Iron (Fe) likely played an important role in the origin of life (Bonfio et al., 2017; Jin et al., 2018) and is fundamental to almost all extant metabolisms (Aguirre et al., 2013) serving as a cofactor in enzymes involved in DNA replication, photosynthesis, cellular respiration, nitrate assimilation, nitrogen fixation, and reactive oxygen species defense (Crichton, 2016). Early anoxic oceans were rich in readily bioavailable ferrous, Fe²⁺, iron, but with the rise of oxygenic photosynthesis and the Great Oxygenation Event (GOE) in the Paleoproterozoic ~2.3 billion years ago followed by the Neoproterozoic Oxygenation Event (NOE) ~1.5 billion years later, most of the ferrous iron oxidized into insoluble ferric, Fe³⁺, minerals which are not bioavailable (Camacho et al., 2017; Knoll et al., 2016; Och and Shields-Zhou, 2012). Conceivably, these large global shifts in ocean chemistry could have had a major role in driving the evolution of novel iron uptake mechanisms.

Iron availability limits primary productivity in high-nutrient, low-chlorophyll (HNLC) regions which cover ~25% of the modern ocean habitat as demonstrated by numerous large-scale Fe fertilization experiments and natural Fe upwelling events invariably resulting in diatom-dominated phytoplankton blooms (Ardyna et al., 2019; de Baar, 2005; Martin et al., 1994). Although dissolved Fe in marine environments is primarily found complexed to organic ligands (Hutchins and Boyd, 2016; Tagliabue et al., 2017) such as bacterially produced siderophores and hemes (Boiteau et al., 2016; Hogle et al., 2014), low (pM) amounts of unchelated labile Fe, Fe’, are crucial for eukaryotic phytoplankton, particularly diatoms (Morel et al., 2008), widespread single-celled microalgae critical to oceanic primary production (Benoiston et al., 2017). Diatoms have convergently evolved phytotransferrin (pTF), which serves as the basis of a high-affinity ferric, Fe³⁺, iron binding, and acquisition pathway (McQuaid et al., 2018; Morrissey et al., 2015). Phytotransferrin sequences have a broad taxonomic distribution and are abundant in marine transcriptomic datasets (Bertrand et al., 2015; Marchetti et al., 2012).

Phytotransferrins are estimated to have emerged concurrently with the NOE exemplifying the link between large environmental changes and molecular innovation (McQuaid et al., 2018). They are phylogenetically related to Fe-assimilation (FEA) domain-containing proteins found in freshwater and marine single-celled algae, and represent functional analogues of iron delivery proteins transferrins (Behnke and LaRoche, 2020; Cheng et al., 2004; McQuaid et al., 2018; Scheiber et al., 2019). pTF (Ensembl ID: Phatr3_J54465, UniProt ID: B7FYL2) from the model marine diatom Phaeodactylum tricornutum (Bowler et al., 2008) was first identified as an iron deficiency-sensitive gene and named ISIP2a (iron starvation induced protein 2a) (Allen et al., 2008), although its expression is relatively high in iron-replete conditions as well (Smith et al., 2016). pTF localizes to the cell surface and intracellular puncta, presumably endosomal vesicles (McQuaid et al., 2018), which is further supported by the presence of an endocytosis motif in the C-terminus of the protein (Lommer et al., 2012). In diatom pTF, dissolved labile ferric iron is coordinated synergistically with carbonate anion (CO₃^2-), a common feature of all transferrins (Cheng et al., 2004; McQuaid et al., 2018; Zak et al., 2002). Thus, decline in seawater carbonate concentrations due to ocean acidification caused by elevated atmospheric CO₂ may negatively impact this prevalent iron uptake system in diatoms and other marine phytoplankton (McQuaid et al., 2018).

While accessing and binding dilute dissolved labile ferric iron on the cell surface is important, its subsequent internalization and delivery to target sites within complex cellular milieu are also critical. Fe³⁺ is highly insoluble and conversion between Fe³⁺ and Fe²⁺ can lead to toxic reactive oxygen species causing damage to proteins, lipids, and nucleic acids (Cheng et al., 2004). Precise control of iron internalization and intracellular trafficking in either of its redox states is thus crucial for maintenance of cellular homeostasis (Philpott and Jadhav, 2019; Wang and Pantopoulos, 2011). Human ferric iron-laden transferrin (Tf) bound to transferrin receptor is internalized via clathrin-mediated endocytosis. Endosome acidification leads to carbonate protonation and Tf conformational change resulting in iron release. Fe³⁺ is then reduced by six-transmembrane epithelial antigen of prostate 3 (STEAP3), exported to the cytoplasm through divalent metal transporter 1 (DMT1; also NRAMP2) or Zrt- and Irt-like protein (ZIP) 14, and offloaded to iron chaperones for distribution to cellular iron sinks (Bogdan et al., 2016; Eckenroth et al., 2011; Ohgami et al., 2005; Philpott and Jadhav, 2019; Wang and Pantopoulos, 2011). In contrast, proteins that mediate intracellular allocation of internalized Fe³⁺ and conduct specific biochemical transformations downstream of diatom pTF are unknown. Elucidation of these pathways will advance our understanding of diatom adaptation to survival in iron-deficient ocean waters and provide insight into how climate change will impact diatom physiology and fitness.

To investigate the proximal proteomic neighborhood of P. tricornutum pTF, we employed APEX2, an engineered soybean ascorbate peroxidase that functions as a dual probe for electron microscopy and proximity proteomics (Hung et al., 2016; Lam et al., 2015; Martell et al., 2017). When fused with a protein of interest (bait protein), the ~27 kDa APEX2 enzyme permits spatially resolved proteomic mapping by oxidizing biotin-phenol to short lived (<1 ms) phenoxyl radicals which can covalently react with electron-rich amino acid residues—primarily tyrosine—on the surface of nearby endogenous proteins. Tagged proteins can then be isolated by purification with streptavidin beads and analyzed using mass spectrometry (MS). While the ‘biotinylation radius’ in APEX2 experiments is estimated to be in the 10–20 nm range, it should rather be seen as a ‘probability gradient’ where the likelihood of tagging decreases with distance away from the APEX2-tagged protein of interest (Gingras et al., 2019; Hung et al., 2016; Lam et al., 2015). Proteomic hits can therefore include vicinal proteins which interact with the APEX2-tagged bait protein indirectly in addition to direct interactors of varying strength (Lundberg and Borner, 2019). Tha ability to capture weakly and transiently interacting proteins in a biological process of interest is a major advantage APEX2 offers over traditional pull-down approaches (Gingras et al., 2019). Swaping biotin-phenol for diaminobendizine (DAB)—another APEX2 substrate—enables one to determine fusion protein localization via high-resolution electron microscopy making APEX2 a powerful bifunctional probe (Martell et al., 2017; Martell et al., 2012).

APEX2 and related enzyme-based chemical biology tools have over the past decade been applied to investigate cellular compartments and processes in diverse model systems including mammalian cell culture, yeast, protists, and plant protoplasts (Gingras et al., 2019; Trinkle-Mulcahy, 2019). APEX2 was recently used to map proteomes associated with post-Golgi vesicle trafficking and endocytosis (Del Olmo et al., 2019; Otsuka et al., 2019) as well as receptor-mediated signaling (Paek et al., 2017), successes that are all directly pertinent to the goals of our study.

Here, we provide further evidence for P. tricornutum phytotransferrin pTF endocytosis, demonstrate correct subcellular localization and activity of its APEX2 fusion, and identify nearly 40 likely proximal proteins through a proximity-dependent proteomic mapping experiment conducted in quintuplicate. To the best of our knowledge, this study represents the first application of APEX2 in a marine microbial model system (Figure 1). We then provide initial characterization, using dual fluorophore protein tagging fluorescence microscopy, phylogenetic, domain, and biochemical analyses, for three proteins expressed from a chromosomal gene cluster (pTF.CREG1, pTF.CatCh1, pTF.ap1) believed to act in association with endocytosed pTF downstream of ferric iron binding at the cell surface. Bacterially expressed recombinant pTF.CREG1 was found to be catalytically dead indicating it may have a regulatory, rather than enzymatic, role in pTF endocytosis, similar to functions proposed for human CREG1. Meanwhile, bioinformatic interrogation of pTF.CatCh1 and its localization pattern suggest it might be a chloroplast-associated iron-binding protein. Finally, we introduce a model for an intracellular ferric iron allocation pathway in P. tricornutum based on our proximity proteomics hits.

Figure 1

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In this study, we used the iron receptor protein phytotransferrin (pTF) to implement APEX2-mediated proximity-dependent proteomic mapping in the model marine diatom Phaeodactylum tricornutum. The resulting data provide several advances regarding the function of pTF and its putative protein interaction partners downstream of iron binding at the cell surface.

pTF (ISIP2a; iron starvation induced protein 2a) was identified in P. tricornutum as a marker for iron (Fe) limitation (Allen et al., 2008). It was subsequently shown to be involved in Fe acquisition (Morrissey et al., 2015) and to be widespread in marine and freshwater microeukaryotic phytoplankton communities (Bertrand et al., 2015; McQuaid et al., 2018; Tara Oceans Coordinators et al., 2018). In a breakthrough study, McQuaid et al., 2018, demonstrated that ISIP2a is a type of transferrin, phytotransferrin (pTF), that is crucial for acquisition of dissolved labile ferric, Fe³⁺, iron, a critical micronutrient for cellular biochemistry. McQuaid et al., 2018, showed that pTF is essential for high-affinity iron uptake in P. tricornutum, and that carbonate, CO₃^2-, and Fe³⁺ interact synergistically to control the iron uptake rate. The finding that carbonate anions are required for the activity of a key diatom iron transport system is noteworthy and suggests that ocean acidification might inhibit microbial iron uptake. The occurrence of transferrin in marine diatoms and other single-celled marine and freshwater phytoplankton raises significant questions concerning its evolutionary origin and cellular role.

McQuaid et al., 2018 observed pTF in intracellular puncta, showed that the clathrin-mediated endocytosis inhibitor Pitstop 2 reduces iron (Fe) uptake rates, visualized vesicles after adding iron to an iron-limited WT P. tricornutum culture stained with the membrane dye FM 1–43, but our MDY-64 labeling data provide the first direct evidence that diatom pTF is indeed associated with intracellular membranous compartments. Further work is needed to elucidate pTF dynamic upon cellular internalization—especially after iron addition to iron-deplete cells which mimicks oceanic iron repletion events—and its trafficking in and potential exit from the endomembrane system in relation to cell cycle phases and light-dark cycles (Mayle et al., 2012; Naslavsky and Caplan, 2018; Smith et al., 2016).

While APEX2 was expected to be active in P. tricornutum given previous heterologous expression of functional horseradish peroxidase (HRP) in the model marine diatom Thalassiosira pseudonana (Sheppard et al., 2012), it was unclear whether endogenous peroxidases would contribute to the background signal in various APEX2 assays. In our Amplex UltraRed assay experiments, an order of magnitude higher signal-to-noise ratio (i.e. resurofin signal in pTF-APEX2 expressing versus WT P. tricornutum cells) was detected when live cells were reacted on ice as opposed to on room temperature, suggesting endogenous P. tricornutum peroxidases, but not exogenous APEX2, are largely inhibited on ice. This is notable as it indicates that performing APEX2 assays on ice may represent a generalizable experimental strategy for mesophilic microeukaryotic phytoplankton model systems.

Identifying Amplex UltraRed assay conditions which minimized WT background was promising, but not a guarantee that some endogenous enzymes would not be able to process other APEX2 substrates, such as those crucial for electron microscopy (3,3’-diaminobenzidine/DAB) and proximity proteomics (biotin-phenol). Indeed, mitochondrial background signal was detected in our electron microscopy experiments both in WT and pTF-APEX2 expressing cells, and our P. tricornutum proteome analysis revealed candidate endogenous APEX2-like peroxidases that could explain this result. Nevertheless, C-terminal tagging of pTF with APEX2 resulted in the expected cell surface and what appeared to be vesicular fusion protein localization away from mitochondria indicating no spatial overlap between endogenous APEX2-like enzymes and pTF-APEX2. Inconsistent size and shape of vesicle-like structures observed in our electron micrographs were conceivably due to a combination of arbitrary cell orientation inside agar blocks (where diatom cells were embedded) and the exact z position of ultramicrotome cut sites. On the whole, APEX2-based imaging is complementary to super-resolution microscopy in diatoms and represents a basis for electron tomography applications (Gröger et al., 2016; Sengupta et al., 2019). Some off-target biotinylation in our proximity labeling experiments was likely due to background mitochondrial peroxidase activity as indicated by the presence of mitochondrial proteins in our mass spectrometry data. Their APEX2/WT ratios close to one give us more confidence that those with elevated ratios above 1.5 were enriched due to APEX2 activity. Many of the latter have functional annotations for proteins we might expect to play a role in endosomal iron assimilation, and many have previously been shown to have iron-sensitive transcripts, both of which further support this notion. Future efforts to express pTF tagged with N-terminal APEX2, which should lead to intravesicular proximity labeling according to our pTF orientation prediction, optimizing conditions for APEX2 reactivation in iron-deplete conditions with hemin chloride, and more cell wall-permeant APEX2 probes (Li et al., 2020) will allow for identification of additional candidate proteins. Considering iron metabolism genes in P. tricornutum, including pTF, are highly expressed at night, proteomic maps with improved spatial and temporal resolution may emerge from proximity-dependent proteomic mapping experiments conducted with synchronized cell lines sampled at different timepoints throughout diel cycle (Huysman et al., 2014; Lundberg and Borner, 2019; Smith et al., 2016). The successful implementation of TurboID—an engineered biotin ligase for proximity proteomics (Branon et al., 2018)—in model plants Arabidopsis thaliana (Kim et al., 2019; Mair et al., 2019) and Nicotiana benthamiana (Zhang et al., 2019) suggests its adoption in existing and emerging single-celled marine phytoplankton model systems, including diatoms, is likely (Faktorová et al., 2020; Falciatore et al., 2020). TurboID is an iron-independent protein and could thus be particuarly valuable to further advance iron metabolism studies in P. tricornutum. These proposed improvements and alternative experimental approaches should cross-validate the proteins identified in this study as well as illuminate new ones.

Functionally uncharacterized gene cluster on chromosome 20 we zoomed in on is transcriptionally upregulated in iron-deplete (Allen et al., 2008; Smith et al., 2016) and silicic-acid-replete (Sapriel et al., 2009) conditions corroborating a known link between iron and silicon metabolism in diatoms (Brzezinski et al., 2015; Durkin et al., 2012; Hutchins and Bruland, 1998; Leynaert et al., 2004). Physically clustered functionally related genes are a hallmark of prokaryotic biology and operon-like genomic elements have been described in fungi and plants (Nützmann et al., 2018; Osbourn and Field, 2009); however, identifying and characterizing them in marine diatom genomes is in its relative infancy. For example, genes encoding light harvesting fucoxanthin-chlorophyll-binding proteins (FCPs) are co-located on chromosome 2 in P. tricornutum (Bhaya and Grossman, 1993), and a compact ~7 kbp gene cluster encoding enzymes for domoic acid neurotoxin production was characterized in a cosmopolitan diatom Pseudo-nitzschia multiseries (Brunson et al., 2018). Given that diatom genomes have likely been extensively shaped by horizontal gene transfer (Bowler et al., 2008; Diner et al., 2017), it is interesting to note that some yeast species acquired iron acquisition-enabling operon-like genomic elements via horizontal operon transfer (HOT) from an ancient bacterium (Kominek et al., 2019). Our phylogenetic analyses suggest all three proteins—pTF.CREG1, pTF.CatCh1, pTF.ap1—encoded by this locus are present in diatoms and other (marine) microeukaryotes, while pTF.CREG1 homologs are found across the tree of life. They, alongside the rest of our proteomic data, paint an emerging view of intracellular ferric, Fe³⁺, iron processing events downstream of its phytotransferrin-mediated binding at the cell surface.

In humans, endosome acidification causes structural rearrangements in the transferrin protein and protonation of synergistically coordinated carbonate anion leading to ferric iron release (Cheng et al., 2004). This event is concurrent with or followed by endosomal reductase STEAP3-catalyzed ferric iron reduction and ferrous, Fe²⁺, iron export through divalent metal transporter DMT1/NRAMP2 or Zrt- and Irt-like protein (ZIP) 14 (Bogdan et al., 2016). Two putative proton, H⁺, pumps were present in our mass spectrometry data: Phatr3_J23497—predicted as a P-type ATPase—and Phatr3_J27923—a vacuolar-type H⁺-ATPase subunit A. Multisubunit V-ATPases regulate pH homeostasis in virtually all eukaryotes and are known to function as endosome acidifying protein complexes (Finbow and Harrison, 1997; Maxson and Grinstein, 2014), which makes Phatr3_J27923 a candidate protein involved in acidification of phytotransferrin-rich endosomes in P. tricornutum. Notably, V-ATPase transcripts were overrepresented in diatoms following iron enrichment, possibly to account for an increase in the number of endosomes associated with pTF-driven iron acquisition (Marchetti et al., 2012), and V-ATPase was shown to be involved in diatom silica deposition vesicle (SDV) acidification (Hildebrand et al., 2018; Yee et al., 2020). Endosome acidification, perhaps via V-ATPase, may facilitate phytotransferrin-bound ferric, Fe³⁺, iron release, possibly for processing by an iron reducing enzyme similar to the human metalloreductase STEAP3.

Human cellular repressor of E1A-stimulated genes 1 (CREG1) is a glycoprotein involved in embryonic development, growth, differentiation, and senescence as part of the endosomal-lysosomal system (Ghobrial et al., 2018; Schähs et al., 2008). Contrary to evolutionarily related flavin mononucleotide (FMN)-binding split barrel fold oxidases and oxidoreductases such as pyridoxine 5′-phosphate (PNP) oxidase, an enzyme involved in vitamin B₆ metabolism (Musayev et al., 2003), CREG1 forms a homodimer unable to bind FMN in vitro suggesting it does not act as an enzyme in vivo (Sacher et al., 2005). The lack of FMN in human CREG1 is due to a short loop protruding into the FMN-binding pocket at the dimer interface, and aspartic acid and methionine amino acid residues replacing the corresponding two yeast PNP oxidase (PDB ID: 1CI0) arginines responsible for binding the terminal FMN phosphate (Sacher et al., 2005).

CREG1-like protein pTF.CREG1 was proposed to be central to sustained growth of iron-limited P. tricornutum cells (Allen et al., 2008) or to serve as a cell surface iron receptor (Lommer et al., 2012). CREG transcripts were more common in diatom transcriptomes from the Southern Ocean as opposed to non-Southern Ocean regions indicating these proteins are important in coping with iron limitation in this major high-nutrient, low-chlorophyll (HNLC) zone (Moreno et al., 2018). This may also explain why more diatom homologs are not present in our phylogenetic tree as transcriptomes from the Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP) database used in our phylogenetic analysis were not obtained from iron-limited cultures (Caron et al., 2017; Keeling et al., 2014). Our protein feature analysis revealed a signal peptide and multiple putative glycosylation sites which suggests that pTF.CREG1 enters the secretory pathway (Bard and Chia, 2016). While the nature of pTF.CREG1 recruitment to and colocalization with pTF-rich endosomes in the vicinity of chloroplast periphery—as suggested by our fluorescence microscopy results—is unclear, it is possible that pTF.CREG1-containing vesicles intersect with pTF-rich endosomes en route through the secretory pathway. Vesicular trafficking is highly dynamic and bidirectional (Naslavsky and Caplan, 2018; Progida and Bakke, 2016), and early endosomal traffic consists of sequential vesicle fusion events (Brenner, 2012), so this is at least in principle plausible. Punctate pTF.CREG1 colocalization with pTF suggests that this protein acts in an endosomal-lysosomal system, and this localization pattern is consistent with the observation that proteins with low confidence ASAFind chloroplast prediction (such as pTF.CREG1) can be associated with ‘blob-like structures’ (BLBs) adjacent to, but not inside, the chloroplast (Gruber et al., 2015; Kilian and Kroth, 2005). Given that our transgenic colocalization P. tricornutum cell lines were neither synchronized nor grown in iron-deplete conditions, it is plausible the protein also acts closer to the cell surface. Likely, but not confirmed, APEX2 orientation away from the cell surface and endosome interior in our proximity labeling experiments, and the reported inability of APEX2-generated phenoxyl radicals to penetrate endomembranes (Rhee et al., 2013), would suggest that pTF.CREG1—at some point during its recruitment to pTF-rich endosomes—has to be exposed to the cytosol to promote efficient biotinylation. Alternatively, it is impossible to rule out the possibility that lipid composition of P. tricornutum endomembranes is permissive to phenoxyl radical diffusion (Zulu et al., 2018).

Domain annotation in pTF.CREG1 (UniProt ID: B7G9B3) encouraged us to perform amino acid sequence and structural alignments with human CREG1 (UniProt ID: O75629, PDB ID: 1XHN). These analyses revealed that pTF.CREG1 lacks at least one arginine amino acid residue required for terminal FMN phosphate binding and likely contains an FMN binding pocket-occluding loop, two key features delineating human CREG1 from the yeast PNP oxidase (Sacher et al., 2005). Our hypothesis that pTF.CREG1 is unable to coordinate FMN was initially supported by our protein purifications which were not yellow as expected from a typical flavoprotein (Theorell, 1935), and was subsequently strengthened by their catalytic inactivity upon supplementation with biologically relevant flavin cofactors. The demonstrated lack of enzymatic activity in pTF.CREG1 and localization data from in vivo fluorescent reporter experiments represent an important extension of the human CREG1 literature to the single-celled eukaryotic phytoplankton field.

Secreted glycosylated human CREG1 was shown to delay G1/S cell cycle phase transition via direct interaction with mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R), a receptor internalized via clathrin-mediated endocytosis (Di Bacco and Gill, 2003; Ghosh et al., 2003; Han et al., 2009). This cellular growth inhibition was demonstrated to, at least partially, depend on a tetrapeptide motif within an FMN binding pocket-occluding loop (Sacher et al., 2005). In conjunction with our amino acid sequence alignments and structural prediction which point to the conservation of these two features in pTF.CREG1, these studies lend additional support to our data suggesting colocalization and potential direct physical association between phytotransferrin and pTF.CREG1 is non-enzymatic in nature. Internalization of the human transferrin-transferrin receptor complex is not a constitutive, but rather a regulated kinase-activity-dependent process (Cao et al., 2016). We currently hypothesize that pTF.CREG1 may serve as a ‘checkpoint’ to keep phytotransferrin endocytosis at a low level in iron-replete conditions, but when iron is scarce instead positively regulate this pathway. Indeed, transcriptomics experiments show that a second transcriptional peak for pTF.CREG1 occurs in iron-deplete medium approximately 8 hr prior to the pTF peak, a transcriptional dynamic which would be consistent with our hypothesis (Figure 5—figure supplement 1; Smith et al., 2016). Given that glycosylation in human CREG1 appears to be important for interaction with its cognate receptor (Han et al., 2011; Sacher et al., 2005; Schähs et al., 2008), posttranslational modifications, perhaps glycosylation(s), of pTF.CREG1 may further drive this regulatory activity. The presence of Phatr3_J10972, an iron-insensitive pTF.CREG1 paralog, in the P. tricornutum genome suggests that CREG-like proteins are implicated in diverse endocytosis pathways in single-celled eukaryotic phytoplankton. However, the association between iron-sensitive pTF.CREG1 and phytotransferrin in P. tricornutum implies a specific pTF.CREG1 function in non-reductive iron uptake. It will be very interesting to define the exact pTF.CREG1 role in further cellular and mechanistic studies and compare it to that of its homologs in multicellular organisms. Conceivably, an ancient protein like pTF.CREG1 could have remained at the core of linking cell cycle progression, cellular growth, and nutrient homeostasis across the tree of life.

In contrast to pTF.CREG1, no functional annotation could be assigned to and no structural homology models were generated for pTF.CatCh1 in the Pfam database (El-Gebali et al., 2019) and by Phyre2, respectively. This protein has a predicted N-terminal signal peptide and numerous N- and O-glycosylation sites suggesting it enters and trafficks along the secretory pathway. The outermost complex plastid membrane in diatoms corresponds to the chloroplast endoplasmic reticulum (cER) and is continuous with the nuclear envelope (Gibbs, 1981; Prihoda et al., 2012). Nuclear-encoded P. tricornutum proteins destined for the periplastidial compartment (vastly reduced endosymbiont cytoplasm between the inner two and the outer two complex plastid membranes) are first cotranslationally imported into the cER lumen (Grosche et al., 2014; Peschke et al., 2013). Our microscopy results show that pTF.CatCh1 is localized on the chloroplast margin, perhaps anchored to the outer chloroplast (cER) membrane with its C-terminal transmembrane domain, thus raising a possibility that pTF.CatCh1 gets to its final destination via cotranslational membrane insertion.

The cysteine-rich domain contains one CXC and one CXXC motifs which are nearly invariably present in pTF.CatCh1 homologs. Two cysteines spaced by two non-cysteine amino acids (the CXXC motif) are commonly found in metal-binding proteins, including those that bind iron (Fomenko et al., 2008; Gladyshev et al., 2004). This motif in pTF.CatCh1 is preceded by an aspartic acid residue resulting in DCXXC which is known to be particularly permissive to binding divalent metal ions (Figure 7—figure supplement 1A; Banci et al., 2002; Banci et al., 2006). Histidine, tyrosine, methionine, aspartic acid, and glutamic acid residues can also be involved in iron cation coordination, but none of these are conserved across pTF.CatCh1 homologs (Figure 7—source data 1; Dokmanić et al., 2008). Given the nearly completely conserved C(X)XC motifs and additional six 100% conserved cysteine residues, it is conceivable they are critical to pTF.CatCh1 activity.

Based on the fluorescence microscopy data, the conserved cysteine amino acid residues and cysteine-containing motifs, and given that disordered protein domains—two are predicted to flank the pTF.CatCh1 transmembrane domain—are mediators of protein-protein interactions and play an important role in the formation of protein complexes (Uversky, 2015b; Uversky, 2016), we are considering two hypotheses for in vivo pTF.CatCh1 function.

First, pTF.CatCh1 may serve as a metallochaperone that binds ferrous, Fe²⁺, iron downstream of the endosomal ferric, Fe³⁺, iron reduction step, and directs it to the chloroplast interior. Metallochaperones are intracellular proteins responsible for protecting and guiding metal ions on their way to correct metalloenzyme sinks (O'Halloran and Culotta, 2000). They engage in protein-protein interactions and help prevent metal-induced toxicity caused by free radical-generating Fenton reactions which are especially common with iron and copper (Philpott and Jadhav, 2019; Rosenzweig, 2002; Valko et al., 2005).

Second, pTF.CatCh1 may recruit pTF to the chloroplast periphery via one of its two C-terminal disordered regions flanking the predicted transmembrane domain, and thus serve as a docking site for iron-laden pTF. It could achieve this via the ‘fly-casting mechanism’ which involves binding-induced protein folding (Shoemaker et al., 2000). The observed colocalization of pTF.CatCh1 with pTF may indicate that Fe³⁺ can be offloaded directly from pTF to pTF.CatCh1 which would imply an additional, non-endosomal, intracellular Fe³⁺ reduction step. Notably, a chloroplast-associated Fe³⁺ reduction pathway mediated by Fe³⁺ chelate reductase FRO7 is present in Arabidopsis thaliana (Jeong et al., 2008). Abberant cellular localization of heterologously expressed fluorescent protein fusions is a widely observed and acknowledged phenomenon (Jensen, 2012), but we note that pTF localization on the chloroplast margin insdistinguishable from that in this study was reported by McQuaid et al., 2018 suggesting it is biologically relevant. Similarly to pTF.CREG1, pTF.CatCh1 exhibits a second transcriptional peak in iron-deplete medium approx. 16 hr prior to the pTF peak (Figure 5—figure supplement 1). It would make sense to synthesize a protein critical for iron-laden pTF recruitment to the chloroplast margin prior to significant increase in basal pTF endocytosis.

All in all, our data suggest pTF.CatCh1 is a chloroplast margin-localized, pTF-associated, P. tricornutum protein with a possible role in chloroplast iron homeostasis. The two hypotheses are put forth here to help frame its further genetic, cellular, biochemical, and structural dissection.

Chloroplasts are the most iron-rich system in plant cells (López-Millán et al., 2016). Iron—in particular in the form of iron–sulfur (Fe–S) clusters—serves as an essential cofactor for the photosynthetic electron transfer chain, chlorophyll biosynthesis, and chloroplast protein import (Balk and Schaedler, 2014; Marchand et al., 2018; Soll and Schleiff, 2004). A complex vesicle-based system possibly in perpetual dynamic exchange with cytosolic vesicles is present in chloroplasts (Hertle et al., 2020; Lindquist and Aronsson, 2018), an endoplasmic reticulum to Golgi to chloroplast protein trafficking pathway exists (Radhamony and Theg, 2006; Villarejo et al., 2005), and additional hypotheses for such specialized vesicle-mediated plastid targeting of proteins are established (Baslam et al., 2016). Therefore, a direct link between endocytic internalization of phytotransferrin-bound iron and its offloading to or adjacent to chloroplasts as suggested by our results seems plausible. ‘Kiss and run’ mechanism that allows erythroid cell mitochondria to access iron via direct interaction with transferrin-rich endosomes further underscores this idea (Hamdi et al., 2016). Analysis of our mass spectrometry (MS) hits that were not co-expressed with pTF-mCherry reveals three additional proteins—ISIP1 (Phatr3_J55031), FBP1 (Phatr3_J46929), and FBAC5 (Phatr3_J41423)—that support the idea of a direct cell surface-to-chloroplast iron trafficking pathway in Phaeodactylum tricornutum.

ISIP1 (iron starvation induced protein 1; Phatr3_J55031) gene expression in P. tricornutum and other marine microeukaryotes is induced by iron limitation (Allen et al., 2008; Kazamia et al., 2018; Marchetti et al., 2012). P. tricornutum ISIP1—the second most enriched protein in our MS dataset—has a role in siderophore-bound iron uptake and colocalizes with phytotransferrin (pTF) adjacently to the chloroplast in an ‘iron-processing compartment’ (Kazamia et al., 2019; Kazamia et al., 2018). FBP1 (Phatr3_J46929)—iron-sensitive siderophore ferrichrome-binding protein with punctate intracellular localization and central to the carbonate-independent iron acquisition pathway in P. tricornutum (Allen et al., 2008; Coale et al., 2019)—was another highly enriched protein in our dataset indicating the internalization pathways for inorganic (i.e. phytotransferrin-bound) and organic (i.e. siderophore-bound) iron in P. tricornutum may be physically coupled intracellularly and directed to iron-accumulating vesicles close to the chloroplast. Metal homeostasis by such discrete membranous compartments is widespread in nature (Blaby-Haas and Merchant, 2014). Copper- and zinc-storing compartments—cuprosomes and zincosomes, respectively—are present in the model chlorophyte microalga Chlamydomonas reinhardtii (Aron et al., 2015; Hong-Hermesdorf et al., 2014; Merchant, 2019), and lipid-bound iron-accumulating ferrosome compartments exist in diverse bacteria and archaea (Grant and Komeili, 2020).

Finally, the most enriched protein in our MS dataset was FBAC5 (Phatr3_J41423)—iron-sensitive iron-independent class I fructose-biophosphate aldolase—which was shown to be localized to the P. tricornutum pyrenoid—proteinaceous RuBisCO-containing subchloroplastic organelle—and proposed to link Calvin-Benson-Bassham cycle activity with the CO₂ concentrating mechanism (Allen et al., 2012; Matsuda et al., 2017). FBAC5 is one of the most reliable markers for diatom iron stress as demonstrated by laboratory culture (Cohen et al., 2018; Lommer et al., 2012), microcosm incubation (Cohen et al., 2017a; Cohen et al., 2017b), and field studies (Bertrand et al., 2015); however, its exact role has remained elusive. Fructose-biphosphate aldolases (FBAs) are glycolytic enzymes known for their ability to moonlight (Boukouris et al., 2016), a term for proteins with multiple physiologically relevant functions encoded by one polypeptide chain (Jeffery, 2018). Moonlighting FBAs have roles in actin cytoskeleton dynamic, cellular growth arrest, and apoptosis (in human cancer cell lines; Gizak et al., 2019), transcriptional regulation (in yeast; Cieśla et al., 2014), and endosome acidification via direct interaction with vacuolar-type ATPase (in a mouse cell line; Merkulova et al., 2011). These reported non-catalytic FBA activities suggest that FBAC5 acting outside of the P. tricornutum chloroplast, perhaps in the endosomal-lysosomal system as our data would imply, is possible.

Our initial characterization of pTF.CREG1 and pTF.CatCh1, together with the discussion on the four other mass spectrometry hits, enable proposition of a model connecting cell surface ferric iron binding, internalization, and intracellular trafficking in Phaeodactylum tricornutum with many outstanding questions ripe for future investigation (Figure 8). Importantly, it remains to be determined which protein is responsible for intracellular ferric, Fe³⁺, iron reduction in P. tricornutum, activity that would be consistent with the non-reductive cell surface ferric, Fe³⁺, iron uptake proposed for phytotransferrin (pTF) (McQuaid et al., 2018; Morrissey et al., 2015). Perhaps notably, neither of the five proteins with clear ferric reductase annotation (FRE1–5, Phatr3 IDs: J54486, J46928, J54940, J54409, J54982) nor additional five hypothetical ferric reductases (Coale et al., 2019; Smith et al., 2016) were present in our mass spectrometry data. Further proximity-dependent proteomic mapping experiments will be essential for determining the full set of proteins in the non-reductive iron acquisition pathway in P. tricornutum as enzymatic Fe³⁺ reduction may happen later in the pathway and/or in a separate subcellular compartment.

Figure 8 with 1 supplement see all

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In conclusion, the identified proteins and the associated model we introduce in this work represent an important advance for the marine phytoplankton field, given that diatoms are central to the study of cellular iron homeostasis and its link to fluctuating iron levels in the ocean. The presented insights are vital as our understanding of intracellular iron trafficking is relatively incomplete even in significantly more established model systems such as mammalian cells, and our knowledge of chloroplast iron uptake is in its infancy (Blaby-Haas and Merchant, 2012; Philpott and Jadhav, 2019). We anticipate further molecular dissection of our proteomics dataset will not only provide details on the transformation dynamic of acquired ferric iron and its cellular sinks but will thereby also illuminate the extent to which phytotransferrin pathway mirrors the metazoan transferrin cycle.

Implementing the use of APEX2 in a model marine diatom is itself a significant and timely advance as the field is expanding and moving from broad -omics-based surveys to detailed molecular studies (Faktorová et al., 2020; Falciatore et al., 2020). APEX2 and related molecular tools hold great promise to dissect additional key subcellular compartments in diatoms such as pyrenoids and silica deposition vesicles (Figure 8—figure supplement 1; Barrett et al., 2021; Hildebrand et al., 2018; Matsuda et al., 2017; Wang and Jonikas, 2020), two exciting future avenues for these single-celled heterokonts uniquely positioned at the nexus of evolutionary cell biology and blue biotechnology.

All data generated or analyzed during this study are included in the manuscript and supporting files.

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Author details

1. Jernej Turnšek

   1. Biological and Biomedical Sciences, The Graduate School of Arts and Sciences, Harvard University, Cambridge, United States
   2. Department of Systems Biology, Harvard Medical School, Boston, United States
   3. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, United States
   4. Integrative Oceanography Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, United States
   5. Center for Research in Biological Systems, University of California San Diego, La Jolla, United States
   6. Microbial and Environmental Genomics, J. Craig Venter Institute, La Jolla, United States

   Present address

   Department of Plant and Microbial Biology, University of California, Berkeley, Howard Hughes Medical Institute, University of California, Berkeley, United States

   Contribution

   Conceptualization, including proposing the idea to implement APEX2 in diatoms, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft preparation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9056-3565

2. John K Brunson

   1. Microbial and Environmental Genomics, J. Craig Venter Institute, La Jolla, United States
   2. Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, La Jolla, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

3. Maria del Pilar Martinez Viedma

   Informatics, J. Craig Venter Institute, La Jolla, United States

   Present address

   PharmaMar S.A., Madrid, Spain

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - review and editing, performed expressing and purifying new, cleaner, pTF.CREG1 batches for biochemical assays

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8885-5008

4. Thomas J Deerinck

   National Center for Microscopy and Imaging Research, University of California San Diego, La Jolla, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology

   Competing interests

   No competing interests declared

5. Aleš Horák

   1. Biology Centre CAS, Institute of Parasitology, České Budějovice, Czech Republic
   2. University of South Bohemia, Faculty of Science, České Budějovice, Czech Republic

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization

   Competing interests

   No competing interests declared

6. Miroslav Oborník

   1. Biology Centre CAS, Institute of Parasitology, České Budějovice, Czech Republic
   2. University of South Bohemia, Faculty of Science, České Budějovice, Czech Republic

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization

   Competing interests

   No competing interests declared

7. Vincent A Bielinski

   Synthetic Biology and Bioenergy, J. Craig Venter Institute, La Jolla, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

8. Andrew Ellis Allen

   1. Integrative Oceanography Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, United States
   2. Microbial and Environmental Genomics, J. Craig Venter Institute, La Jolla, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Project administration, Writing - review and editing

   For correspondence

   aallen@ucsd.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5911-6081