Carbonate-sensitive phytotransferrin controls high-affinity iron uptake in diatoms

Abstract

In vast areas of the ocean, the scarcity of iron controls the growth and productivity of phytoplankton1,2. Although most dissolved iron in the marine environment is complexed with organic molecules3, picomolar amounts of labile inorganic iron species (labile iron) are maintained within the euphotic zone4 and serve as an important source of iron for eukaryotic phytoplankton and particularly for diatoms5. Genome-enabled studies of labile iron utilization by diatoms have previously revealed novel iron-responsive transcripts6,7, including the ferric iron-concentrating protein ISIP2A8, but the mechanism behind the acquisition of picomolar labile iron remains unknown. Here we show that ISIP2A is a phytotransferrin that independently and convergently evolved carbonate ion-coordinated ferric iron binding. Deletion of ISIP2A disrupts high-affinity iron uptake in the diatom Phaeodactylum tricornutum, and uptake is restored by complementation with human transferrin. ISIP2A is internalized by endocytosis, and manipulation of the seawater carbonic acid system reveals a second-order dependence on the concentrations of labile iron and carbonate ions. In P. tricornutum, the synergistic interaction of labile iron and carbonate ions occurs at environmentally relevant concentrations, revealing that carbonate availability co-limits iron uptake. Phytotransferrin sequences have a broad taxonomic distribution8 and are abundant in marine environmental genomic datasets9,10, suggesting that acidification-driven declines in the concentration of seawater carbonate ions will have a negative effect on this globally important eukaryotic iron acquisition mechanism.

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Figure 1: Transferrin and phytotransferrin are functional analogues with a common origin.
Figure 2: Characterization of phytotransferrin ISIP2A (P. tricornutum).
Figure 3: The synergistic interaction of Fe′ and CO32− determines Fe′ uptake rates.
Figure 4: pH and CO2 manipulation of P. tricornutum induces expression of ISIP2A.

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Acknowledgements

We thank J. Badger for early contributions to phylogenetic analyses, A. Dickson for pH analysis, K. Forsch for CSV measurements and E. Bertrand for trace-metal clean techniques. This study was supported by the National Science Foundation (NSF-MCB-1024913, NSF-ANT-1043671 and NSF-OCE-0727997), United States Department of Energy Genomics Science program (DE-SC00006719 and DE-SC0008593), and the Gordon and Betty Moore Foundation grant GBMF3828 (A.E.A.); NSF-1557928 (A.B.K.); and the Czech Science Foundation, project 15-17643S (M.O. and A.H.).

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J.B.M., A.B.K., M.O. and A.E.A. designed the study and interpreted the results. J.B.M., M.O. and A.H. generated and analysed phylogenetic and molecular clock data. J.B.M. and B.J.K. generated mutant cell lines, J.B.M. and A.B.K. with assistance from K.A.B. performed physiology experiments. J.B.M. performed microscopy, and H.Z. performed western blots. T.K. and A.J.A. analysed inorganic carbon species. J.B.M. and J.P.M. conducted statistical analyses. J.B.M. wrote the paper with input from A.E.A., A.B.K., M.O., J.P.M, A.J.A. and K.A.B. All authors discussed the results and commented on the manuscript.

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Correspondence to Andrew E. Allen.

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Reviewer Information Nature thanks S. Amin, E. DeLong and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Conservation of active site amino acids.

a, Left, conservation of putative iron-coordinating (red) and carbonate-coordinating (green) amino acids among phylogenetic groups, filled circles indicate >85% conservation, unfilled circles indicate <20% conservation; arginine and lysine were permitted at the carbonate-binding site11. Right, logo tag detailing alignment conservation at the anion-binding region. Phosphonate-binding proteins (Bacteria and Thaumarchaea) retain the S/T-rich phosphonate-binding area, whereas transferrin (Euryarchaea and Metazoa) and phytotransferrin have the carbonate-coordinating K/R insertion. A downstream SAG region used to stabilize carbonate in transferrin24 shows some conservation in phytotransferrin, but an upstream threonine (−4 amino acids from the conserved Arg) is not present. b, Active site homology among ISIP2A (PT54465) and two transmembrane-anchored P. tricornutum homologues, PT45708 and PT54466. Amino acid distances based on PT54465, red/green triangles are iron- and carbonate-coordinating amino acids.

Extended Data Figure 2 Estimated divergence times between transferrin, phytotransferrin and PBPs.

Analyses were carried out using PhyloBayes (top) and BEAST (bottom). For the fossil calibration points used in generating the minimum and maximum constraints, see Supplementary Table 1. MYA, million years ago.

Extended Data Figure 3 Bayesian (PhyloBayes) phylogenetic tree contrasted with alternate topology derived using maximum likelihood.

Prasinococcus capsulatus, Pyraminomonas obovata and other chlorophyte algae have copies of transferrin and phytotransferrin, whereas all other non-chlorophyte algae have only phytotransferrin. Branches are colour-coded by phylogenetic group. Scale bar, 0.5 substitutions per position.

Extended Data Figure 4 Characterization of ISIP2A.

a, Western blot of wild-type P. tricornutum compared to ISIP2A-knockout strains (ΔISIP2A). The estimated mass of ISIP2A protein is 57 kDa. b, Specific growth rates of wild type compared to Δ ISIP2A. c, Uptake of 59Fe-ferrioxamine B is unaffected in Δ ISIP2A, suggesting an alternate pathway for uptake of iron–siderophore complexes. d, Effect of a clathrin-mediated endocytosis (CME) inhibitor on short-term 59Fe′ uptake rates, wild-type P. tricornutum. e, Upon addition of iron to iron-limited cells, the membrane-impermeable FM1-43 stain is internalized into vesicle-like inclusions. Scale bar, 5 μm. Pink is plastid auto-fluorescence. b, Data are mean ± s.e.m.; two-sided heteroscedastic t-test, n = biological replicates for wild-type and ISIP2A are indicated in brackets as (WT, ISIP2A) and P values are indicated. 12 pM Fe′ (5, 3), P = 0.008; 22 pM Fe′ (7, 5), P = 0.003; 43 pM Fe′ (6, 4), P = 0.006; 83 pM Fe′ (4, 4), P = 0.007; 165 pM Fe′ (3, 3), P = 0.07. d, Data are mean ± s.e.m.; n = 3 biological replicates; two-sided t-test, P = 0.009.

Extended Data Figure 5 Reconciliation of the carbonate effect with the influence of acidification on seawater iron chemistry.

a, Linearized representation of Fig. 3a, with high iron uptake rates (blue triangles) plotted on the secondary y axis, and pH values for each [CO32−] listed at top. Fe–FOB uptake rates decrease with pH, consistent with the findings of ref. 26, whereas Fe′ uptake rates increase with decreasing pH, inconsistent with the hypothesized effects of acidification on iron–EDTA chemistry26. This inconsistency is resolved when uptake rates are plotted as a function of the synergistic interaction between Fe′ and CO32− (Fig. 3b). b, c, Under CO2-induced acidification, the strong influence of pH on [Fe′] results in a significant correlation of uptake to [Fe′], although the interaction Fe′ and CO32− results in a better fit (Fig. 4a, solid line). d, e, When the change in [Fe′] is constrained relative to pH and [CO32−], uptake rates are positively correlated with [CO32−] and uncorrelated to [Fe′], revealing the influence of the carbonate ion on Fe′ uptake rates. For statistical analyses of the linear regressions, see Extended Data Table 4.

Extended Data Figure 6 Derivation of second-order and constitutive rate constants from P. tricornutum resuspended in NaHCO3-manipulated medium.

a, Regression of the interaction product of Fe′ and CO32−. Regression excludes the two observations in which the medium was not supplemented with NaHCO3. b, 59Fe uptake rates for treatments incubated with low (2–5 pM Fe′, open symbols) and high (20–50 pM Fe′, closed symbols) 59Fe versus [CO32−]. c, Demonstration of reproducibility at low [Fe′]. Data are mean ± s.e.m.; n = 3 biological replicates. d, Uptake rates normalized to [Fe′], plotted against [CO32−]. The slope has units of mol Fe cell−1 h−1 (M Fe′)−1 (M CO32−)−1, equivalent to the pseudo-first-order uptake rate with respect to carbonate. e, Rates of uptake calculated as a function of [Fe′] and [CO32−] versus measured rates.

Extended Data Table 1 Uptake rates compared to the measured (in bold) and derived concentrations of Fe′ and carbonic acid species, in Aquil uptake medium
Extended Data Table 2 Uptake rates compared to measured (in bold) and derived concentrations for both Fe′ and carbonic acid species
Extended Data Table 3 Measured (in bold) and derived values for CO2 and pH manipulation experiments
Extended Data Table 4 Statistical analyses of NaHCO3 and pH and CO2 manipulations

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McQuaid, J., Kustka, A., Oborník, M. et al. Carbonate-sensitive phytotransferrin controls high-affinity iron uptake in diatoms. Nature 555, 534–537 (2018). https://doi.org/10.1038/nature25982

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