Plastids of diatoms and related algae with complex plastids of red algal origin are surrounded by four membranes, which also define the periplastidic compartment (PPC), the space between the second and third membranes. Metabolic reactions as well as cell biological processes take place in the PPC; however, genome-wide predictions of the proteins targeted to this compartment were so far based on manual annotation work. Using published experimental protein localizations as reference data, we developed the first automatic prediction method for PPC proteins, which we included as a new feature in an updated version of the plastid protein predictor ASAFind. With our method, at least a subset of the PPC proteins can be predicted with high specificity, with an estimate of at least 81 proteins (0.7% of the predicted proteome) targeted to the PPC in the model diatom Phaeodactylum tricornutum. The proportion of PPC proteins varies, since 180 PPC proteins (1.3% of the predicted proteome) were predicted in the genome of the diatom Thalassiosira pseudonana. The new ASAFind version can also generate a newly designed graphical output that visualizes the contribution of each position in the sequence to the score and accepts the output of the recent versions of SignalP (5.0) and TargetP (2.0) as input data. Furthermore, we release a script to calculate custom scoring matrices that can be used for predictions in a simplified score cut-off mode. This allows for adjustments of the method to other groups of algae.

• Klíčová slova
• chloroplast, diatoms, evolution, gene transfer, genome annotation, mitochondria, organelle, periplastidic compartment, protein transport, secretory pathway, technical advance,
• MeSH
• bílkoviny řas * metabolismus   MeSH
• plastidy * metabolismus   MeSH
• proteom MeSH
• Rhodophyta metabolismus   MeSH
• rozsivky * metabolismus   genetika   MeSH
• software * MeSH
• výpočetní biologie * metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bílkoviny řas * MeSH
• proteom MeSH

Complete plastid loss seems to be very rare among secondarily non-photosynthetic eukaryotes. Leukarachnion sp. PRA-24, an amoeboid colourless protist related to the photosynthetic algal class Synchromophyceae (Ochrophyta), is a candidate for such a case based on a previous investigation by transmission electron microscopy. Here, we characterize this organism in further detail and describe it as Leucomyxa plasmidifera gen. et sp. nov., additionally demonstrating it is the first known representative of a broader clade of non-photosynthetic ochrophytes. We recovered its complete plastid genome, exhibiting a reduced gene set similar to plastomes of other non-photosynthetic ochrophytes, yet being even more extreme in sequence divergence. Identification of components of the plastid protein import machinery in the L. plasmidifera transcriptome assembly corroborated that the organism possesses a cryptic plastid organelle. According to our bioinformatic reconstruction, the plastid contains a unique combination of biosynthetic pathways producing haem, a folate precursor and tocotrienols. As another twist to its organellar biology, L. plasmidifera turned out to contain an unusual long insertion in its mitogenome related to a newly discovered mitochondrial plasmid exhibiting unprecedented features in terms of its size and coding capacity. Combined, our work uncovered further striking outcomes of the evolutionary course of semiautonomous organelles in protists.

• Klíčová slova
• Leukarachnion, mitochondrial plasmids, non-photosynthetic plastid, plastid evolution, plastid genome, stramenopiles,
• MeSH
• fylogeneze * MeSH
• genom mitochondriální MeSH
• genom plastidový * MeSH
• mitochondrie genetika   metabolismus   MeSH
• molekulární evoluce MeSH
• plastidy * genetika   metabolismus   MeSH
• plazmidy * genetika   MeSH
• Publikační typ
• časopisecké články MeSH

Stramenopiles represent a significant proportion of aquatic and terrestrial biota. Most biologists can name a few, but these are limited to the phototrophic (e.g. diatoms and kelp) or parasitic species (e.g. oomycetes, Blastocystis), with free-living heterotrophs largely overlooked. Though our attention is slowly turning towards heterotrophs, we have only a limited understanding of their biology due to a lack of cultured models. Recent metagenomic and single-cell investigations have revealed the species richness and ecological importance of stramenopiles-especially heterotrophs. However, our lack of knowledge of the cell biology and behaviour of these organisms leads to our inability to match species to their particular ecological functions. Because photosynthetic stramenopiles are studied independently of their heterotrophic relatives, they are often treated separately in the literature. Here, we present stramenopiles as a unified group with shared synapomorphies and evolutionary history. We introduce the main lineages, describe their important biological and ecological traits, and provide a concise update on the origin of the ochrophyte plastid. We highlight the crucial role of heterotrophs and mixotrophs in our understanding of stramenopiles with the goal of inspiring future investigations in taxonomy and life history. To understand each of the many diversifications within stramenopiles-towards autotrophy, osmotrophy, or parasitism-we must understand the ancestral heterotrophic flagellate from which they each evolved. We hope the following will serve as a primer for new stramenopile researchers or as an integrative refresher to those already in the field.

• Klíčová slova
• chromalveolate hypothesis, heterotrophic flagellates, microbial ecology and evolution, plastid evolution, protistology, rhodoplex hypothesis, stramenopiles,
• MeSH
• biologická evoluce MeSH
• fylogeneze MeSH
• Heterokontophyta * klasifikace   genetika   MeSH
• heterotrofní procesy * MeSH
• plastidy genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Heme biosynthesis is essential for almost all living organisms. Despite its conserved function, the pathway's enzymes can be located in a remarkable diversity of cellular compartments in different organisms. This location does not always reflect their evolutionary origins, as might be expected from the history of their acquisition through endosymbiosis. Instead, the final subcellular localization of the enzyme reflects multiple factors, including evolutionary origin, demand for the product, availability of the substrate, and mechanism of pathway regulation. The biosynthesis of heme in the apicomonad Chromera velia follows a chimeric pathway combining heme elements from the ancient algal symbiont and the host. Computational analyses using different algorithms predict complex targeting patterns, placing enzymes in the mitochondrion, plastid, endoplasmic reticulum, or the cytoplasm. We employed heterologous reporter gene expression in the apicomplexan parasite Toxoplasma gondii and the diatom Phaeodactylum tricornutum to experimentally test these predictions. 5-aminolevulinate synthase was located in the mitochondria in both transfection systems. In T. gondii, the two 5-aminolevulinate dehydratases were located in the cytosol, uroporphyrinogen synthase in the mitochondrion, and the two ferrochelatases in the plastid. In P. tricornutum, all remaining enzymes, from ALA-dehydratase to ferrochelatase, were placed either in the endoplasmic reticulum or in the periplastidial space.

• Klíčová slova
• Chromera velia, heterologous expression, predictions, tetrapyrrole biosynthesis,
• MeSH
• Alveolata fyziologie   MeSH
• Apicomplexa metabolismus   MeSH
• biologický transport MeSH
• hem metabolismus   MeSH
• metabolické sítě a dráhy * MeSH
• mitochondrie genetika   metabolismus   ultrastruktura   MeSH
• molekulární evoluce MeSH
• protozoální proteiny chemie   genetika   metabolismus   MeSH
• regulace genové exprese enzymů MeSH
• rozsivky metabolismus   MeSH
• sekvence aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• hem MeSH
• protozoální proteiny MeSH

BACKGROUND: Comparative analyses have indicated that the mitochondrion of the last eukaryotic common ancestor likely possessed all the key core structures and functions that are widely conserved throughout the domain Eucarya. To date, such studies have largely focused on animals, fungi, and land plants (primarily multicellular eukaryotes); relatively few mitochondrial proteomes from protists (primarily unicellular eukaryotic microbes) have been examined. To gauge the full extent of mitochondrial structural and functional complexity and to identify potential evolutionary trends in mitochondrial proteomes, more comprehensive explorations of phylogenetically diverse mitochondrial proteomes are required. In this regard, a key group is the jakobids, a clade of protists belonging to the eukaryotic supergroup Discoba, distinguished by having the most gene-rich and most bacteria-like mitochondrial genomes discovered to date. RESULTS: In this study, we assembled the draft nuclear genome sequence for the jakobid Andalucia godoyi and used a comprehensive in silico approach to infer the nucleus-encoded portion of the mitochondrial proteome of this protist, identifying 864 candidate mitochondrial proteins. The A. godoyi mitochondrial proteome has a complexity that parallels that of other eukaryotes, while exhibiting an unusually large number of ancestral features that have been lost particularly in opisthokont (animal and fungal) mitochondria. Notably, we find no evidence that the A. godoyi nuclear genome has or had a gene encoding a single-subunit, T3/T7 bacteriophage-like RNA polymerase, which functions as the mitochondrial transcriptase in all eukaryotes except the jakobids. CONCLUSIONS: As genome and mitochondrial proteome data have become more widely available, a strikingly punctuate phylogenetic distribution of different mitochondrial components has been revealed, emphasizing that the pathways of mitochondrial proteome evolution are likely complex and lineage-specific. Unraveling this complexity will require comprehensive comparative analyses of mitochondrial proteomes from a phylogenetically broad range of eukaryotes, especially protists. The systematic in silico approach described here offers a valuable adjunct to direct proteomic analysis (e.g., via mass spectrometry), particularly in cases where the latter approach is constrained by sample limitation or other practical considerations.

• Klíčová slova
• Andalucia godoyi, Jakobids, Mitochondrial evolution, Mitochondrial genome, Mitochondrial proteome, Mitochondrion, Protist,
• MeSH
• buněčné jádro genetika   MeSH
• Eukaryota genetika   MeSH
• genom mitochondriální * MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• proteom * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální proteiny MeSH
• proteom * MeSH

Plastids, organelles that evolved from cyanobacteria via endosymbiosis in eukaryotes, provide carbohydrates for the formation of biomass and for mitochondrial energy production to the cell. They generate their own energy in the form of the nucleotide adenosine triphosphate (ATP). However, plastids of non-photosynthetic tissues, or during the dark, depend on external supply of ATP. A dedicated antiporter that exchanges ATP against adenosine diphosphate (ADP) plus inorganic phosphate (Pi) takes over this function in most photosynthetic eukaryotes. Additional forms of such nucleotide transporters (NTTs), with deviating activities, are found in intracellular bacteria, and, surprisingly, also in diatoms, a group of algae that acquired their plastids from other eukaryotes via one (or even several) additional endosymbioses compared to algae with primary plastids and higher plants. In this review, we summarize what is known about the nucleotide synthesis and transport pathways in diatom cells, and discuss the evolutionary implications of the presence of the additional NTTs in diatoms, as well as their applications in biotechnology.

• Klíčová slova
• adenosine triphosphate (ATP), endosymbiosis, evolution, photosynthesis, plastid, synthetic biology, transport,
• MeSH
• biologická evoluce MeSH
• biologický transport MeSH
• biotechnologie MeSH
• membránové transportní proteiny chemie   metabolismus   MeSH
• nukleotidy biosyntéza   metabolismus   MeSH
• rozsivky metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• membránové transportní proteiny MeSH
• nukleotidy MeSH

Diatoms are unicellular algae and evolved by secondary endosymbiosis, a process in which a red alga-like eukaryote was engulfed by a heterotrophic eukaryotic cell. This gave rise to plastids of remarkable complex architecture and ultrastructure that require elaborate protein importing, trafficking, signaling and intracellular cross-talk pathways. Studying both plastids and mitochondria and their distinctive physiological pathways in organello may greatly contribute to our understanding of photosynthesis, mitochondrial respiration and diatom evolution. The isolation of such complex organelles, however, is still demanding, and existing protocols are either limited to a few species (for plastids) or have not been reported for diatoms so far (for mitochondria). In this work, we present the first isolation protocol for mitochondria from the model diatom Thalassiosira pseudonana. Apart from that, we extended the protocol so that it is also applicable for the purification of a high-quality plastids fraction, and provide detailed structural and physiological characterizations of the resulting organelles. Isolated mitochondria were structurally intact, showed clear evidence of mitochondrial respiration, but the fractions still contained residual cell fragments. In contrast, plastid isolates were virtually free of cellular contaminants, featured structurally preserved thylakoids performing electron transport, but lost most of their stromal components as concluded from Western blots and mass spectrometry. Liquid chromatography electrospray-ionization mass spectrometry studies on mitochondria and thylakoids, moreover, allowed detailed proteome analyses which resulted in extensive proteome maps for both plastids and mitochondria thus helping us to broaden our understanding of organelle metabolism and functionality in diatoms.

• Klíčová slova
• Chloroplast, Organelle isolation, Photosynthesis, Proteomics, Respiration, Thylakoids,
• MeSH
• mitochondrie metabolismus   MeSH
• plastidy metabolismus   MeSH
• proteom metabolismus   MeSH
• rozsivky metabolismus   MeSH
• tylakoidy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteom MeSH