, the causative agent for African trypanosomiasis, possesses a single mitochondrion that imports hundreds of proteins from the cytosol. energy metabolism, redox balance, autophagy and apoptosis [7C10]. However, the role of mitochondria in a cellular stress response is less understood in parasitic protozoa like are nuclear-encoded. Therefore these proteins are imported into mitochondria in order to perform their functions. In fungi and 1197958-12-5 higher eukaryotes, there are three major protein translocases in mitochondria, the translocase of the mitochondrial outer membrane (TOM), and two translocases of the mitochondrial inner membrane, TIM23 and TIM22 [11, 12]. TOM and TIMs are multi-subunit protein complexes that have been extensively characterized in fungi and later in animals and plants. Interestingly, trypanosomatids do not have any homologs of the TOM subunits. Instead, an archaic -barrel protein, ATOM, serves the function of Tom40, the major component of the fungal TOM complex . Similarly, homologs of the TIM22 components could not be detected in this parasite, but it possesses a few homologs of the fungal TIM23 subunits, such as Tim17 and Tim50 [14, 15]. We have shown previously that Tim17 (TbTim17) is essential for cell survival and is critical for mitochondrial protein import . TbTim17 is present in a larger molecular mass protein complex and it is associated with several novel trypanosome-specific proteins . We also have characterized Tim50 in (TbTim50), which interacts with TbTim17 . TbTim50 possesses a mitochondrial-targeting signal (MTS) at its N-terminus and a characteristic phosphatase motif at its C-terminus, which shows similarity to the transcription factor II (TFII)-stimulated RNA polymerase II C-terminal domain (CTD)-phosphatase. Knockdown (KD) of TbTim50 inhibits import of proteins into mitochondria that contain an N-terminal MTS, while the recombinant TbTim50 possesses a dual-specificity phosphatase activity . Increasing evidence indicates that besides mitochondrial protein import, these Tom and Tim proteins are also involved in other functions, such as maintaining mitochondrial morphology, regulation of fission and fusion of the organelle and recruitment of anti-apoptotic and autophagy proteins [17C19]. Tom proteins are phosphorylated by cytosolic kinases to control mitochondrial protein biogenesis as a means of regulating mitochondrial activities [20C22]. In addition Tim50 is also known to be involved in developmental regulation and apoptosis in zebra fish, drosophila, and human [23, 24], although the mechanisms responsible for these actions are not well understood. Here we 1197958-12-5 discovered that TbTim50 plays a role in the stress response pathway in to peroxide treatment. Whereas, TbTim50 overexpression hyperpolarized the mitochondrial inner membrane, increased ROS production and promoted cell apoptosis. These results further indicate the important role of the mitochondrion and its protein translocator in the maintenance of cellular homeostasis. 2. Materials and methods 2.1. Strains, media, cell growth and isolation of mitochondria The procyclic form of 1197958-12-5 427 cells was grown in SDM-79 medium containing 10% heat inactivated fetal bovine serum [15, 16]. cells expressing TbTim50 RNAi (TbTim50 KD) and TbTim50 with a C-terminally 3x hemagglutinine (HA) tag (TbTim50 OE) were developed as previously reported . These cell lines were maintained in the same medium supplemented with hygromycin (50 g/ml), G418 (15 g/ml) and phleomycin (2.5 g/ml). Cell growth was assessed by inoculating the procyclic form at a cell density of 2 106/ml in fresh medium containing antibiotics in the presence or absence of doxycycline (1 g/ml). Cells were counted at different growth time points with a Neubauer hemocytometer. The log of the cumulative cell number was plotted against Rab7 time of incubation in culture. Mitochondria were isolated from the parasite after lysis by nitrogen cavitation in isotonic buffer [14, 15]. The isolated mitochondria were stored at a protein concentration of 10 mg/ml in SME buffer (250 mM sucrose, 20 mM MOPS/KOH, 2 mM EDTA, pH 7.4) containing 50% glycerol 1197958-12-5 at ?70C. Before use, mitochondria were washed twice with 9 volumes of SME buffer to remove glycerol. 2.2. Isolation of RNA and northern analyses Total RNA was isolated from the procyclic form of using Trizol (Invitrogen) according to the manufacturers protocol. Northern blot analysis was performed as described [14, 15]. Specific probes were made using a random primer labeling protocol (Invitrogen) from cDNA fragments, which were generated by PCR using sequence-specific primers designed from the coding regions of TbTim50 and actin. 2.3. SDS-PAGE and immunoblot Proteins from isolated mitochondria were resolved on a 10% SDS/PAGE skin gels and then transferred to nitrocellulose membranes (Bio-Rad) [14, 15]. The antigens were visualized by using an enhanced chemiluminescence kit (ECL; Amersham). Antibodies against TbTim50 , HA-epitope (Clone 12CA5, Roche Applied Technology), and -tubulin  were used as probes. The secondary antibodies used were either anti-rabbit or anti-mouse immunoglobulins linked to horseradish peroxidase (Amersham Existence Technology). 2.3. Hydrogen peroxide treatment TbTim50 RNAi and TbTim50-3HA were caused for four days with doxycycline (1 g/ml). Parental 427 cells were also cultivated in parallel for 4 days. Cells were then revealed to different concentration of hydrogen peroxide (0C800 M) for 2 h. Treated cells were immediately used for further analysis. 2.4. Live/deceased assay parental 427, TbTim50 RNAi.