Mitochondria play a key role in iron metabolism in that they synthesize heme, assemble iron-sulfur (Fe/S) proteins, and participate in cellular iron regulation. Here, we review the latter two topics and their intimate connection. The mitochondrial Fe/S cluster (ISC) assembly machinery consists of 17 proteins that operate in three major steps of the maturation process. First, the cysteine desulfurase complex Nfs1-Isd11 as the sulfur donor cooperates with ferredoxin-ferredoxin reductase acting as an electron transfer chain, and frataxin to synthesize an [2Fe-2S] cluster on the scaffold protein Isu1. Second, the cluster is released from Isu1 and transferred toward apoproteins with the help of a dedicated Hsp70 chaperone system and the glutaredoxin Grx5. Finally, various specialized ISC components assist in the generation of [4Fe-4S] clusters and cluster insertion into specific target apoproteins. Functional defects of the core ISC assembly machinery are signaled to cytosolic or nuclear iron regulatory systems resulting in increased cellular iron acquisition and mitochondrial iron accumulation. In fungi, regulation is achieved by iron-responsive transcription factors controlling the expression of genes involved in iron uptake and intracellular distribution. They are assisted by cytosolic multidomain glutaredoxins which use a bound Fe/S cluster as iron sensor and additionally perform an essential role in intracellular iron delivery to target metalloproteins. In mammalian cells, the iron regulatory proteins IRP1, an Fe/S protein, and IRP2 act in a post-transcriptional fashion to adjust the cellular needs for iron. Thus, Fe/S protein biogenesis and cellular iron metabolism are tightly linked to coordinate iron supply and utilization. This article is part of a Special Issue entitled: Cell Biology of Metals.

Balance of physiological levels of iron is essential for every organism. In Aspergillus fumigatus and other fungal pathogens, the transcription factor HapX mediates adaptation to iron limitation and consequently virulence by repressing iron consumption and activating iron uptake. Here, we demonstrate that HapX is also essential for iron resistance via activating vacuolar iron storage. We identified HapX protein domains that are essential for HapX functions during either iron starvation or high-iron conditions. The evolutionary conservation of these domains indicates their wide-spread role in iron sensing. We further demonstrate that a HapX homodimer and the CCAAT-binding complex (CBC) cooperatively bind an evolutionary conserved DNA motif in a target promoter. The latter reveals the mode of discrimination between general CBC and specific HapX/CBC target genes. Collectively, our study uncovers a novel regulatory mechanism mediating both iron resistance and adaptation to iron starvation by the same transcription factor complex with activating and repressing functions depending on ambient iron availability.

Heme is an essential prosthetic group in proteins that reside in virtually every subcellular compartment performing diverse biological functions. Irrespective of whether heme is synthesized in the mitochondria or imported from the environment, this hydrophobic and potentially toxic metalloporphyrin has to be trafficked across membrane barriers, a concept heretofore poorly understood. Here we show, using subcellular-targeted, genetically encoded hemoprotein peroxidase reporters, that both extracellular and endogenous heme contribute to cellular labile heme and that extracellular heme can be transported and used in toto by hemoproteins in all six subcellular compartments examined. The reporters are robust, show large signal-to-background ratio, and provide sufficient range to detect changes in intracellular labile heme. Restoration of reporter activity by heme is organelle-specific, with the Golgi and endoplasmic reticulum being important sites for both exogenous and endogenous heme trafficking. Expression of peroxidase reporters in Caenorhabditiselegans shows that environmental heme influences labile heme in a tissue-dependent manner; reporter activity in the intestine shows a linear increase compared with muscle or hypodermis, with the lowest heme threshold in neurons. Our results demonstrate that the trafficking pathways for exogenous and endogenous heme are distinct, with intrinsic preference for specific subcellular compartments. We anticipate our results will serve as a heuristic paradigm for more sophisticated studies on heme trafficking in cellular and whole-animal models.

Iron is an essential, yet at elevated concentrations toxic trace element. To date, the mechanisms of iron sensing by eukaryotic iron-responsive transcription factors are poorly understood. The Saccharomyces cerevisiae transcription factor Yap5, a member of the Yap family of bZIP stress response regulators, administrates the adaptive response to high-iron conditions. Despite the central role of the iron-sensing process for cell viability, the molecule perceived by Yap5 and the underlying regulatory mechanisms are unknown. Here, we show that Yap5 senses high-iron conditions by two Fe/S clusters bound to its activator domain (Yap5-AD). The more stable iron-regulatory Fe/S cluster at the N-terminal cysteine-rich domain (n-CRD) of Yap5 is detected in vivo and in vitro. Iron is a key trace element for virtually all organisms. It functions as an essential cofactor in electron transfer, metabolite biosynthesis, DNA metabolism, and protein translation. Although iron is highly abundant, its bioavailability is low due to its poor solubility under ambient conditions (1, 2). For microbial pathogens, iron acquisition from their host cells is frequently crucial for their virulence. Hence, mutations in microbial genes involved in iron acquisition or utilization are associated with the loss of pathogenicity (3, 4). On the other hand, high intracellular iron levels are both a source and an amplifier of reactive oxygen species and thus highly toxic.Iron acquisition, transport and storage need to be tightly regulated, and disruption or deregulation of iron-related molecules can have significant health consequences. Hence, organisms have developed sophisticated and largely divergent strategies to acquire appropriate cellular iron levels and to avoid iron overloading (5-7). Except for vertebrates, which regulate cellular iron homeostasis mainly on the level of translation by the iron-regulatory proteins IRP1 and IRP2, transcriptional control of iron-responsive gene expression is widespread (7). In a large number of fungi, iron-responsive gene expression is conferred by the interplay of two conserved transcriptional repressors. Of these, the GATAtype transcription factor SreA regulates the expression of genes involved in iron uptake, while the basic leucine zipper (bZIP) transcription factor HapX regulates the iron-responsive expression of genes involved in iron-consuming pathways through interaction with the CCAAT-binding complex CBC (4,8).The model organism Saccharomyces cerevisiae utilizes three iron-responsive transcription factors. Aft1 and Aft2 play a central role in iron-dependent transcription activation of genes encoding components involved in cellular uptake and intracellular distribution of iron (9, 10). Yap5 is involved in the detoxification of excess iron by inducing the transcription of, e.g., CCC1 encoding the only known iron importer into the vacuole, the major metal storage organelle in fungi (11,12). Yap5 belongs to a fungus-specific family of stress response regulators that contains eight members in S. cerevisiae (...

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