The human mitochondrial ISCA 1 , ISCA 2 , and IBA 57 proteins are required for [ 4 Fe-4 S ] protein maturation

Members of the bacterial and mitochondrial iron–sulfur cluster (ISC) assembly machinery include the so-called A-type ISC proteins, which support the assembly of a subset of Fe/S apoproteins. The human genome encodes two A-type proteins, termed ISCA1 and ISCA2, which are related to Saccharomyces cerevisiae Isa1 and Isa2, respectively. An additional protein, Iba57, physically interacts with Isa1 and Isa2 in yeast. To test the cellular role of human ISCA1, ISCA2, and IBA57, HeLa cells were depleted for any of these proteins by RNA interference technology. Depleted cells contained massively swollen and enlarged mitochondria that were virtually devoid of cristae membranes, demonstrating the importance of these proteins for mitochondrial biogenesis. The activities of mitochondrial [4Fe-4S] proteins, including aconitase, respiratory complex I, and lipoic acid synthase, were diminished following depletion of the three proteins. In contrast, the mitochondrial [2Fe-2S] enzyme ferrochelatase and cellular heme content were unaffected. We further provide evidence against a localization and direct Fe/S protein maturation function of ISCA1 and ISCA2 in the cytosol. Taken together, our data suggest that ISCA1, ISCA2, and IBA57 are specifically involved in the maturation of mitochondrial [4Fe-4S] proteins functioning late in the ISC assembly pathway. INTRODUCTION Owing to the ancient character (Huber et al., 2003) of Fe/S clusters, homologues to the bacterial proteins dedicated to the formation of these cofactors are found in mitochondria of eukaryotes. In humans, Fe/S protein biogenesis (reviewed in Rouault and Tong, 2008, and Sheftel et al., 2010a) is initiated in the mitochondria with the removal of sulfur from cysteine by the pyridoxal phosphate–dependent cysteine desulfurase complex Nfs1-Isd11. The persulfide intermediate must be further reduced to sulfide, which is probably accomplished through a ferredoxin electron transport system comprising ferredoxin reductase and ferredoxin 2 (Sheftel et al., 2010b). Sulfide is then combined to form a [2Fe-2S] cluster with ferrous iron (Fe2+) on the scaffold protein IscU (Tong and Rouault, 2006). The molecular progression of nascent cluster transfer from IscU to target apoproteins has not been fully elucidated. However, the initial steps in this transfer require an Hsp70-based chaperone system (Knight et al., 1998; Uhrigshardt et al., 2010) and the monothiol Grx5 (RodriguezManzaneque et al., 2002; Ye et al., 2010). Components involved in the later dissemination of de novo assembled clusters to specific apoproteins are only beginning to be characterized. Among other components of Fe/S protein assembly, the isc and suf operons of bacteria encode the so-called A-type iron–sulfur cluster (ISC) proteins IscA and SufA, respectively, which are involved in the maturation of bacterial [4Fe-4S] proteins (Johnson et al., 2005; Tan et al., 2009). An additional member of this family, ErpA, is specifically involved in isoprenoid biosynthesis (Loiseau et al., 2007). The molecular function of these proteins in Fe/S biogenesis has only been partially resolved, and some differing views have been proposed. Several reports suggest the A-type ISC proteins act as alternative scaffolds for de novo Fe/S cluster synthesis (Krebs et al., 2001; Ollagnier-de-Choudens et al., 2001; Gupta et al., 2009), whereas Monitoring Editor Thomas D. Fox Cornell University Received: Sep 9, 2011 Revised: Dec 21, 2011 Accepted: Jan 30, 2012 This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E11-09-0772) on February 9, 2012. *These authors contributed equally to the work. Address correspondence to: Roland Lill (lill@staff.uni-marburg.de). © 2012 Sheftel et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,“ “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology. Abbreviations used: cAco, cytosolic aconitase; COX, cytochrome c oxidase; DPYD, dihydropyrimidine dehydrogenase; EM, electron microscopy; FC, ferrochelatase; GPAT, glutamate phosphoribosylpyrophosphate amidotransferase; IRE, iron-responsive element; IRP, iron regulatory protein; ISC, iron-sulfur cluster; LDH, lactate dehydrogenase; mAco, mitochondrial aconitase; RNAi, RNA interference; SAM, S-adenosyl methionine; SDH, succinate dehydrogenase; UTR, untranslated region. http://www.molbiolcell.org/content/suppl/2012/02/06/mbc.E11-09-0772.DC1 Supplemental Material can be found at: