Dre2, a Conserved Eukaryotic Fe/S Cluster Protein, Functions in Cytosolic Fe/S Protein Biogenesis

Abstract

In a forward genetic screen for interaction with mitochondrial iron carrier proteins in Saccharomyces cerevisiae, a hypomorphic mutation of the essential DRE2 gene was found to confer lethality when combined with ⌬mrs3 and ⌬mrs4. The dre2 mutant or Dre2-depleted cells were deficient in cytosolic Fe/S cluster protein activities while maintaining mitochondrial Fe/S clusters. The Dre2 amino acid sequence was evolutionarily conserved, and cysteine motifs (CX 2 CXC and twin CX 2 C) in human and yeast proteins were perfectly aligned. The human Dre2 homolog (implicated in blocking apoptosis and called CIAPIN1 or anamorsin) was able to complement the nonviability of a ⌬dre2 deletion strain. The Dre2 protein with triple hemagglutinin tag was located in the cytoplasm and in the mitochondrial intermembrane space. Yeast Dre2 overexpressed and purified from bacteria was brown and exhibited signature absorption and electron paramagnetic resonance spectra, indicating the presence of both [2Fe-2S] and [4Fe-4S] clusters. Thus, Dre2 is an essential conserved Fe/S cluster protein implicated in extramitochondrial Fe/S cluster assembly, similar to other components of the so-called CIA (cytoplasmic Fe/S cluster assembly) pathway although partially localized to the mitochondrial intermembrane space.

Requirements of the cytosolic iron–sulfur cluster assembly pathway in Arabidopsis

“…Reduction of the protein sample revealed an electron paramagnetic resonance (EPR) spectrum characteristic of a rhombic [2Fe-2S] 1þ cluster, which was detectable up to 84 K (figure 3d). The g values as determined by simulation of the EPR spectrum (1.998, 1.959 and 1.916) are similar to those reported for the purified yeast Dre2 protein [43,44]. Since chemical analysis detected approximately 1.3 Fe and S per polypeptide, we anaerobically reconstituted AtDRE2.…”

Analysis of iron–sulfur protein maturation in eukaryotes

“…This methodology allows convenient determination of soluble enzyme activities. Examples include cytosolic human aconitase (IRP1) in a yeast deletion mutant lacking mitochondrial aconitase 50 , cytosolic 15 or mitochondria-targeted 51 isopropylmalate isomerase (Leu1) in a yeast cell lacking or with low cytosolic Leu1. In addition to the introduction of suitable marker Fe/S proteins, such experiments show that central parts of both the ISC and CIA machineries do not exhibit pronounced target specificity for Fe/S proteins of one given compartment.…”

“…14). Recently, the Fe/S protein Dre2 was found to be another crucial member of the CIA machinery but its function is still unclear 15 .…”

Molecular view of an electron transfer process essential for iron–sulfur protein biogenesis

“…The CIA machinery comprises several proteins (10,11), among which one named Dre2 has been recently identified in yeast (12). The C-terminal domain of Dre2 (residues 228-348) is able to bind two ISCs, a [2Fe-2S] and a [4Fe-4S] (12,13).…”

“…The C-terminal domain of Dre2 (residues 228-348) is able to bind two ISCs, a [2Fe-2S] and a [4Fe-4S] (12,13). The [2Fe-2S] cluster of Dre2 receives electrons from a cytosolic diflavin reductase, termed Tah18 (13), which contains a FAD and a FMN prosthetic group, respectively, bound in two distinct structural domains, to accept electrons from NADPH (14).…”