Abstract 2001 The yeast [2Fe-2S] mitochondrial protein Aim32 supports cytochrome c oxidase biogenesis

Mitochondrial respiratory complexes, the ATP synthase, and mitoribosomes have a genetic hybrid origin. Most of their components are encoded by the nuclear genome and a few by the mitochondrial DNA (mtDNA). Mitochondrial gene expression is finely regulated, and yeast studies have unveiled several regulatory events in the translation of mitochondrial encoded proteins (Tang et al., 2020). Mitochondrial ribosomes are functionally specialized for the synthesis of several essential inner membrane proteins of the respiratory chain. Extensive interactions of ribosomes with factors involved in all steps of posttranscriptional gene expression occurs in higher-order complexes termed as mitochondrial organization of gene expression (MIOREX) complexes (Kehrein et al, 2015). Recent work has demonstrated that translational activators coordinate mitochondrial and nuclear gene expression to facilitate biogenesis of the oxidative phosphorylation (OXPHOS) complexes (Micketal, 2011). In S. cerevisiae, Altered inheritance of Mitochondria 32 (Aim32) is a thioredoxin-like ferredoxin mitochondrial protein dual-localized to the matrix and intermembrane space (Zhang et al., 2021).Deletion of AIM32 (Δaim32) results in poor growth on respiratory media and accumulation of proteins with aberrant disulfide linkages (Zhang et al., 2021). In this work, we identify Aim32 as a new regulatory component required for the final stoichiometry of the OXPHOS complexes. We demonstrate that cells need Aim32 to adapt efficiently from fermentative to respiratory carbon sources. Western blot analysis indicated substantially lowered steady-state levels of mitochondrially encoded subunits of cytochrome c oxidase in fermenting Δaim32 cells. To explore if formation of OXPHOS complexes is impacted, we examined the organization and activity of the Respiratory SuperComplexes (RSCs) using a combination of colorless native and blue native -PAGE. In yeast, CIII and CIV further assemble into RSC structures consisting of a dimer of CIII and one or two copies of CIV [III2+IV1-2], which enhances the efficiency of electron transfer between complexes. Although reductions in CIV activity were not observed, we detected more of the active CIV monomer and prevalence of the smaller RSC (III2IV > III2IV2) and increased pool of free dimeric Complex III in the Δaim32 mitochondrial extracts. Finally, immunoprecipitations (IP) of Aim32 tagged with FLAG followed by mass spectrometry analysis revealed several mitochondrial ribosome proteins as binding partners, including mitochondrial acidic matrix protein (Mam33) which is a known COX1 translational activator in yeast mitochondria. We were additionally able to confirm the interaction of Mam33 by IP/western blot analysis. Future studies will address the possibility if Aim32 may work alongside Mam33 during respiration. Funding for these studies was generously provided by the William McLauglin endowment grant (LMU Biology).