Trypanosoma brucei Tim17 uses a unique characteristic mitochondrial targeting signal

Chauncey Darden¹ , Joseph Donkor, Olga Korolkova, Muhammad Younas Khan Barozai, Tanu Rana, and Minu Chaudhuri²*

¹Department of Biochemistry, Cancer Biology, Neuroscience, and Pharmacology, Meharry Medical
College, 1005 Dr. D. B. Todd Jr. Blvd., Nashville, TN 37208 and ²Department of Microbiology, Immunology, and Physiology, Meharry Medical College, 1005 Dr. D. B. Todd Jr. Blvd., Nashville, TN 37208. * corresponding author

Mitochondria are highly versatile organelles involved in many cellular processes and linked to multiple diseases such as cancer, heart disease, diabetes, and neurodegeneration. To perform its essential functions, mitochondria utilize hundreds of proteins, however only around 1% of these proteins come from the mitochondrial genome. Using an elaborate multi-protein import machinery (Translocases) mitochondria transport nuclear encoded proteins from the cytosol to different compartments within the mitochondria. Proteins destined to the matrix and single spanning inner membrane proteins utilize an N-terminal localization signal with characteristic features. However, the localization signal within multispanning mitochondrial proteins such as Tim17/22/23 is not well characterized. Here we analyze one of the main components of the translocase in Trypanosoma brucei, Tim17 (TbTim17). This protein consists of four transmembrane domains with its N- and C- termini facing the intermembrane space. Using deletion mutants of TbTim17 where we removed the N-termini, C-termini, and each transmembrane domain (TMD) successively, we found that TbTim17 may possess more than one internal targeting signal (ITS) within TMDs 1 and 4. To validate if TM1 contained a signal we created N-terminal fragments consisting of either N-termini only, TM1 only, or both. We further created additional C-terminal deletion mutants which deleted two successive amino acids at a time to narrow down individual amino acid residues of TM4 required for localization. Overall, it was determined that both TM1 and a portion of TM4 between amino acid residues 121 and 134 are necessary for mitochondrial localization.