Abstract
Iron-sulfur clusters are essential cofactors of proteins. In eukaryotes, iron-sulfur cluster biogenesis requires a mitochondrial iron-sulfur cluster machinery (ISC) and a cytoplasmic iron-sulfur protein assembly machinery (CIA). Here we used mitochondria and cytoplasm isolated from yeast cells, and [35S]cysteine to detect cytoplasmic Fe-35S cluster assembly on a purified apoprotein substrate. We showed that mitochondria generate an intermediate, called (Fe-S)int, needed for cytoplasmic iron-sulfur cluster assembly. The mitochondrial biosynthesis of (Fe-S)int required ISC components such as Nfs1 cysteine desulfurase, Isu1/2 scaffold, and Ssq1 chaperone. Mitochondria then exported (Fe-S)int via the Atm1 transporter in the inner membrane, and we detected (Fe-S)int in active form. When (Fe-S)int was added to cytoplasm, CIA utilized it for iron-sulfur cluster assembly without any further help from the mitochondria. We found that both iron and sulfur for cytoplasmic iron-sulfur cluster assembly originate from the mitochondria, revealing a surprising and novel mitochondrial role. Mitochondrial (Fe-S)int export was most efficient in the presence of cytoplasm containing an apoprotein substrate, suggesting that mitochondria respond to the cytoplasmic demand for iron-sulfur cluster synthesis. Of note, the (Fe-S)int is distinct from the sulfur intermediate called Sint, which is also made and exported by mitochondria but is instead used for cytoplasmic tRNA thiolation. In summary, our findings establish a direct and vital role of mitochondria in cytoplasmic iron-sulfur cluster assembly in yeast cells.
Highlights
Iron–sulfur clusters are essential cofactors of proteins
We showed that mitochondria generate an intermediate, called (Fe–S)int, needed for cytoplasmic iron–sulfur cluster assembly
Mitochondria isolated from yeast cells contain a complete iron–sulfur cluster machinery (ISC) machinery, and isolated mitochondria by themselves are capable of forming iron–sulfur clusters when supplemented with cysteine, iron, and nucleotides (GTP, NADH, and ATP) [19, 22, 23]
Summary
Sulfur intermediate; (Fe–S)int, iron–sulfur intermediate; ⌬N60 Yah, the N-terminal 60 amino acids including the mitochondrial targeting signal removed from the Yah precursor protein; ISC, mitochondrial iron–sulfur cluster machinery; CIA, cytoplasmic iron– sulfur protein assembly machinery; PMSF, phenylmethylsulfonyl fluoride. The CIA machinery is apparently incomplete, requiring a contribution from mitochondria to function as an assembly apparatus for cytoplasmic iron–sulfur clusters This idea was originally hinted at by in vivo experiments in which mitochondrial Nfs cysteine desulfurase was depleted from yeast cells using a regulated promoter. A key feature that emerges from this study is that iron for (Fe–S)int originates in mitochondria
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