Unravelling the role of Tim17 in maintaining the architecture of presequence translocase and stability of mitochondrial DNA Srujan Kumar Matta1, Gautam Pareek1, Kondalarao Bankapalli1, Anjaneya O1 and Patrick D'Silva1 1Department of Biochemistry, New Biological Sciences Building, Indian Institute of Science, C V Raman Avenue, Bangalore-560012, India Mitochondrial biogenesis is an intricate cellular process which predominantly depends on efficient protein import through translocases of outer and inner membrane. Tim17 is an essential component of the mitochondrial presequence translocase (TIM23-complex) and presumed to maintain the architecture of channel by recruiting terminal subunits of the translocase. Tim17 dysfunction has been largely attributed to destabilization of the TIM23-channel in addition to defective import. However, none of the previous studies highlight the mechanistic insights into how Tim17 coordinates these regulatory events within the complex. We demonstrate that, Tim17 interacts with Tim23 via conserved G/AXXXG/A motifs and plays a crucial role in the translocase assembly. Tandem motifs are highly essential as most of the amino-acid substitutions lead to inviability due to complete destabilization of the TIM23-channel. Importantly, these motifs act as a scaffold for the dynamic recruitment of PAM sub-complex to aid matrix import and could anchor Tim21 to form TIM23 (SORT)-complex thus facilitates lateral insertion of preproteins. Although, Tim17 has been shown to prevent mtDNA loss indirectly, we provide conclusive evidence for the first-time highlighting that conditional mutants of Tim17 are petite-negative and exhibited aberrant nucleoid quantity with coalescent morphology thus signifying the salient role of Tim17 in maintaining stability of mitochondrial genome. Remarkably, the conditional mutants showed compromised membrane polarity and accumulation of excessive ROS leading to mitochondrial fragmentation. In summary, our findings reveal previously elusive important functional attributes of Tim17 protein as a key regulator of Tim23-complex architecture, voltage gating and inner membrane polarity, protein turnover, mitochondrial morphology, and its genome maintenance.