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.

• Keywords
• chloroplast, diatoms, evolution, gene transfer, genome annotation, mitochondria, organelle, periplastidic compartment, protein transport, secretory pathway, technical advance,
• MeSH
• Algal Proteins * metabolism   MeSH
• Plastids * metabolism   MeSH
• Proteome MeSH
• Rhodophyta metabolism   MeSH
• Diatoms * metabolism   genetics   MeSH
• Software * MeSH
• Computational Biology * methods   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Algal Proteins * MeSH
• Proteome 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.

• Keywords
• adenosine triphosphate (ATP), endosymbiosis, evolution, photosynthesis, plastid, synthetic biology, transport,
• MeSH
• Biological Evolution MeSH
• Biological Transport MeSH
• Biotechnology MeSH
• Membrane Transport Proteins chemistry   metabolism   MeSH
• Nucleotides biosynthesis   metabolism   MeSH
• Diatoms metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Membrane Transport Proteins MeSH
• Nucleotides 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.

• Keywords
• Chloroplast, Organelle isolation, Photosynthesis, Proteomics, Respiration, Thylakoids,
• MeSH
• Mitochondria metabolism   MeSH
• Plastids metabolism   MeSH
• Proteome metabolism   MeSH
• Diatoms metabolism   MeSH
• Thylakoids metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Proteome MeSH

The establishment of the mitochondrion is seen as a transformational step in the origin of eukaryotes. With the mitochondrion came bioenergetic freedom to explore novel evolutionary space leading to the eukaryotic radiation known today. The tight integration of the bacterial endosymbiont with its archaeal host was accompanied by a massive endosymbiotic gene transfer resulting in a small mitochondrial genome which is just a ghost of the original incoming bacterial genome. This endosymbiotic gene transfer resulted in the loss of many genes, both from the bacterial symbiont as well the archaeal host. Loss of genes encoding redundant functions resulted in a replacement of the bulk of the host's metabolism for those originating from the endosymbiont. Glycolysis is one such metabolic pathway in which the original archaeal enzymes have been replaced by bacterial enzymes from the endosymbiont. Glycolysis is a major catabolic pathway that provides cellular energy from the breakdown of glucose. The glycolytic pathway of eukaryotes appears to be bacterial in origin, and in well-studied model eukaryotes it takes place in the cytosol. In contrast, here we demonstrate that the latter stages of glycolysis take place in the mitochondria of stramenopiles, a diverse and ecologically important lineage of eukaryotes. Although our work is based on a limited sample of stramenopiles, it leaves open the possibility that the mitochondrial targeting of glycolytic enzymes in stramenopiles might represent the ancestral state for eukaryotes.

• MeSH
• Biological Evolution MeSH
• Blastocystis cytology   enzymology   genetics   metabolism   MeSH
• Energy Metabolism MeSH
• Genome, Mitochondrial MeSH
• Glycolysis * MeSH
• Mitochondria genetics   metabolism   MeSH
• Diatoms cytology   enzymology   genetics   metabolism   MeSH
• Symbiosis MeSH
• Transformation, Genetic MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH