 with the translocation pore. Therefore, phosphorylation serves as an inhibitor for the association of Toc34 with other components of the complex and phosphorylation can now be discussed as a mechanism to exchange different isoforms of Toc34 within this ensemble

The proteome of the heterocyst cell wall in Anabaena sp. PCC 7120

Anabaena sp. PCC 7120 is a filamentous cyanobacterium that serves as a model to analyze prokaryotic cell differentiation, evolutionary development of plastids, and the regulation of nitrogen fixation. The cell wall is the cellular structure in contact with the surrounding medium. To understand the dynamics of the cell wall proteome during cell differentiation, the cell wall from Anabaena heterocysts was enriched and analyzed. In line with the recently proposed continuity of the outer membrane along the Anabaena filament, most of the proteins identified in the heterocyst cell-wall fraction are also present in the cell wall of vegetative cells, even though the lipid content of both membranes is different

The protein translocation systems in plants - composition and variability on the example of Solanum lycopersicum

Background: Protein translocation across membranes is a central process in all cells. In the past decades the molecular composition of the translocation systems in the membranes of the endoplasmic reticulum, peroxisomes, mitochondria and chloroplasts have been established based on the analysis of model organisms. Today, these results have to be transferred to other plant species. We bioinformatically determined the inventory of putative translocation factors in tomato (Solanum lycopersicum) by orthologue search and domain architecture analyses. In addition, we investigated the diversity of such systems by comparing our findings to the model organisms Saccharomyces cerevisiae, Arabidopsis thaliana and 12 other plant species. Results: The literature search end up in a total of 130 translocation components in yeast and A. thaliana, which are either experimentally confirmed or homologous to experimentally confirmed factors. From our bioinformatic analysis (PGAP and OrthoMCL), we identified (co-)orthologues in plants, which in combination yielded 148 and 143 orthologues in A. thaliana and S. lycopersicum, respectively. Interestingly, we traced 82% overlap in findings from both approaches though we did not find any orthologues for 27% of the factors by either procedure. In turn, 29% of the factors displayed the presence of more than one (co-)orthologue in tomato. Moreover, our analysis revealed that the genomic composition of the translocation machineries in the bryophyte Physcomitrella patens resemble more to higher plants than to single celled green algae. The monocots (Z. mays and O. sativa) follow more or less a similar conservation pattern for encoding the translocon components. In contrast, a diverse pattern was observed in different eudicots. Conclusions: The orthologue search shows in most cases a clear conservation of components of the translocation pathways/machineries. Only the Get-dependent integration of tail-anchored proteins seems to be distinct. Further, the complexity of the translocation pathway in terms of existing orthologues seems to vary among plant species. This might be the consequence of palaeoploidisation during evolution in plants; lineage specific whole genome duplications in Arabidopsis thaliana and triplications in Solanum lycopersicum

Approximate Hermitian-Yang-Mills structures and semistability for Higgs bundles. II: Higgs sheaves and admissible structures

We study the basic properties of Higgs sheaves over compact K\"ahler manifolds and we establish some results concerning the notion of semistability; in particular, we show that any extension of semistable Higgs sheaves with equal slopes is semistable. Then, we use the flattening theorem to construct a regularization of any torsion-free Higgs sheaf and we show that it is in fact a Higgs bundle. Using this, we prove that any Hermitian metric on a regularization of a torsion-free Higgs sheaf induces an admissible structure on the Higgs sheaf. Finally, using admissible structures we proved some properties of semistable Higgs sheaves.Comment: 18 pages; some typos correcte

TonB-dependent transporters and their occurrence in cyanobacteria

Background Different iron transport systems evolved in Gram-negative bacteria during evolution. Most of the transport systems depend on outer membrane localized TonB-dependent transporters (TBDTs), a periplasma-facing TonB protein and a plasma membrane localized machinery (ExbBD). So far, iron chelators (siderophores), oligosaccharides and polypeptides have been identified as substrates of TBDTs. For iron transport, three uptake systems are defined: the lactoferrin/transferrin binding proteins, the porphyrin-dependent transporters and the siderophore-dependent transporters. However, for cyanobacteria almost nothing is known about possible TonB-dependent uptake systems for iron or other substrates. Results We have screened all publicly available eubacterial genomes for sequences representing (putative) TBDTs. Based on sequence similarity, we identified 195 clusters, where elements of one cluster may possibly recognize similar substrates. For Anabaena sp. PCC 7120 we identified 22 genes as putative TBDTs covering almost all known TBDT subclasses. This is a high number of TBDTs compared to other cyanobacteria. The expression of the 22 putative TBDTs individually depends on the presence of iron, copper or nitrogen. Conclusions We exemplified on TBDTs the power of CLANS-based classification, which demonstrates its importance for future application in systems biology. In addition, the tentative substrate assignment based on characterized proteins will stimulate the research of TBDTs in different species. For cyanobacteria, the atypical dependence of TBDT gene expression on different nutrition points to a yet unknown regulatory mechanism. In addition, we were able to clarify a hypothesis of the absence of TonB in cyanobacteria by the identification of according sequences

Chloroplast signal length requirement reflects the outer membrane and TOC complex dimension

Background and Purpose: The evolution of an efficient preprotein targeting and translocation system was a central prerequisite for the endosymbiotic integration of a-proteobacteria and cyanobacteria as cellular organelles. Today, it is widely accepted that during evolution most (pre-)proteins destined for these two organelles were equipped with an N-terminal targeting signal for localization. While multiple modes of evolution of these extensions are currently discussed, all evolved signals serve the same function – forming a signal for targeting to the correct organelle and translocation across both membranes. We aimed to generalize the current idea for the length requirement of the N-terminal extension for efficient translocation. Methods: To explore translocation efficiency in vivo we used protoplasts isolated from different plant sources. We compared the behavior of native and artificial precursor proteins in this plant cell system by fluorescence microscopy. Results: We demonstrate that the minimal length of the N-terminal amino acid stretch in a loosely folded conformation of a precursor of a chloroplast protein is about 60 amino acid residues. This amino acid stretch in a loosely folded state is prerequisite that a preprotein can traverse the outer membrane in vivo. Conclusion: We generalize the evidence that two distinct prerequisites framed the evolutionary process of development of targeting signals for chloroplast translocation. (i) The emerging signal had to be sufficiently distinct to signals existing for targeting to other cellular compartments. (ii) The N-terminal signal had to evolve with physico-chemical properties that serve both purposes: targeting and translocation. With respect to the latter, the length of the unfolded polypeptide is defined by the dimension of the translocon and the resulting distance between the cytosolically exposed receptors acting on the cis side of the membrane and the molecular machinery energizing translocation acting in trans – in the intermembrane space

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

Structural and functional analysis of the archaeal endonuclease Nob1

Eukaryotic ribosome biogenesis requires the concerted action of numerous ribosome assembly factors, for most of which structural and functional information is currently lacking. Nob1, which can be identified in eukaryotes and archaea, is required for the final maturation of the small subunit ribosomal RNA in yeast by catalyzing cleavage at site D after export of the preribosomal subunit into the cytoplasm. Here, we show that this also holds true for Nob1 from the archaeon Pyrococcus horikoshii, which efficiently cleaves RNA-substrates containing the D-site of the preribosomal RNA in a manganese-dependent manner. The structure of PhNob1 solved by nuclear magnetic resonance spectroscopy revealed a PIN domain common with many nucleases and a zinc ribbon domain, which are structurally connected by a flexible linker. We show that amino acid residues required for substrate binding reside in the PIN domain whereas the zinc ribbon domain alone is sufficient to bind helix 40 of the small subunit rRNA. This suggests that the zinc ribbon domain acts as an anchor point for the protein on the nascent subunit positioning it in the proximity of the cleavage site