Functional characterization of mitochondrial transporters reveals TMEM65 as a potential component of the biogenesis machinery of respiratory complexes I and III

The active transport of metabolites, ions, and other molecules across the inner mitochondrial membrane is a crucial process for maintaining mitochondrial functionality and, consequently, cellular homeostasis. Despite notable progress in the characterization of these transporters, key questions remain regarding their direct functional interplay with the OXPHOS machinery. For this reason, this thesis investigated the effect of the loss of function of previously characterized and putative mitochondrial transporters—namely mitoKATP, SLC25A32, SLC25A51 and TMEM65—on OXPHOS complexes function and integrity. The bioenergetic characterization of KO cell models showed that the recently described putative calcium transporter TMEM65 has a role in regulating the stability of respiratory complexes I (CI) and III (CIII). By dissecting TMEM65 interactome we revealed that the protein is in physical proximity to the biogenesis machinery of CI and CIII, composed by assembly factors and components of the TIM23 protein import system and interactors of the insertase OXA1L. Furthermore, accumulation of specific CI early assembly intermediates and impairment in the assembly of CIII subunit CYC1 were observed in the absence of TMEM65, further highlighting its role in the biogenesis of these two complexes. Notably, we also observed that TMEM65 KO induces instability of the mitoribosome large subunit with a mechanism that needs to be elucidated. In conclusion, this thesis identified TMEM65 as a potential component of the biogenesis machinery of CI and CIII, providing new insights into its molecular function and its contribution to mitochondrial organization and energy production.