1. Joint Public Review:

    Hsiang-Chun Chang et al. investigated the role of ALR, component of the mitochondrial MIA40/ALR protein import apparatus, in cytosolic Fe/S cluster biogenesis performing loss-of-function (silencing) and gain-of-function (over-expression) experiments with MEFs (mouse embryonic fibroblast) and HEK293 (human embryonic kidney) cells. They find that downregulation of ALR impairs maturation of cytosolic Fe/S cluster proteins, while activities of mitochondrial Fe/S cluster proteins such as complex I and II are unaffected. Furthermore by reducing ALR expression cells up-regulate cellular iron transporter transferrin receptor 1 (Tfrc) and consequently cellular iron levels increase. The authors reveal that ALR down-regulation post-transcriptionally regulates Trfc through stabilization of Trfc mRNA mediated by IRP1, which is activated by absence of its mature Fe/S cluster. Additionally they demonstrate that only over- expression of full-length ALR, mainly located in the mitochondria and not the cytosolic short from ALR can reverse cytosolic Fe/S cluster maturation and therefore IRP1 activity and cellular iron levels. In the last part of their manuscript the authors present evidence about the mechanism by which ALR carries out this function. They find that ALR enables mitochondrial import of ABCB8 but not ABCB7, two mitochondrial proteins involved in the maturation of cytoplasmic Fe/S clusters. This transport into mitochondria requires functional MIA40/ALR in the IMS and further the TIM23 complex to the inner mitochondrial membrane. ABCB8 interacts directly with MIA40 by 5 cysteines (difulfide bond formation) and therefore these conserved cysteins are necessary for recognition and binding, which is not the case for ABCB7. These data add an interesting view on how ALR expression is linked to Fe/S cluster protein maturation, cellular iron homeostasis and their potential impact on related dieases.

    The strength of the manuscript are the well designed and performed experiments presenting evidence of how mitochondrial function of ALR is linked to the sulfur redox homeostasis and cellular iron regulation. Interestingly, reduction in cytsolic Fe/S cluster maturation and therefore increased cellular iron levels is also associated with increased sensitivity of cells to oxidative stress and this might be a plausible explanation for the previously described impact of full length ALR expression on oxidative stress in various disease models (PMID: 30579845).

    The drawn conclusions that the mechanistic studies about the role of ALR for Fe/S cluster maturation and cellular iron uptake may parallel the disease phenotype of patients with mutations in ALR gene GFER may be in parts speculative. The reported ALR mutations are varying and result either in partial functional or truncated protein expression (PMID: 20593814, PMID: 25269795). ALR is expressed in several isoforms (varying between two or three depending on the organ) of different size (15kDa, 21kDa, 23kDa). Most of the data showing the short form ALR (15kDa) solely in the cytosol and the full length ALR (23 kDa) as wells a second immuno-reactive band of 21 kDa ALR, both in cytosol and mitochondria (PMID: 30579845). While over-expressing full length ALR the authors show in the manuscript higher expression level in the cytosol than in the mitochondria fraction (w-blot, which is not reflected in the graph of Fig. S3 B). It was reported earlier that continuous over-expression of full length ALR in mammalian cells leads to the accumulation of full length ALR not only in the mitochondria but also in the cytosol (PMID: 23676665), which is also in agreement to observations of cytosolic occurrence of full length ALR (see above). This raises the question whether the conclusions made in the manuscript may be due to its cytosolic accumulation rather than or in addition to its mitochondrial localization. The presented study refers at several points to a study by Lange et al 2001 demonstrating that ALR rescues cytoplasmic Fe/S cluster maturation defects in Erv1- null yeast. There has been contradictory evidence published about the role of ALR in the maturation and export of cytosolic Fe-S cluster proteins. Lange et al. claimed that ALR interacts with Atm1 (an ABC transporter in the inner membrane of the mitochondria) and facilitates the export of Fe-S proteins to the cytosol. However, later it was suggested that, in yeast cells, ALR plays neither a direct nor an indirect role in cytosolic Fe-S cluster assembly and iron homeostasis. It is claimed that Iron homeostasis is independent of Erv1/Mia40 function in various yeast strains (Erv1 mutant) and that the finding by Lange et al. is based on only one Erv1 mutant strain, mainly due to strongly decreased glutathione (GSH) levels (PMID: 26396185).

    Additionally, this statement is reinforced by a study in human cells, demonstrating that depletion of ALR does not impact the maturation of cytosolic Fe-S proteins assembled via the CIA pathway (PMID: 25012650). Furthermore, this study in mammalian cells has pointed out the role of ALR in exporting MitoNEEt to the outer mitochondrial membrane (OMM). MitoNEEt is a Fe-S protein that is synthesized in the mitochondrial matrix. Upon synthesis, MitoNEEt translocates through the inner membrane of the mitochondria by ABCB7 and then through the IMS by ALR to the OMM where it contributes to cell proliferation (PMID: 25012650).

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  2. Evaluation Summary:

    This is an interesting manuscript and experiments generally make their point on Alr effects. However, additional data would strengthen the paper with respect to the relative roles of cytoplasmic vs mitochondrial isoforms as would mitochondrial function studies.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

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