Oliver Mirus

The molecular chaperone Hsp90 delivers precursor proteins to the chloroplast import receptor Toc64

Precursor protein targeting toward organellar surfaces is assisted by different cytosolic chaperones. We demonstrate that the chloroplast protein translocon subunit Toc64 is the docking site for Hsp90 affiliated preproteins. Thereby, Hsp90 is recognised by the clamp type TPR domain of Toc64. The subsequent transfer of the preprotein from Toc64 to the major receptor of the Toc complex, namely Toc34, is affinity driven and nucleotide dependent. We propose that Toc64 acts as an initial docking site for Hsp90 associated precursor proteins. We outline a mechanism in which chaperones are recruited for a specific targeting event by a membrane‐inserted receptor.

Prediction of the plant β-barrel proteome: A case study of the chloroplast outer envelope

In the postgenomic era, the transformation of genetic information into biochemical meaning is required. We have analyzed the proteome of the chloroplast outer envelope membrane by an in silico and a proteomic approach. Based on its evolutionary relation to the outer membrane of Gram‐negative bacteria, the outer envelope membrane should contain a large number of β‐barrel proteins. We therefore calculated the probability for the existence of β‐sheet, β‐barrel, and hairpin structures among all proteins of the Arabidopsis thaliana genome. According to the existence of these structures, a number of candidates were selected. This protein pool was analyzed by TargetP to discard sequences with signals that would direct the protein to other organelles different from chloroplasts. In addition, the pool was manually controlled for the presence of proteins known to function outside of the chloroplast envelope. The approach developed here can be used to predict the topology of β‐barrel proteins. For the proteomic approach, proteins of highly purified outer envelope membranes of chloroplasts from Pisum sativum were analyzed by ESI‐MS/MS mass spectrometry. In addition to the known components, four new proteins of the outer envelope membranes were identified in this study.

Hiding behind Hydrophobicity TRANSMEMBRANE SEGMENTS IN MASS SPECTROMETRY

Proteomics of membrane proteins is essential for the understanding of cellular function. However, mass spectrometric analysis of membrane proteomes has been less successful than the proteomic determination of soluble proteins. To elucidate the mystery of transmembrane proteins in mass spectrometry, we present a detailed statistical analysis of experimental data derived from chloroplast membranes. This approach was further accomplished by the analysis of the Arabidopsis thaliana proteome after in silico digestion. We demonstrate that both the length and the hydrophobicity of the proteolytic fragments containing transmembrane segments are major determinants for detection by mass spectrometry. Based on a comparative analysis, we discuss possibilities to overcome the problem and provide possible protocols to shift the hydrophobicity of transmembrane segment-containing peptides to facilitate their detection.

Conserved pore-forming regions in polypeptide-transporting proteins

Transport of solutes and polypeptides across membranes is an essential process for every cell. In the past, much focus has been placed on helical transporters. Recently, the β‐barrel‐shaped transporters have also attracted some attention. The members of this family are found in the outer bacterial membrane and the outer membrane of endosymbiotically derived organelles. Here we analyze the features and the evolutionary development of a specified translocator family, namely the β‐barrel‐shaped polypeptide‐transporters. We identified sequence motifs, which characterize all transporters of this family, as well as motifs specific for a certain subgroup of proteins of this class. The general motifs are related to the structural composition of the pores. Further analysis revealed a defined distance of two motifs to the C‐terminal portion of the proteins. Furthermore, the evolutionary relationship of the proteins and the motifs are discussed.

Filling the Gap, Evolutionarily Conserved Omp85 in Plastids of Chromalveolates

Chromalveolates are a diverse group of protists that include many ecologically and medically relevant organisms such as diatoms and apicomplexan parasites. They possess plastids generally surrounded by four membranes, which evolved by engulfment of a red alga. Today, most plastid proteins must be imported, but many aspects of protein import into complex plastids are still cryptic. In particular, how proteins cross the third outermost membrane has remained unexplained. We identified a protein in the third outermost membrane of the diatom Phaeodactylum tricornutum with properties comparable to those of the Omp85 family. We demonstrate that the targeting route of P. tricornutum Omp85 parallels that of the translocation channel of the outer envelope membrane of chloroplasts, Toc75. In addition, the electrophysiological properties are similar to those of the Omp85 proteins involved in protein translocation. This supports the hypothesis that P. tricornutum Omp85 is involved in precursor protein translocation, which would close a gap in the fundamental understanding of the evolutionary origin and function of protein import in secondary plastids.

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 β-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 β-barrel domain. In plants, two phylogenetically distinct and essential Omp85s 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.

TonB-dependent transporters and their occurrence in cyanobacteria

BackgroundDifferent 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.ResultsWe 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.ConclusionWe 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.

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.

The evolution of the ribosome biogenesis pathway from a yeast perspective

Ribosome biogenesis is fundamental for cellular life, but surprisingly little is known about the underlying pathway. In eukaryotes a comprehensive collection of experimentally verified ribosome biogenesis factors (RBFs) exists only for Saccharomyces cerevisiae. Far less is known for other fungi, animals or plants, and insights are even more limited for archaea. Starting from 255 yeast RBFs, we integrated ortholog searches, domain architecture comparisons and, in part, manual curation to investigate the inventories of RBF candidates in 261 eukaryotes, 26 archaea and 57 bacteria. The resulting phylogenetic profiles reveal the evolutionary ancestry of the yeast pathway. The oldest core comprising 20 RBF lineages dates back to the last universal common ancestor, while the youngest 20 factors are confined to the Saccharomycotina. On this basis, we outline similarities and differences of ribosome biogenesis across contemporary species. Archaea, so far a rather uncharted domain, possess 38 well-supported RBF candidates of which some are known to form functional sub-complexes in yeast. This provides initial evidence that ribosome biogenesis in eukaryotes and archaea follows similar principles. Within eukaryotes, RBF repertoires vary considerably. A comparison of yeast and human reveals that lineage-specific adaptation via RBF exclusion and addition characterizes the evolution of this ancient pathway.

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 β-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.