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  1. eLife assessment

    This study makes an important contribution to the function of the iron transporter Ferroportin (Fpn). By using a combination of proteoliposome assays, mutagenesis and structural studies by cryo EM, the authors are able to demonstrate that the H+-driven transporter for Fe2+-efflux is also capable of passive Ca2+ influx. The evidence supporting the conclusions is convincing, but the rate of Ca2+ influx and the physiological relevance of Ca2+ entry is yet to be established. The work will be of broad interest to cell biologists and biochemists.

  2. Reviewer #1 (Public Review):

    Ferroportin (Fpn) is a Fe2+/H+ antiporter that extrudes Fe2+ from cells and is important for iron homeostasis. Using a combination of proteoliposome assays, mutagenesis and structural studies by cryo EM the authors are aiming to demonstrate that the Fpn-transporter is also capable of Ca2+ influx, indicating a novel route for Ca2+ entry into cells.

    Strengthens: The paper combines a number of different methods to robustly demonstrate the interaction of Ca2+ with the iron transporter and to show translocation of Ca2+ is not pH dependent.

    Weaknesses: Fpn uses proton-gradient to drive Fe2+ efflux. The proposal is that the antiporter can also passively uptake Ca2+. This means that after Ca2+ release on the inside, Fpn would need to spontaneously rest to the outside again, which it has not evolved to do in the absence of Fe2+. To provide further support for Ca2+ uptake it is important to show that there are mutiple turnovers of the transporter, i.e., more kinetic information is needed,

    The impact of this paper is the demonstration that transporters (exchangers) can also operate as facilitative transporters for other substrates. The study also implies that Ca2+ can enter cells by this pathway, but if so the physiological context of this entry route needs further investigation and/or justification.

  3. Reviewer #2 (Public Review):

    In this manuscript the authors build upon their previous work describing the structure of the iron efflux pump ferroportin. Here they examine ferroportin's capacity to bind and transport calcium ions. Previous studies indicated a binding site for calcium in Fpn and suggested that calcium binding was needed for iron efflux, while other studies found no requirement for calcium in iron efflux. Here the authors use cryo-EM to structurally characterize a calcium-bound form of Fpn and compare this form to iron- and hepcicin-bound Fpn structures. Using site directed mutagenesis, they functionally characterized the calcium binding site and kinetics of calcium transport and its effects on Fe(II) and Co(II) transport. They report that Ca2+ is transported by Fpn proteoliposomes and Fpn-overexpressing HEK cells, that Ca2+ uptake has little effect on Fe/Co transport, and that Fe/Co efflux inhibits Ca transport.

    The data reported here appear to be of high quality and are convincing; the experimental design is excellent and the necessary controls are appropriately employed. A couple of issues need clarification in the text. FPN is clearly an iron efflux pump and these studies make clear that Fpn can also import Ca2+, although it does not appear to function as an Fe2+-Ca2+ antiporter. What is less clear is whether Fpn will transport calcium bi-directionally. A further question that needs explaining is why bind and transport calcium? Cells have a high capacity for calcium flux independent of Fpn. Is there a physiological importance to this activity?

  4. Reviewer #3 (Public Review):

    The major strength of this work is that the authors take a complementary approach to understand Ca2+ binding to ferroportin. Importantly, the following lines of evidence are used to establish Ca2+ binding - transport assays, cryo-EM structure, mutagenesis studies (using both transport of Ca2+ and ITC to measure direct binding). These all convincingly indicate that Ca2+ can indeed bind to ferroportin. The authors go on to show that Co2+ can inhibit binding of Ca2+ but not the converse. The authors need to take into account some prior in interpreting their data.

    I suggest the following considerations to improve the manuscript:

    1. Line 38-39 - the authors state that the S2 site has a more prominent role in iron transport than the S1 site. Billesboelle et al 2020 argues the converse based on the fact that mutations in the S2 site lead to iron overload diseases, suggesting that the S2 site cannot be the key site for iron transport. This is also seen in mutagenesis studies by Bonaccorsi etal FEBS J 2014, which reported that mutation of the S1 site completely abolished iron transport. The authors should consider these alternative models in addition to citing their prior work.

    2. It is unclear from the introduction/study why it is important to understand Ca2+ transport by ferroportin. Deshpande et al 2018 established that Ca2+ can regulate Fe2+ transport by ferroportin. While it is clear that Ca2+ binds ferroportin, I am not clear on why this is important from a biological perspective. The authors state that Ca2+ binding may integrate signaling with ferroportin activity, but this is not clearly explored, either with prior studies or in this study. If ferroportin acts as a uniporter, how would it be regulated to prevent inappropriate Ca2+ influx? Is there a clear reason why Ca2+ influx would integrate with iron biology? Overall, the premise of the study seems confusing to me despite the well done biochemistry/structural biology.

    3. Can the authors reliably exclude the ability of this new site to bind and/or transport other metals? The CryoEM structure at this resolution cannot reliably distinguish other possibilities (e.g. Zn2+), and it is possible that the observed effects are not specific to Ca2+.

    4. The maximal concentration of Ca2+ tested in Figure 4 is 500 micromolar - based on this, the authors indicate that Ca2+ has no effect on Fe2+ transport. This stands in contrast to work by Deshpande et al 2018 and Billesboelle et al 2020 which show that there is a Ca2+ effect on Fe2+ and Co2+ transport (though at higher concentrations). Have the authors tested higher Ca2+ concentrations? Given the extracellular concentration of Ca2+ (2 mM), this seems important.