In this paper, we describe the identification and initial characterization of the essential protein Cia1 as a novel component of the CIA machinery. Cia1 is required for cytosolic and nuclear Fe/S protein assembly and interacts with Nar1, another recently identified member of the CIA machinery. Depletion of Cia1 resulted in the loss of the enzyme activities of the cytosolic Fe/S proteins isopropylmalate isomerase (Leu1) and sulfite reductase. The decrease in the enzyme activities is explained by a requirement of Cia1 in the de novo assembly of the Fe/S cofactor in Leu1 as shown by in vivo 55
Fe labeling studies. The same experimental approach revealed that Fe/S cluster assembly was strongly decreased in the mostly cytosolic Rli1 and the nuclear Ntg2 Fe/S proteins. As expected from the extramitochondrial location of Cia1, both the enzyme activities and 55
Fe incorporation of mitochondrial Fe/S proteins were not negatively affected. Surprisingly, 55
Fe incorporation into the predominantly cytosolic Fe/S proteins Nar1 and Nbp35 was also not decreased following depletion of Cia1. How may this latter observation be explained? Nar1 and Nbp35 were previously shown to be required for Fe/S protein assembly outside mitochondria. In particular, Nar1 depletion resulted in diminished 55
Fe incorporation into Nbp35 (4
), and depletion of Nbp35 abolished 55
Fe incorporation into Nar1 (9
). Thus, Cia1 behaves differently from these proteins in that it is required only for “true” Fe/S protein targets such as Leu1 and Rli1 (16
), but not for the Fe/S protein members of the CIA machinery. These in vivo iron-labeling data place the function of Cia1 clearly after that of Nar1 and Nbp35. The fact that Nar1 and Nbp35 can assemble their Fe/S clusters independently of Cia1 suggests that these cofactors may bind transiently to Nbp35 and Nar1, and the function of Cia1 may be, e.g., in the delivery of the clusters to the apoproteins. It is therefore tempting to speculate that these proteins serve as scaffolds for Fe/S cluster assembly in the cytosol. In support of these ideas, the Fe/S clusters bound to Nbp35 are unstable in vivo compared to those of target Fe/S proteins such as Leu1 (P. Smith and R. Lill, unpublished). Rigorous testing of these ideas will require successful in vitro reconstitution of the assembly process of Fe/S clusters in the eukaryotic cytosol.
We demonstrate a specific and stable protein interaction of Cia1 with Nar1, but not with Cfd1 or Nbp35, suggesting a close collaboration of the former components in Fe/S protein biogenesis. While Cfd1, Nbp35, and Nar1 are predominately located in the cytosol with only a minor fraction in the nucleus (3
), a large proportion of Cia1 is localized in the nucleus. Further, Cia1 appears to be at least 10-fold higher in its cellular concentration than Cfd1, Nbp35, and Nar1 (5
). These localization and expression differences between Cfd1/Nbp35/Nar1 and Cia1 may indicate a second function of the latter protein in the nucleus. Interestingly, the human protein with the highest sequence similarity to Cia1 was found to interact with a zinc finger transcription factor, WT1, the Wilms' tumor suppressor protein (14
). This human WD40 repeat protein named Ciao1 (Chinese for “bridge”) modulates the mobility of the WT1-DNA complex in gel shift assays and decreases the transcriptional activity of WT1. Although Cia1 and Ciao1 exhibit 64% amino acid similarity, no obvious sequence homologue of WT1 can be found in the yeast genome. Nevertheless, other yeast zinc-finger protein or proteins may interact with Cia1. For instance, Cia1 was found to interact with the zinc-dependent transcription factor Hms1 using systematic yeast two-hybrid studies (10
Cia1 did not show a stable protein interaction with Cfd1 (or Nbp35) under our experimental conditions. This was somewhat surprising, since in Schizosaccharomyces pombe the CFD1 and CIA1 sequence homologues are linked as a fusion gene. Now that more genome sequences have become available, it appears that this gene arrangement is unique for S. pombe. It is important to note that there is no experimental evidence at this point for the existence of a fusion protein in S. pombe, since posttranslational cleavage may occur. Nevertheless, the presence of a fusion gene in S. pombe points to a common function of both encoded proteins.
The function of Cia1 in the incorporation of the essential Fe/S clusters at the N terminus of Rli1 explains why Cia1 is indispensable for cell growth. Rli1 plays a crucial role in ribosome biogenesis, and its depletion results in the accumulation of ribosomal subunits in the cell nucleus (16
). The function of Cia1 in Fe/S cluster assembly on Rli1 also satisfactorily explains why Cia1 was needed for ribosome biogenesis, as demonstrated by the requirement of Cia1 for export of the large ribosomal subunit from the nucleus, visualized using an Rpl25-GFP fusion protein. Even though the function of Cia1 in nuclear export of ribosomes appears to be indirect, this phenotype has also been observed after down-regulation of Cfd1, Nbp35, and Nar1 (16
) and thus may be a general, yet secondary, consequence of defects in cytosolic Fe/S protein biogenesis. These data support the previous notion of an intimate link between cellular Fe/S protein biogenesis and protein synthesis, two evolutionarily ancient and essential processes (16
The WD40 repeat domain is a common structural module in eukaryotes and is found as part of many protein complexes exhibiting diverse functions. The WD40 repeat proteins form characteristic β-propeller structures. Specificity of each of the WD40 repeat proteins for its binding target and cellular process is thought to be defined by the protein loops protruding from one side of the propeller blades (31
). Crystallographic studies are currently being undertaken to structurally define these loops in Cia1. This information can then aid in mutagenesis studies to unravel the precise molecular function of Cia1 and its interaction with Nar1. Furthermore, modeling of the human Ciao1 protein on the crystal structure of Cia1 could give an indication of whether these two proteins may be functional homologues.
Depletion of Cia1 did not lead to the stimulation of iron uptake into the cell nor to hyperaccumulation of iron in mitochondria, unlike defects in the mitochondrial ISC components involved in Fe/S protein assembly (15
). It appears that none of the known cytosolic Fe/S protein assembly components (Cfd1, Nbp35, Nar1, and Cia1) is critically involved in regulating cellular iron homeostasis via the Aft1/Aft2 transcription factors (25
). Moreover, decreased iron uptake under iron-limiting conditions does not necessarily lead to a (cytosolic) Fe/S cluster assembly defect, as growth in iron-depleted medium did not affect cytosolic Fe/S protein assembly (J. Balk, unpublished). Hence, regulation of cellular and particularly mitochondrial iron homeostasis in yeast does not require active Fe/S protein assembly in the cytosol. Together, these data underline the importance of yeast mitochondria and the ISC system for cellular iron homeostasis and distinguish them from the cytosolic Fe/S protein assembly apparatus.
The past 2 years have led to the discovery of four extramitochondrial components that are functionally involved in Fe/S protein assembly in cytosolic and nuclear apoproteins in yeast. Currently, the mechanism by which these CIA components assist the assembly process is unknown, apart from the finding that Cia1 acts late in biogenesis. Further progress can now be made by a combination of in vivo and in vitro approaches to unravel the precise mode of action of the individual proteins and by identifying even more members of the CIA system. Yeast is an ideal model system for developing a more and more complete picture of Fe/S protein assembly in the eukaryotic cell.