Mechanistic and structural analysis of the function of the mitochondrial ABC transporter Atm1 in cellular iron-sulfur and iron metabolism
Final Report Abstract
Cellular iron-sulfur (Fe/S) protein fulfil important tasks in respiration, metabolism, protein translation, regulation, antiviral response, DNA synthesis and DNA repair. Assembly of the Fe/S cofactors and their insertion into apoproteins requires complex machinery. In mitochondria, the iron-sulfur cluster assembly (ISC) machinery matures all intra-organellar Fe/S proteins, yet is also indispensable for cytosolic and nuclear Fe/S protein assembly. The ISC system synthesizes a sulfur-, glutathione (GSH)- and iron-containing factor termed X-S that is exported by the mitochondrial ABC transporter Atm1 (human ortholog ABCB7) to the cytosol where X-S is used by early-acting components of the cytosolic iron-sulfur protein assembly (CIA) machinery for maturation of cytosolic and nuclear Fe/S proteins. The functional deficiency of Atm1 is not only associated with defects in cytosolic-nuclear Fe/S proteins but also a deregulation of cellular iron homeostasis showing that cells use the efficiency of the mitochondrial core ISC system and Atm1 to regulate cellular iron supply. Mutations in human ABCB7 cause the iron-storage disease X-linked sideroblastic anemia and cerebellar ataxia (XLSA/A). The phenotypes of this disease can be partly understood on the basis of the previous functional studies. The solved crystal structure of nucleotide-free Atm1 with and without bound glutathione provided avenues for studying the molecular function of Atm1 in more detail. Here, we have combined in vitro and in vivo studies to gain new insights into the biochemical mechanism of Atm1-mediated transport and into new aspects of its physiological role within the cell. In an attempt to better define the physiological substrate of Atm1, we show that maturation of cytosolic [2Fe-2S] proteins, a process poorly understood so far, require the core ISC machinery, glutathione and Atm1, but none of the CIA components. This is consistent with the export of a Fe-, sulfur-, and GSH- containing species by Atm1. In this view the CIA system is required to convert this species into a [4Fe-4S] cluster and specifically insert it into cytosolic and nuclear apoproteins. Despite extensive biochemical and mass spectroscopic approaches, the transported substrate could not be precisely defined, but the efforts are continuing. Structural studies on yeast Atm1 were expanded by using a combination of X-ray crystallography, cryogenic electron microscopy (cryo EM), and Hydrogen Deuterium exchange Mass Spectrometry (HDX-MS) to better understand the structural and mechanistic aspects and the dynamics of Atm1 function during the transport cycle. A new conformation of Atm1 in the inward-closed arrangement with bound AMP-PNP was obtained by cryo EM. This structure allowed the precise definition of the substrate binding pocket separated from the ATP binding site, and opened ongoing studies to model chemical compounds into the cavity to help theoretically defining the physiological substrate. The structural changes during the ATP-dependent closure of the nucleotide binding domains (NBDs) were closely reflected in HDX-MS experiments showing the interaction of the NBDs thereby preventing HDX. At the same time, parts of the transmembrane domain became more accessible because of structural rearrangements. The effects of GSH on HDX were minor indicating that this molecule has a tendency to binding in the cavity, but the binding specificity is low. Additional physiological roles of Atm1 were analyzed in human cells and in the pathogenic fungus Cryptococcus neoformans. Despite several reports that Atm1/ABCB7 executes an (ill-defined) role in heme metabolism and the apparent heme defect in XLSA/A, we could show that ABCB7-depleted cultured cells can generate heme normally to mature the hemedependent catalase and cytochromes. We therefore propose that the heme defect observable in Atm1/ABCB7 cells is indirect and is caused by the iron accumulation and oxidative stress condition in these cells. A new role of Atm1 in protection against copper toxicity was defined in C. neoformans. This organism overexpresses Atm1 in response to high copper sequestered by macrophages during infection. The increased Atm1 levels protect the fungal cytosolic Fe/S proteins, in particular those of the CIA machinery from copper toxicity defining a new physiological role of Atm1 in pathogenic fungi.
Publications
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(See online at https://doi.org/10.1042/bst20150118) - (2015). The role of mitochondria and the CIA machinery in the maturation of cytosolic and nuclear iron-sulfur proteins. Eur. J. Cell Biol. 94, 280-291
Lill, R., Dutkiewicz, R., Freibert, S.A., Heidenreich, T., Mascarenhas, J., Netz, D.J., Paul, V.D., Pierik, A.J., Richter, N., Stümpfig, N., Srinivasan, V., Stehling, O., & Mühlenhoff, U.
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Marcellino T., and Srinivasan V.
(See online at https://doi.org/10.1515/hsz-2016-0244) - (2017). Cryptococcus neoformans Iron-Sulfur Protein Biogenesis Machinery Is a Novel Layer of Protection against Cu Stress. mBio 8, e01742-17
Garcia-Santamarina, S., Uzarska, M.A., Festa, R.A., Lill, R., & Thiele, D.J.
(See online at https://doi.org/10.1128/mBio.01742-17) - (2017). Evolutionary conservation and in vitro reconstitution of microsporidian iron-sulfur cluster biosynthesis. Nat. Commun. 8, 13932
Freibert, S.A., Goldberg, A.V., Hacker, C., Molik, S., Dean, P., Williams, T.A., Nakjang, S., Long, S., Sendra, K., Bill, E., Heinz, E., Hirt, R.P., Lucocq, J.M., Embley, T.M., & Lill, R.
(See online at https://doi.org/10.1038/ncomms13932) - (2017). Iron-sulfur cluster biogenesis and trafficking in mitochondria. J. Biol. Chem. 292, 12754–12763
Braymer, J.J. & Lill, R.
(See online at https://doi.org/10.1074/jbc.R117.787101) - (2019). Depletion of thiol reducing capacity impairs cytosolic but not mitochondrial Fe/S protein assembly machineries. Biochim. Biophys. Acta 1866, 240-251
Braymer, J.J., Stümpfig, M., Thelen, S., Mühlenhoff, U., & Lill, R.
(See online at https://doi.org/10.1016/j.bbamcr.2018.11.003) - (2020). Mechanisms of mitochondrial iron-sulfur protein biogenesis. Annu. Rev. Biochem. 89, 471-499
Lill, R. & Freibert, S.A.
(See online at https://doi.org/10.1146/annurev-biochem-013118-111540) - (2020). Structural and functional diversity calls for a new classification of ABC transporters. FEBS Lett. 594, 3767– 3775
Thomas, C., Aller, S.G., Beis, K., Carpenter, E.P., Chang, G., Chen, L., Dassa, E., Dean, M., Duong Van Hoa, F., Ekiert, D., Ford, R., Gaudet, R., Gong, X., Holland, I.B., Huang, Y., Kahne, D.K., Kato, H., Koronakis, V., Koth, C.M., Lee, Y., Lewinson, O., Lill, R., Martinoia, E., Murakami, S., Pinkett, H.W., Poolman, B., Rosenbaum, D., Sarkadi, B., Schmitt, L., Schneider, E., Shi, Y., Shyng, S.L., Slotboom, D.J., Tajkhorshid, E., Tieleman, D.P., Ueda, K., Váradi, A., Wen, P.C., Yan, N., Zhang, P., Zheng, H., Zimmer, J., & Tampé, R.
(See online at https://doi.org/10.1002/1873-3468.13935) - (2021). Mechanistic concepts of ironsulfur protein biogenesis in Biology. Biochim Biophys Acta Mol Cell Res.
Braymer, J.J., Freibert, S.A., Rakwalska-Bange, M., & Lill, R.
(See online at https://doi.org/10.1016/j.bbamcr.2020.118863)
