Gruber, Ansgar


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Intracellular distribution of the reductive and oxidative pentose phosphate pathways in two diatoms

2009, Gruber, Ansgar, Weber, Till, Río Bártulos, Carolina, Vugrinec, Sascha, Kroth, Peter G.

Diatoms contribute a large proportion to the worldwide primary production and are particularly effective in fixing carbon dioxide. Possibly because diatom plastids originate from a secondary endocytobiosis, their cellular structure is more complex and metabolic pathways are rearranged within diatom cells compared to cells containing primary plastids. We annotated genes encoding isozymes of the reductive and oxidative pentose phosphate pathways in the genomes of the centric diatom Thalassiosira pseudonana and the pennate diatom Phaeodactylum tricornutum and bioinformatically inferred their intracellular distribution. Prediction results were confirmed by fusion of selected presequences to Green Fluorescent Protein and expression of these constructs in P. tricornutum. Calvin cycle enzymes for the carbon fixation and reduction of 3-phosphoglycerate are present in single isoforms, while we found multiple isoenzymes involved in the regeneration of ribulose-1,5-bisphosphate. We only identified one cytosolic sedoheptulose-1,7-bisphosphatase in both investigated diatoms. The oxidative pentose phosphate pathway seems to be restricted to the cytosol in diatoms, since we did not find stromal glucose-6-phosphate dehydrogenase and 6-phosphogluconolactone dehydrogenase isoforms. However, the two species apparently possess a plastidic phosphogluconolactonase. A 6-phosphogluconolactone dehydrogenase is apparently plastid associated in P. tricornutum and might be active in the periplastidic compartment, suggesting that this compartment might be involved in metabolic processes in diatoms.


Molecular Characterisation of Diatom Plastids

2008, Gruber, Ansgar

Diatoms are photoautotrophic, unicellular organisms, found in many aquatic habitats. Diatom plastids most likely evolved by secondary endocytobiosis, the uptake of a eukaryotic alga into another eukaryotic host cell and the subsequent evolutionary reduction and specialisation of this endosymbiont to a cell organelle. Diatom plastids differ from plastids of higher plants in many characteristics, e.g. they are surrounded by four (instead of two) membranes.
Plastids and mitochondria contain independent genomes that trace back to the genomes of their free living ancestors, cyanobacteria and Alphaproteobacteria, respectively. The ratio of plastid and mitochondrial genome copies to nuclear genome copies was determined via quantitative real-time polymerase chain reaction (qPCR). Fusion of a plastid targeted recombinase (RecA) to the green fluorescent protein (GFP) leads to selective labelling of plastid nucleoids in the diatom Phaeodactylum tricornutum.
Many essential photosynthesis enzymes are encoded in the nuclear genome, the gene products thus have to be imported into the plastids. A conserved sequence motif of unknown function ( ASAFAP -motif) within the N-terminal presequence of plastid preproteins is particularly important for the import reaction. The molecular characterisation of the conserved presequence motif allows conclusions on the targeting mechanisms involved and facilitates the prediction of plastid localised proteins on a genomic scale.
The genomes of the model diatoms P. tricornutum and Thalassiosira pseudonana have been sequenced completely. By analysis of the putative intracellular localisations of enzymes based on identified presequences, indications for a C4-like photosynthesis in P. tricornutum were found, and models for carbon concentrating mechanisms and CO2 fixation in P. tricornutum and T. pseudonana have been inferred. Peculiarly, multiple isoforms of enzymes of the Calvin cycle and glycolysis are present in the investigated diatoms. The isoforms differ in their presequences and are putatively active in the plastids, the mitochondria and the cytosol.
The exchange of metabolites between stroma, cytosol and other organelles is crucial for plastid function. Eight and six putative plastidic nucleotide transporters are encoded in the genomes of T. pseudonana and P. tricornutum respectively. Fusion proteins of nucleotide transporter presequences or full length fusions to GFP show that the investigated nucleotide transporters are plastid associated.


Protein targeting into complex diatom plastids: functional characterization of a specific targeting motif

2007, Gruber, Ansgar, Vugrinec, Sascha, Hempel, Franziska, Gould, Sven B., Maier, Uwe-G., Kroth, Peter G.

Plastids of diatoms and related algae evolved by secondary endocytobiosis, the uptake of a eukaryotic alga into a eukaryotic host cell and its subsequent reduction into an organelle. As a result diatom plastids are surrounded by four membranes. Protein targeting of nucleus encoded plastid proteins across these membranes depends on N-terminal bipartite presequences consisting of a signal and a transit peptide-like domain. Diatoms and cryptophytes share a conserved amino acid motif of unknown function at the cleavage site of the signal peptides (ASAFAP), which is particularly important for successful plastid targeting. Screening genomic databases we found that in rare cases the very conserved phenylalanine within the motif may be replaced by tryptophan, tyrosine or leucine. To test such unusual presequences for functionality and to better understand the role of the motif and putative receptor proteins involved in targeting, we constructed presequence: GFP fusion proteins with or without modifications of the "ASAFAP"-motif and expressed them in the diatom Phaeodactylum tricornutum. In this comprehensive mutational analysis we found that only the aromatic amino acids phenylalanine, tryptophan, tyrosine and the bulky amino acid leucine at the +1 position of the predicted signal peptidase cleavage site allow plastid import, as expected from the sequence comparison of native plastid targeting presequences of P. tricornutum and the cryptophyte Guillardia theta. Deletions within the signal peptide domains also impaired plastid import, showing that the presence of F at the N-terminus of the transit peptide together with a cleavable signal peptide is crucial for plastid import.