We have identified a novel component of the mitochondrial ISC assembly system, Iba57p (previously termed Caf17p), in a genome-wide screen for S. cerevisiae
mutants that carry a coupled lysine and glutamate auxotrophy, which is indicative of defects of aconitase and homoaconitase maturation. Iba57p was demonstrated to be crucial for de novo Fe/S cluster incorporation into these mitochondrial aconitase-type Fe/S proteins. In addition, Iba57p is required for the in vivo enzymatic functions of the mitochondrial radical-SAM Fe/S proteins biotin synthase and lipoic acid synthase. Iba57p interacts with Isa1p and Isa2p, a finding consistent with the virtually identical phenotypes of isa1Δ
). No defects in the maturation of other Fe/S proteins were detected in cells depleted for Iba57p or Isa1p/Isa2p (Mühlenhoff et al., unpublished). In addition, the deregulated iron homeostasis that is typical of cells with general defects in the mitochondrial ISC assembly and export systems was not found in cells depleted of Iba57p or Isa1p/Isa2p. These observations strongly indicate that these three proteins are specialized ISC assembly proteins dedicated to the maturation of aconitase-type Fe/S proteins and the functional activation of the radical SAM proteins Bio2p and Lip5p only. The auxotrophies of the isa1/2
mutants described here and in earlier investigations can be fully explained by the functional defects in these classes of Fe/S proteins (29
). Loss of mtDNA is also observed upon deletion of either LIP5
. The lack of involvement in the maturation of essential cytosolic Fe/S proteins can account for why isa1/2
mutants are viable, unlike most components of the mitochondrial ISC systems.
Thus, our work on Iba57p and Isa1/2p introduces a novel aspect to our understanding of eukaryotic Fe/S assembly, that of substrate-specific assembly factors, since all previously identified ISC proteins are universally required for mitochondrial Fe/S protein assembly. The complex of Isa1p/Isa2p and Iba57p represents the first example of such a specialized Fe/S protein maturation system in eukaryotes (Mühlenhoff et al., unpublished). This property of Isa1/2p and Iba57p is, however, reminiscent of the substrate specificity of the Isa1/2p ortholog ErpA of E. coli
, an essential member of the IscA protein family with a role in isoprenoid synthesis (39
). It is possible that this particular class of Fe/S assembly proteins act as specificity factors. It will be interesting to see whether other members of the bacterial IscA family are shown to perform a specific task in Fe/S protein maturation and whether the bacterial Iba57p relative functionally cooperates with this protein family. The dedicated Iba57p/Isa1/2p protein assembly system works in collaboration with the general ISC assembly machinery of mitochondria, distinguishing it from the dedicated NIF system of nitrogen-fixing bacteria which functions as an independent unit. Since our genome-wide screen failed to identify further viable mutants sharing the isa
-specific growth defects, most likely there are no further components involved in this specialized assembly task.
Iba57p shows low sequence similarity to aminomethyl transferase of the GDC complex and contains the highly conserved motif KGCY/FXGQE that characterizes a protein family of unknown function (PTHR22602) that is widely represented across eubacterial and eukaryotic taxa. This family includes YgfZ, an E. coli
protein whose crystal structure is highly similar to aminomethyl transferase, DMGO, and related THF-binding enzymes, a class of proteins not previously associated with Fe/S cluster maturation. Disruption of YgfZ
in E. coli
resulted in decreased methylthiolation of N6
A) tRNA (47
). This reaction is catalyzed by MiaB, a radical-SAM Fe/S enzyme that likely shares a catalytic mechanism with its relatives biotin synthase and lipoic acid synthase (49
). As documented here, enzymatic function of these two proteins require Iba57p and the Isa1p/Isa2p complex. Therefore, the function of Iba57p in the activation of radical SAM proteins may be conserved between bacteria and eukaryotes. In addition, human Iba57 complemented the growth defect of the iba57Δ
yeast mutant, suggesting that the function of this protein is also conserved across eukaryotes, including mammals.
Both Isa1p and Isa2p are essential for biotin synthase activity, without being required for de novo synthesis of its Fe/S cofactors (45
). Here, we have shown that the same is true for Iba57p. Moreover, Isa1p, Isa2p, and Iba57p are required for lipoic acid biosynthesis, indicating that these proteins are also required for the function of lipoic acid synthase and thus may play a general role in the activation of radical SAM Fe/S proteins. The role of Iba57p and Isa1/2p is restricted to the functional activation of lipoic acid synthase, since the maturation of its Fe/S cofactors is not affected in the absence of each of these proteins. Both biotin synthase and lipoic acid synthase have been suggested to donate sulfur from one of their two Fe/S clusters directly to their substrates (4
). Thus, the complex of Isa1p, Isa2p and Iba57p may be involved in the catalytic cycle of sulfur-donating radical-SAM enzymes, probably in the process of Fe/S cluster regeneration after donation of one of the intrinsic sulfide ions to the substrate.
In humans, defects in the mitochondrial branched-chain α-keto acid dehydrogenase complex, which uses lipoic acid as a cofactor, causes maple syrup urine disease or branched-chain ketoaciduria, an autosomal-recessive disease characterized by the accumulation of unprocessed keto acid in blood and urine causing severe ketoacidosis, seizures, and physical and mental retardation (11
). Some mutations associated with the disease affect genes encoding the dehydrogenase complex (6
); however, not all disease-causing mutations have been identified. Our finding that Isa1p, Isa2p, and Iba57p are required for the last step in lipoic acid biosynthesis in vivo identifies the orthologous human ISA1
genes as candidates for mutations causing maple syrup urine disease.
In summary, we have identified a new member of a group of specialized mitochondrial ISC assembly proteins whose function is confined to the maturation of aconitase-type Fe/S proteins and the activation of mitochondrial radical-SAM Fe/S proteins. These specialized assembly factors are needed in addition to the general members of the ISC assembly apparatus. In the case of radical SAM Fe/S enzymes Iba57p and Isa1/2p act after Fe/S cluster insertion by the general ISC apparatus. A molecular explanation for why aconitase-type and radical SAM Fe/S proteins specifically depend on additional maturation factors will require dedicated in vitro reconstitution of the maturation process using purified proteins. However, it is tempting to speculate that the unifying feature for Iba57p/Isa1/2p requirement may be the presence of a solvent-exposed non-cysteinyl-liganded iron that is sensitive to oxidation, such as that found in aconitases (35
). An oxidant-sensitive nonliganded iron is also present in the [4Fe-4S] cluster that binds to the SAM molecule in radical-SAM enzymes (3
). In addition, the catalytic [2Fe-2S] cluster of biotin synthase becomes labile after insertion of sulfur into the substrate.
This investigation and our previous studies on Isa1p and Isa2p have comprehensively defined the physiological role of Iba57p and the Isa proteins in the eukaryotic cell and thus extend our model of this biosynthetic process. These insights pave the way for future studies that will unravel the precise mechanisms underlying the molecular function of Iba57p and the Isa proteins in the maturation of mitochondrial aconitases and activation of radical SAM proteins in eukaryotes. It seems likely to us that the bacterial homolog of Iba57p also plays a specific role in Fe/S protein biogenesis that, due to its specialized nature, has thus far escaped identification.