Understanding the structural organization of the carrier translocase machinery in regulating mitochondrial biogenesis and organelle quality control

Abstract

Mitochondria are essential eukaryotic organelles required for diverse cellular functions including, energy homeostasis, iron-sulfur cluster biogenesis, and signaling. Therefore, the maintenance of organelle quality control is critical for cell survival, and any impairment in mitochondrial function is detrimental to cells. Proper mitochondrial functioning requires accurate and efficient transport of approximately 99% of proteins from the cytosol to their precise location into the mitochondria. This protein import is performed by sophisticated protein machinery present at the outer and inner mitochondrial membranes. The outer membrane contains the TOM complex, which serves as a general entry gate for most of the mitochondrial proteins. The inner membrane harbors two distinct import machinery types, namely, the presequence translocase (TIM23 complex) and the carrier translocase (TIM22 complex). The TIM23 complex is dedicated machinery for importing proteins containing N-terminal cleavable targeting signals into the matrix and inner membrane. On the other hand, the TIM22 complex facilitates the import of polytopic inner membrane proteins having internal hydrophobic targeting sequences. Tim22 forms the central channel of the carrier translocase with a twin pore structure. However, the role of the central channel forming Tim22 protein in modulating the assembly process of carrier translocase machinery coupled to protein import remains still elusive. In the present study, we report a novel set of conditional mutants isolated by an unbiased genetic screen from different regions of Tim22. Our genetic and biochemical analyses revealed a distinct functional role for different segments of Tim22 in the assembly of carrier translocase machinery. Further, we demonstrated that impairment in the TIM22 complex assembly process influences its translocase activity, the mitochondrial network, and the viability of cells lacking mitochondrial DNA. Overall, our results provide compelling evidence highlighting the functional significa...