Mitoregulin self-associates to form likely homo-oligomeric pore-like complexes.

We and others previously found that a misannotated long noncoding RNA encodes for a conserved mitochondrial transmembrane microprotein named Mitoregulin (Mtln). Beyond an established role for Mtln in lipid metabolism, Mtln has been shown to broadly influence mitochondria, boosting respiratory efficiency and Ca retention capacity, while lowering ROS, yet the underlying mechanisms remain unresolved. Prior studies have identified possible Mtln protein interaction partners; however, a lack of consensus persists, and no claims have been made about Mtln's structure. We noted two key published observations that seemingly remained overlooked: 1) endogenous Mtln co-immunoprecipitates with epitope-tagged Mtln at high efficiency, and 2) Mtln primarily appears to exist in a ∼66 kDa complex. To investigate if Mtln may self-oligomerize into higher-order complexes, we performed co-immunoprecipitation, computational modeling, and native gel assessments of Mtln-containing complexes in cells and tissues and tested whether synthetic Mtln protein itself forms oligomeric complexes. Our combined results provide strong support that Mtln self-associates and likely forms a hexameric pore-like structure.