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.

Vesicle transport is a central process to ensure protein and lipid distribution in eukaryotic cells. The current knowledge on the molecular components and mechanisms of this process is majorly based on studies in Saccharomyces cerevisiae and Arabidopsis thaliana, which revealed 240 different proteinaceous factors either experimentally proven or predicted to be involved in vesicle transport. In here, we performed an orthologue search using two different algorithms to identify the components of the secretory pathway in yeast and 14 plant genomes by using the ’core-set’ of 240 factors as bait. We identified 4021 orthologues and (co-)orthologues in the discussed plant species accounting for components of COP-II, COP-I, Clathrin Coated Vesicles, Retromers and ESCRTs, Rab GTPases, Tethering factors and SNAREs. In plants, we observed a significantly higher number of (co-)orthologues than yeast, while only 8 tethering factors from yeast seem to be absent in the analyzed plant genomes. To link the identified (co-)orthologues to vesicle transport, the domain architecture of the proteins from yeast, genetic model plant A. thaliana and agriculturally relevant crop Solanum lycopersicum has been inspected. For the orthologous groups containing (co-)orthologues from yeast, A. thaliana and S. lycopersicum, we observed the same domain architecture for 79% (416/527) of the (co-)orthologues, which documents a very high conservation of this process. Further, publically available tissue-specific expression profiles for a subset of (co-)orthologues found in A. thaliana and S. lycopersicum suggest that some (co-)orthologues are involved in tissue-specific functions. Inspection of localization of the (co-)orthologues based on available proteome data or localization predictions lead to the assignment of plastid- as well as mitochondrial localized (co-)orthologues of vesicle transport factors and the relevance of this is discussed.

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.

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.

Ribosome biogenesis is well described in Saccharomyces cerevisiae. In contrast only very little information is available on this pathway in plants. This study presents the characterization of five putative protein co-factors of ribosome biogenesis in Arabidopsis thaliana, namely Rrp5, Pwp2, Nob1, Enp1 and Noc4. The characterization of the proteins in respect to localization, enzymatic activity and association with pre-ribosomal complexes is shown. Additionally, analyses of T-DNA insertion mutants aimed to reveal an involvement of the plant co-factors in ribosome biogenesis. The investigated proteins localize mainly to the nucleolus or the nucleus, and atEnp1 and atNob1 co-migrate with 40S pre-ribosomal complexes. The analysis of T-DNA insertion lines revealed that all proteins are essential in Arabidopsis thaliana and mutant plants show alterations of rRNA intermediate abundance already in the heterozygous state. The most significant alteration was observed in the NOB1 T-DNA insertion line where the P-A3 fragment, a 23S-like rRNA precursor, accumulated. The transmission of the T-DNA through the male and female gametophyte was strongly inhibited indicating a high importance of ribosome co-factor genes in the haploid stages of plant development. Additionally impaired embryogenesis was observed in some mutant plant lines. All results support an involvement of the analyzed proteins in ribosome biogenesis but differences in rRNA processing, gametophyte and embryo development suggested an alternative regulation in plants.

Protein translocation of cytosolically synthesized proteins requires signals for both targeting of precursor proteins to the surface of the respective compartment and their transfer across its membrane. In contrast to signals for peroxisomal and endoplasmic reticulum translocation, the signals for mitochondrial and chloroplast transport are less well defined with respect to length and amino acid requirements. To study the properties of signals required for translocation into chloroplasts in vitro and in vivo, we used fusion proteins composed of transit peptides and the Ig-like module of the muscle protein titin as passenger. We observed that about 60 amino acids-longer than the transit peptide length of many experimentally confirmed chloroplast proteins-are required for efficient translocation. However, within native chloroplast precursor proteins with transit peptides shorter than 60 amino acids, extension appears to be present as they are efficiently imported into organelles. In addition, the interaction of an unfolded polypeptide stretch of 60 or more amino acids with receptors at the chloroplast surface results in the unidirectionality of protein translocation into chloroplasts even in the presence of a competing C-terminal peroxisomal targeting signal. These findings prove the existing ideas that initial targeting is defined by the N-terminal signal and that the C-terminal signal is sensed only subsequently.