The Iron-Sulfur Cluster Proteins Isa1 and Isa2 Are Required for the Function but Not for the De Novo Synthesis of the Fe/S Clusters of Biotin Synthase in Saccharomyces cerevisiae

Abstract

The yeast Saccharomyces cerevisiae is able to use some biotin precursors for biotin biosynthesis. Insertion of a sulfur atom into desthiobiotin, the final step in the biosynthetic pathway, is catalyzed by biotin synthase (Bio2). This mitochondrial protein contains two iron-sulfur (Fe/S) clusters that catalyze the reaction and are thought to act as a sulfur donor. To identify new components of biotin metabolism, we performed a genetic screen and found that Isa2, a mitochondrial protein involved in the formation of Fe/S proteins, is necessary for the conversion of desthiobiotin to biotin. Depletion of Isa2 or the related Isa1, however, did not prevent the de novo synthesis of any of the two Fe/S centers of Bio2. In contrast, Fe/S cluster assembly on Bio2 strongly depended on the Isu1 and Isu2 proteins. Both isa mutants contained low levels of Bio2. This phenotype was also found in other mutants impaired in mitochondrial Fe/S protein assembly and in wild-type cells grown under iron limitation. Low Bio2 levels, however, did not cause the inability of isa mutants to utilize desthiobiotin, since this defect was not cured by overexpression of BIO2. Thus, the Isa proteins are crucial for the in vivo function of biotin synthase but not for the de novo synthesis of its Fe/S clusters. Our data demonstrate that the Isa proteins are essential for the catalytic activity of Bio2 in vivo.

Specialized Function of Yeast Isa1 and Isa2 Proteins in the Maturation of Mitochondrial [4Fe-4S] Proteins

“…Following the radiolabeling, Isu1 was immunoprecipitated from cell lysates with specific antibodies. 55 Fe bound to the immunobeads reflects the amount of de novo Fe/S cluster assembly on Isu1 and was quantified by scintillation counting (19). Remarkably, the amount of 55 Fe co-immunoprecipitated with Isu1 from these cells slightly increased in both Gal-ISA1 and Gal-ISA2 under depleting conditions, indicating that the maturation of Isu1 can occur independently of the Isa proteins ( Fig.…”

“…Miscellaneous Methods-In vivo radiolabeling of yeast cells with 55 FeCl 3 (ICN) and measurement of 55 Fe incorporation into Fe/S proteins by immunoprecipitation and scintillation counting were carried out as described previously (44,45). Antibodies were raised in rabbits against recombinant proteins expressed in E. coli.…”

“…Alternatively, the proteins were demonstrated to bind mononuclear iron, and thus they were suggested to serve as iron chaperones promoting Fe/S cluster assembly on the U-type ISC scaffold proteins (30, 32, 40 -42). In order to test the relevance of these observations for the yeast Isa proteins, we analyzed the de novo synthesis of the Fe/S cluster of Isu1 by 55 Fe radiolabeling of Gal-ISA1 and Gal-ISA2 cells overproducing Isu1. In these Gal strains, the expression of ISA1 and ISA2 can be repressed upon cultivation in glucose (see supplemental Table SII).…”

“…2A). Because Isu1 binds a [2Fe-2S] cluster, we compared these findings for 55 Fe binding to the mitochondrial [2Fe-2S] ferredoxin Yah1 by in vivo radiolabeling of wild-type, isa1⌬, isa2⌬, and isa1/2⌬ cells upon overexpression of Yah1. The amounts of 55 Fe associated with Yah1 were 1.5-fold higher in cells lacking the Isa proteins compared with wild-type cells ( .…”

Loss of iron–sulfur clusters from biotin synthase as a result of catalysis promotes unfolding and degradation

“…First, the protein could obtain a new [2Fe-2S] 2+ cluster and return to catalysis. Although there is no data indicating how this occurs in E. coli, genetic experiments in S. cerevisiae suggest that the Isc iron-sulfur cluster assembly system, and IscU in particular, is essential for maintaining the FeS clusters in biotin synthase [28,29]. When E. coli are incubated with excess dethiobiotin, measurements of BioB turnover and biotin production clearly indicate that each polypeptide is involved in multiple rounds of catalysis [21], and therefore E. coli must be capable of restoring the FeS cluster complement of BioB following each turnover when sufficient iron is available.…”