title: Tim50 in Trypanosoma brucei possesses a dual specificity phosphatase activity and is critical for mitochondrial protein import.
abstract: In eukaryotes, proteins are imported into mitochondria via multiprotein translocases of the mitochondrial outer and inner membranes, TOM and TIM, respectively. Trypanosoma brucei, a hemoflagellated parasitic protozoan and the causative agent of African trypanosomiasis, imports about a thousand proteins into the mitochondrion; however, the mitochondrial protein import machinery in this organism is largely unidentified. Here, we characterized a homolog of Tim50 that is localized in the mitochondrial membrane in T. brucei. Similar to Tim50 proteins from fungi and mammals, Tim50 in T. brucei (TbTim50) possesses a mitochondrial targeting signal at its N terminus and a C-terminal domain phosphatase motif at its C terminus. Knockdown of TbTim50 reduced cell growth and inhibited import of proteins that contain N-terminal targeting signals. Co-immunoprecipitation analysis revealed that TbTim50 interacts with TbTim17. Unlike its fungal counterpart but similar to the human homolog of Tim50, recombinant TbTim50 possesses a dual specificity phosphatase activity with a greater affinity for protein tyrosine phosphate than for protein serine/threonine phosphate. Mutation of the aspartic acid residues to alanine in the C-terminal domain phosphatase motif (242)DXDX(V/T)(246) abolished activity for both type of substrates. TbTim50 knockdown increased and its overexpression decreased the level of voltage-dependent anion channel (VDAC). However, the VDAC level was unaltered when the phosphatase-inactive mutant of TbTim50 was overexpressed, suggesting that the phosphatase activity of TbTim50 plays a role in regulation of VDAC expression. In contrast, phosphatase activity of the TbTim50 is required neither for mitochondrial protein import nor for its interaction with TbTim17. Overall, our results show that TbTim50 plays additional roles in mitochondrial activities besides preprotein translocation.
authors.affiliation: Department of Microbiology and Immunology, Meharry Medical College, Nashville, Tennessee 37208, USA.
authors.forename: Melanie R
authors.initials: MR
authors.lastname: Duncan
authors.forename: Marjorie
authors.initials: M
authors.lastname: Fullerton
authors.forename: Minu
authors.initials: M
authors.lastname: Chaudhuri
mesh_terms: D001224:Aspartic Acid / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D054637:Dual-Specificity Phosphatases / Q000737:chemistry / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D047468:Immunoprecipitation; D053078:Membrane Potential, Mitochondrial; D008928:Mitochondria / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D021382:Protein Sorting Signals; D017433:Protein Structure, Secondary; D021381:Protein Transport; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000201:enzymology; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics; D050994:Voltage-Dependent Anion Channels / Q000378:metabolism
chemical_list: D024101:Mitochondrial Proteins; D021382:Protein Sorting Signals; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D050994:Voltage-Dependent Anion Channels; D001224:Aspartic Acid; D054637:Dual-Specificity Phosphatases