Peroxisomal-derived ether phospholipids link nucleotides to respirasome assembly

The protein complexes of the mitochondrial electron transport chain exist in isolation and higher order assemblies termed supercomplexes (SCs) or respirasomes (SC I+III(2)+IV). The association of complexes I, III, and IV into the respirasome is regulated by unknown mechanisms. Here, we designed a Nanoluciferase complementation reporter for complex III and IV proximity to determine in vivo respirasome levels. In a chemical screen, we found that inhibitors of the de novo pyrimidine synthesis enzyme dihydroorotate dehydrogenase (DHODH) potently increased respirasome assembly and activity. Bypassing DHODH inhibition via uridine supplementation decreases SC assembly by altering mitochondrial phospholipid composition, specifically elevated peroxisomal-derived ether phospholipids. Cell growth rates upon DHODH inhibition depends on ether lipid synthesis and SC assembly. These data reveal that nucleotide pools signal to peroxisomes to modulate synthesis and transport of ether phospholipids to mitochondria for SC assembly that are necessary for optimal cell growth in conditions of nucleotide limitation.