2011, Vugrinec, Sascha, Gruber, Ansgar, Kroth, Peter G.

Plastids of diatoms are surrounded by four membranes. The outermost membrane is continuous with the endoplasmic reticulum and therefore is termed chloroplast ER (CER) membrane. The complex ultra structure of diatom plastids naturally requires more transport steps to import nucleus encoded proteins into the plastid compared to higher plant plastids which possess only two envelope membranes.
Several hypothetic models for the import of preproteins into the complex plastids of diatoms are discussed. Common to all these models is the postulation of a first cotranslational transport step into the chloroplast endoplasmic reticulum lumen via the Sec61 translocon. Furthermore, all models postulate transport via a translocator in the innermost membrane similar to the Tic complex (translocon of the inner chloroplasts envelope) of higher plant plastids. The models differ, however, with respect to their explanation of transport out of the CERlumen and into the interenvelope space: either translocators, vesicles crossing the periplastidic space or putative membrane channels connecting CERlumen and the interenvelope space have been proposed. To investigate the presence of such a hypothetic connection between the CERlumen and the interenvelope space, we expressed different preproteins in the diatom Phaeodactylum tricornutum that were fused to selfassembling fragments of GFP (GFP110 and GFP11). Complementary fragments were fused to marker proteins of the CERlumen and the interenvelope space, respectively. Our data indicate that the GFP110 and GFP11 fusion proteins are located in two separate compartments which are not connected to each other.

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.

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.