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All of the mentioned proteins are not predicted to be plastid targeted and were interestingly only disclosed in the Tig19 cell lysate fraction and neither in the PF nor in the MF, arguing for a redox regulatory role rather in the cytosol or the nucleus instead of serving in serving in regulation of photosynthesis.
Our work provides a novel tool to investigate mitochondrial respiration and photosynthesis of diatoms in organello , which will help us to broaden our understanding of diatom metabolism and its compartmentalization. Studies that may profit from the described methodology might address questions concerning organelle-specific metabolite uptake and release, pigment composition, post-transcriptional modifications or investigation of organellar DNA and gene regulation.
Also, comparative proteomics, metabolomics, transcriptomics or lipidomics on organelles derived from cultures grown with varying nutrition or illumination are imaginable and thus might help us to shed further light on organellar fate and functionality in diatoms. Moreover, we would like to raise the idea that organelle proteomes provided here might be used to improve meta-analyses that benefit from the report of experimental data sets.
As some of these proteins might be essential for photosynthetic organisms and the function of some is still unknown, future work should concentrate on revealing their role. The T.
Digestion of both flanking KpnI restriction sites allowed cloning into the T. Cell line number 19 TIG19 was used for further experiments. Four liters of early-stationary T. Cells were resuspended in 9 ml isolation buffer IB; Table 1 and resulting cell suspension was loaded to a pre-chilled French Press mini cell.
The homogenate was diluted and cautiously inverted for 30 s in a total volume of approximately 20 ml of washing buffer WB; Table 1. Cells and cell debris were sedimented by centrifugation using a swing-out bucket rotor TS This step was typically repeated 3 times during mitochondria isolation and once when plastids were isolated. Starting from a pooled organelle extract see Fig. Each pellet was gently resuspended in 2—4 ml WB see Table 1 before being pooled to a single SS34 centrifuge tube.
A maximum of 1—2 ml of organelle suspension was loaded on top of two continuous Percoll gradients each. The separated bands were evaluated by fluorescence microscopy and mitochondria of highest purity were extracted. The supernatant was discarded and the pellet was again dissolved carefully with a fine paint-brush in 2—4 ml of RB.
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Pooled organelle extract see Fig. The supernatant was discarded and the pellet was dissolved gently in 2—4 ml WB using a fine paint-brush.
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The supernatant was discarded and the pellet containing the final PF was dissolved carefully with a fine paint-brush in 2—4 ml of RB see Table 1. Glass beads with three different diameters 0. Homogenate was kept on ice for 5 min in between every cycle.
After homogenization, 0. Chlorophyll a Chl a content of either cells or isolated plastids was carried out spectrophotometrically with an Ultrospec Pro GE healthcare, Munich, Germany according to the method described by Jeffrey and Humphrey Protein yields were determined colorimetrically according to the Bradford protein assay kit catalog B, Sigma Aldrich; Munich, Germany, Bradford , Compton and Jones Relative absorbance was recorded at wavelength ranging from to nm with a bandwidth set to 1 nm.
Plastids and mitochondria isolation was monitored by fluorescence and light microscopy during the whole isolation process. Particular attention was paid to the French Press eluates and the Percoll gradient fractions. Microscopy was performed with a BX51 epifluorescence microscope Olympus; Tokyo, Japan at excitation and emission wavelengths for GFP of nm and nm, respectively. Chlorophyll autofluorescence was observed at wavelengths of nm excitation and nm emission. Each step was followed by a subsequent incubation step of 15 min on ice.
Prior to every experiment, the luminous source was adjusted, and the oxygen electrode was calibrated under consideration of daily atmospheric pressure. Mitochondrial respiration mitochondria fraction was measured in complete darkness in order to avoid light-driven oxygen production performed by residual thylakoidal contaminants. Therefore, 0. Light-dependent oxygen evolution PF was determined in the same buffer, but complemented with 40 mM of the artificial electron-acceptor potassium ferricyanide K 3 [Fe CN 6 ].
If not stated otherwise, final concentrations of substrates and inhibitors applied were: 5 mM malate, 5 mM succinate, 0. The O-J-I-P fluorescence transient measuring routine describes the stepwise increase in fluorescence during electron transport rate saturation upon application of a saturation multi turnover pulse, from the minimum fluorescence F o O via the inflection points J and I towards the maximum fluorescence F m P , and is characteristic for the reduction of the PSII electron acceptor site components Neubauer and Schreiber , Schreiber and Neubauer , Strasser and Srivastava When plastids were measured, 5 mM of dipotassium phosphate was added to the RB.
Proteins were transferred to a 0. After a successful blotting control with 0. Proteins were separated and stained as described above. Mitochondria samples were analyzed as whole lanes, while plastid and cell samples were cut into four separate gel slices 0—25 kDa, 25—35 kDa, 35—55 kDa, rest that were measured individually. A workflow of the annotation procedure is illustrated in Supplementary Fig. Mass spectrometric raw data were matched against the optimized, de-replicated T. Protein models that encoded identical proteins on different genomes e. Proteins that are potentially targeted to the mitochondria, the secretory or other pathways were predicted using the TargetP 1.
We thank Doris Ballert for performing biolistic bombardment and for routine cell cultivation, Dr. Joachim Hentschel, Dr. Michael Laumann, Dr. Further we grateful acknowledge Dr. Andreas Marquardt from the University of Konstanz Proteomics Centre PC for the mass spectrometric analysis of organelle and cell fractions. Oxford University Press is a department of the University of Oxford.
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Corresponding author: E-mail, Alexander. Schober uni-konstanz. Oxford Academic. Google Scholar. Annsophie Bischoff. Bernard Lepetit. Ansgar Gruber. Peter G Kroth. Alexander F. Cite Citation. Permissions Icon Permissions. Abstract 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. Open in new tab Download slide.
Table 1. Open in new tab. Evolution and metabolic significance of the urea cycle in photosynthetic diatoms. Search ADS. Whole-cell response of the pennate diatom Phaeodactylum tricornutum to iron starvation. Lateral heterogeneity in the distribution of chlorophyll-protein complexes of the thylakoid membranes of spinach chloroplasts.
The genome of the diatom Thalassiosira pseudonana : ecology, evolution, and metabolism. Energetic coupling between plastids and mitochondria drives CO 2 assimilation in diatoms. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Respiratory enzymes in oxidative phosphorylation: IV. The respiratory chain. The nature of the CO 2 -concentrating mechanisms in a marine diatom, Thalassiosira pseudonana.
High light stress triggers distinct proteomic responses in the marine diatom Thalassiosira pseudonana. Photosynthesis by isolated protoplasts, protoplast extracts, and chloroplasts of wheat: influence of orthophosphate, pyrophosphate, and adenylates. Isolation, purification, and characterization of mitochondria from Chlamydomonas reinhardtii. The intracellular distribution of inorganic carbon fixing enzymes does not support the presence of a C4 pathway in the diatom Phaeodactylum tricornutum.
The metabolic blueprint of Phaeodactylum tricornutum reveals a eukaryotic Entner-Doudoroff glycolytic pathway. Peptide and protein molecular weight determination by electrophoresis using a high-molarity tris buffer system without urea. Changes in relative thylakoid protein abundance induced by fluctuating light in the diatom Thalassiosira pseudonana. The thylakoid membrane proteome of two marine diatoms outlines both diatom-specific and species-specific features of the photosynthetic machinery. Google Preview.
Intracellular metabolic pathway distribution in diatoms and tools for genome-enabled experimental diatom research. Plastid proteome prediction for diatoms and other algae with secondary plastids of the red lineage. Cation-dependent changes in the thylakoid membrane appression of the diatom Thalassiosira pseudonana. New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton. An optimized protocol for the preparation of oxygen-evolving thylakoid membranes from Cyclotella meneghiniana provides a tool for the investigation of diatom plastidic electron transport.
The GreenCut2 resource, a phylogenomically derived inventory of proteins specific to the plant lineage. A model for carbohydrate metabolism in the diatom Phaeodactylum tricornutum deduced from comparative whole genome analysis. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Molecular dynamics of the diatom thylakoid membrane under different light conditions. Spectroscopic and molecular characterization of the oligomeric antenna of the diatom Phaeodactylum tricornutum. Proteins from each organelle co-fractionate and therefore exhibit similar distributions in the gradient. Protein distributions can be determined through a series of pair-wise comparisons of gradient fractions, using cleavable ICAT to enable relative quantitation of protein levels by MS. The localization of novel proteins is determined using multivariate data analysis techniques to match their distributions to those of proteins that are known to reside in specific organelles.
Using this approach, we have simultaneously demonstrated the localization of membrane proteins in both the endoplasmic reticulum and the Golgi apparatus in Arabidopsis. Localization of organelle proteins by isotope tagging is a new tool for high-throughput protein localization, which is applicable to a wide range of research areas such as the study of organelle function and protein trafficking.
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Pipeline for interpretation and storage of organelle proteomics data
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