Interaction between the reductase Tah18 and highly conserved Fe‐S containing Dre2 C‐terminus is essential for yeast viability

Abstract

SummaryTah18-Dre2 is a recently identified yeast protein complex, which is highly conserved in human and has been implicated in the regulation of oxidative stress induced cell death and in cytosolic Fe-S proteins synthesis. Tah18 is a diflavin oxido-reductase with binding sites for flavin mononucleotide, flavin adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, which is able to transfer electrons to Dre2 Fe-S clusters. In this work we characterized in details the interaction between Tah18 and Dre2, and analysed how it conditions yeast viability. We show that Dre2 C-terminus interacts in vivo and in vitro with the flavin mononucleotide-and flavin adenine dinucleotide-binding sites of Tah18. Neither the absence of the electron donor nicotinamide adenine dinucleotide phosphate-binding domain in purified Tah18 nor the absence of Fe-S in aerobically purified Dre2 prevents the binding in vitro. In vivo, when this interaction is affected in a dre2 mutant, yeast viability is reduced. Conversely, enhancing artificially the interaction between mutated Dre2 and Tah18 restores cellular viability despite still reduced cytosolic Fe-S cluster biosynthesis. We conclude that Tah18-Dre2 interaction in vivo is essential for yeast viability. Our study may provide new insight into the survival/death switch involving this complex in yeast and in human cells.

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

“…S4). This result is consistent with the absence of any interaction between the N-terminal domain of Dre2 and Tah18, as monitored both in vitro and in vivo (16). When the anamorsin/FMN-Ndor1 interaction was monitored on a 13 C, 15 Nlabeled oxidized [2Fe-2S]-anamorsin sample by 13 C-direct detection CON, CACO, and CBCACO NMR experiments (Fig.…”

“…Although the molecular targets of the electron transfer flow generated by this protein-protein complex are not yet defined, suggestions for the targets of the electron flow include the conversion of the sulfur of cysteine (formally S 0 ) to the sulfide (S 2− ) present in Fe/S clusters and/or the reductive coupling of two [2Fe-2S] clusters to form a [4Fe-4S] cluster (13). This nondissociative electron transfer process might also rationalize how anamorsin regulates cell survival/death mechanisms in human cells (35) and why a stable Dre2-Tah18 interaction is essential for yeast viability (16). Indeed, the disruption of the stable interaction between anamorsin and Ndor1 might provoke the interruption of the electron flow between the two proteins within the cell and, as a result of that, its essential function for cellular survival is abolished and consequently cell death mechanisms might be activated.…”

“…This is based on the structural characterization of the FMN-binding domain of Ndor1 and of the C-terminal region (linker and CIAPIN1 domain) of anamorsin containing the [2Fe-2S] cluster in the oxidized state ([2Fe-2S]-anamorsin, hereafter) and on the identification of the protein regions between [2Fe-2S]-anamorsin and the FMN-binding domain of Ndor1 responsible for the formation of their stable complex and of those regions interacting in the electron transfer process. The molecular model of the electron transfer process proposed here provides significant information on the functional processes in which the anamorsinNdor1 interaction has been implicated, i.e., the assembly of ISCs (13) and diferric (17) proteins and the regulation of cell survival/ death mechanisms (15,16). This article is a PNAS Direct Submission.…”

“…The C-terminal region of Dre2 (residues 173-348) is fundamental to form, both in vivo and in vitro, the stable complex with Tah18 (16). The Tah18-Dre2 interaction is essential for yeast viability and it has been implicated in cellular death regulation mechanisms (16). This complex was proposed to provide electrons necessary for the CIA machinery (13) and for assembling the diferric tyrosyl radical cofactor of ribonucleotide reductase, Rnr2 (17).…”

“…Dre2 and Tah18 are protein partners forming a stable complex in vivo (13,15). The C-terminal region of Dre2 (residues 173-348) is fundamental to form, both in vivo and in vitro, the stable complex with Tah18 (16). The Tah18-Dre2 interaction is essential for yeast viability and it has been implicated in cellular death regulation mechanisms (16).…”