Isa2p is essential in <i>Saccharomyces cerevisiae</i> for mitochondrial function and stress resistance by allowing Rip1p subunit assembly into cytochrome <i>bc</i>₁ complex.

In <i>Saccharomyces cerevisiae</i>, the iron-sulfur cluster biogenesis late-acting subsystem (Fe-S-IBG) comprises the mitochondrial glutaredoxin (Grx5), Isa1, Isa2, and iron-sulfur cluster assembly factor IBA57 (Iba57) proteins. The Fe-S-IBG subsystem assists in inserting [4Fe-4S] clusters into apoproteins, some of which belong to the electron transport chain (ETC). However, whether the Fe-S-IBG subsystem indirectly stabilizes respiratory supercomplexes and proper ETC function via insertion of [Fe-S] proteins into ETC complexes remains to be elucidated. We compared the effects of <i>ISA2</i>- and <i>GRX5</i>-independent mutations on the insertion of Rip1p, a [2Fe-2S]-containing protein involved in both electron transfer in the <i>bc</i>₁ complex and the formation of respiratory supercomplexes. The levels of Rip1p, supercomplex assembly, ETC function, oxidative stress, and resistance of yeast to ethanol stress were evaluated on haploid <i>S. cerevisiae</i> cells with independent mutations of the <i>ISA2</i> and <i>GRX5</i> genes. Susceptibility to ethanol was increased in the <i>isa2Δ</i> and <i>grx5Δ</i> mutants, which was associated with enhanced glutathione oxidation due to higher levels of free iron and increased oxidants. Furthermore, the <i>isa2Δ</i> mutant showed decreased Rip1p expression, respiratory dysfunction, and defective respiratory supercomplex formation, which was restored by <i>ISA2</i> complementation. These results suggest that Isa2p is essential for proper respiratory chain function and resistance to oxidative stress by stabilizing supercomplexes in a manner dependent on Rip1p insertion in the cytochrome <i>bc</i>₁ complex.