Proximity proteomics in a marine diatom reveals a putative cell surface-to-chloroplast iron trafficking pathway

• Published in: bioRxiv
• Main Authors: Turnšek, Jernej, Brunson, John K, Maria Del Pilar Martinez Viedma, Deerinck, Thomas J, Horák, Aleš, Oborník, Miroslav, Bielinski, Vincent A, Allen, Andrew E
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 04.02.2021
• Subjects: Ascorbic acid; Cell surface; Chloroplasts; Endocytosis; Iron; L-Ascorbate peroxidase; Nitrogen fixation; Peroxidase; Photosynthesis; Phylogeny; Proteins; Proteomics; Reactive oxygen species
• DOI: 10.1101/806539

Abstract Iron is a biochemically critical metal cofactor in enzymes involved in photosynthesis, cellular respiration, nitrate assimilation, nitrogen fixation, and reactive oxygen species defense. Marine microeukaryotes have evolved a phytotransferrin-based iron uptake system to cope with iron scarcity, a major factor limiting primary productivity in the global ocean. Diatom phytotransferrin is endocytosed, however proteins downstream of this environmentally ubiquitous iron receptor are unknown. We applied engineered ascorbate peroxidase APEX2-based subcellular proteomics to catalog proximal proteins of phytotransferrin in the model marine diatom Phaeodactylum tricornutum. Proteins encoded by poorly characterized iron-sensitive genes were identified including three that are expressed from a chromosomal gene cluster. Two of them showed unambiguous colocalization with phytotransferrin adjacent to the chloroplast. Further phylogenetic, domain, and biochemical analyses suggest their involvement in intracellular iron processing. Proximity proteomics holds enormous potential to glean new insights into iron acquisition pathways and beyond in these evolutionarily, ecologically, and biotechnologically important microalgae. Competing Interest Statement The authors have declared no competing interest. Footnotes * Additional enzymatic control experiments and structural comparison insights lead us to propose a non-enzymatic role for pTF.CREG1. This is in contrast to our initial submission where we hint at a possibility that pTF.CREG1 is an FMN-dependent reductase. * Glossary AA/AAs amino acid residue/amino acid residues APEX engineered ascorbate peroxidase (APX) CREG cellular repressor of E1A-stimulated genes DAB 3,3’-diaminobenzidine EYFP enhanced yellow fluorescent protein Fe’ dissolved labile iron (all unchelated iron species) FMN flavin mononucleotide HNLC high-nutrient, low-chlorophyll ISIP iron starvation induced protein MS mass spectrometry PBS phosphate-buffered saline pTF phytotransferrin RT room temperature TEM transmission electron microscopy TM transmembrane TMT tandem mass tag UTR untranslated region V-ATPase vacuolar-type H⁺-ATPase WT wild type