Iron-sulfur (Fe-S) protein biogenesis in eukaryotes is mediated by two different machineries-one in the mitochondria and another in the cytoplasm. Glutaredoxin 5 (Grx5) is a component of the mitochondrial iron-sulfur cluster machinery. Here, we define the roles of Grx5 in maintaining overall mitochondrial/cellular Fe-S protein biogenesis, utilizing mitochondria and cytoplasm isolated from Saccharomyces cerevisiae cells. We previously demonstrated that isolated wild-type (WT) mitochondria themselves can synthesize new Fe-S clusters, but isolated WT cytoplasm alone cannot do so unless it is mixed with WT mitochondria. WT mitochondria generate an intermediate, called (Fe-S)_int, that is exported to the cytoplasm and utilized for cytoplasmic Fe-S cluster assembly. We here show that mitochondria lacking endogenous Grx5 (Grx5↓) failed to synthesize Fe-S clusters for proteins within the organelle. Similarly, Grx5↓ mitochondria were unable to synthesize (Fe-S)_int, as judged by their inability to promote Fe-S cluster biosynthesis in WT cytoplasm. Most importantly, purified Grx5 precursor protein, imported into isolated Grx5↓ mitochondria, rescued these Fe-S cluster synthesis/trafficking defects. Notably, mitochondria lacking immediate downstream components of the mitochondrial iron-sulfur cluster machinery (Isa1 or Isa2) could synthesize [2Fe-2S] but not [4Fe-4S] clusters within the organelle. Isa1↓ (or Isa2↓) mitochondria could still support Fe-S cluster biosynthesis in WT cytoplasm. These results provide evidence for Grx5 serving as a central hub for Fe-S cluster intermediate trafficking within mitochondria and export to the cytoplasm. Grx5 is conserved from yeast to humans, and deficiency or mutation causes fatal human diseases. Data as presented here will be informative for human physiology.

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Journal Article

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Pandey AK, Pain J, Singh P, Dancis A, Pain D

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