title: Tim62, a Novel Mitochondrial Protein in Trypanosoma brucei, Is Essential for Assembly and Stability of the TbTim17 Protein Complex.
abstract: Trypanosoma brucei, the causative agent of human African trypanosomiasis, possesses non-canonical mitochondrial protein import machinery. Previously, we characterized the essential translocase of the mitochondrial inner membrane (TIM) consisting of Tim17 in T. brucei. TbTim17 is associated with TbTim62. Here we show that TbTim62, a novel protein, is localized in the mitochondrial inner membrane, and its import into mitochondria depends on TbTim17. Knockdown (KD) of TbTim62 decreased the steady-state levels of TbTim17 post-transcriptionally. Further analysis showed that import of TbTim17 into mitochondria was not inhibited, but its half-life was reduced >4-fold due to TbTim62 KD. Blue-native gel electrophoresis revealed that TbTim62 is present primarily in ∼150-kDa and also in ∼1100-kDa protein complexes, whereas TbTim17 is present in multiple complexes within the range of ∼300 to ∼1100 kDa. TbTim62 KD reduced the levels of both TbTim62 as well as TbTim17 protein complexes. Interestingly, TbTim17 was accumulated as lower molecular mass complexes in TbTim62 KD mitochondria. Furthermore, depletion of TbTim62 hampered the assembly of the ectopically expressed TbTim17-2X-myc into TbTim17 protein complex. Co-immunoprecipitation analysis revealed that association of TbTim17 with mHSP70 was markedly reduced in TbTim62 KD mitochondria. All together our results demonstrate that TbTim62, a unique mitochondrial protein in T. brucei, is required for the formation of a stable TbTim17 protein complex. TbTim62 KD destabilizes this complex, and unassembled TbTim17 degrades. Therefore, TbTim62 acts as a novel regulatory factor to maintain the levels of TIM in T. brucei mitochondria.
authors.affiliation: From the Department of Microbiology and Immunology, Meharry Medical College, Nashville, Tennessee 37208.
authors.forename: Ujjal K
authors.initials: UK
authors.lastname: Singha
authors.affiliation: From the Department of Microbiology and Immunology, Meharry Medical College, Nashville, Tennessee 37208.
authors.forename: VaNae
authors.initials: V
authors.lastname: Hamilton
authors.affiliation: From the Department of Microbiology and Immunology, Meharry Medical College, Nashville, Tennessee 37208 mchaudhuri@mmc.edu.
authors.forename: Minu
authors.initials: M
authors.lastname: Chaudhuri
mesh_terms: D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism*; D014353:Trypanosomiasis, African
chemical_list: D018840:HSP70 Heat-Shock Proteins; D033681:Mitochondrial Membrane Transport Proteins; D046912:Multiprotein Complexes; D015800:Protozoan Proteins
keywords: Hsp70; TIM complex; Tim17; Tim62; Trypanosoma brucei; mitochondria; protein assembly; protein import; translocation