Towards Subcellular Proteomic Maps in Model Marine Diatoms
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CitationTurnšek, Jernej. 2020. Towards Subcellular Proteomic Maps in Model Marine Diatoms. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
AbstractMarine phytoplankton forms the base of oceanic food webs and is integral to biogeochemical cycles that maintain Earth’s habitability. Marine diatoms are a diverse lineage of single-celled microalgae responsible for 20% of planetary primary production. Genome sequencing and genetic engineering techniques have established diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana as marine microbiology “yeasts” making them invaluable to modern research on cellular mechanisms of elemental cycling in marine environments. We here describe advances on fundamental aspects of diatom cell biology related to iron, carbon, and silicon metabolism.
We first present the foundational work to use engineered soybean ascorbate peroxidase APEX2 for subcellular proteomic profiling in diatoms. APEX2 permits spatially resolved proteomic mapping by oxidizing biotin-phenol to phenoxyl radicals which can covalently react with nearby proteins. Tagged proteins can be isolated and analyzed using mass spectrometry. Using APEX2-mediated proteomics, we cataloged vicinal proteins of Phaeodactylum tricornutum phytotransferrin, an environmentally ubiquitous endocytosed iron receptor. Proteins encoded by iron-sensitive genes expressed from a chromosomal gene cluster were identified and two of them colocalized with phytotransferrin adjacent to the chloroplast. This, along with their phylogenetic, domain, and biochemical analyses, suggests they are involved in intracellular iron processing.
Next, using fluorescence microscopy and circular dichroism we confirmed that PtEPYC1, an iron- and CO2-sensitive gene in P. tricornutum, encodes a chloroplast-localized disordered protein. Expression of its APEX2 fusion revealed a proteolytic pattern consistent with spacing of the identified protease cleavage sites. We hypothesize PtEPYC1 is involved in pyrenoid—RuBisCO-containing subchloroplastic organelle crucial for CO2 fixation—organization in P. tricornutum and provide insights that suggest this role is coupled to cell cycle control mechanisms.
Finally, we used bacterial conjugation to localize a biosilica-associated protein in Thalassiosira pseudonana and provide evidence for its expression as an APEX2 fusion. Biomineral morphogenesis proceeds inside a silica deposition vesicle (SDV), a hallmark, yet elusive, diatom compartment. T. pseudonana is a prime model system to study biosilicification which makes this result a valuable advance for the diatom community.
In summary, proximity proteomics holds enormous potential to glean new insights into subcellular proteomic composition in these evolutionarily, ecologically, and biotechnologically relevant microalgae.
Citable link to this pagehttps://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37365158
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