PLoS Pathog 6:e1000812. restored by the expression of ScTim17 but was not complemented by the expression of either ScTim23 or ScTim22. Together, these results suggest that TbTim17 is divergent compared to ScTim23 but that its function is closer to that of ScTim17. In addition, ScTim22 could not be sorted properly in the mitochondrion and thus failed to complement the function of TbTim17. INTRODUCTION A majority of proteins in the mitochondria are encoded by nuclear DNA. These proteins are NTRK2 imported by the translocase of the mitochondrial outer membrane (TOM) and the translocase of the mitochondrial inner membrane (TIM) (1, 2). The TOMs and TIMs are multiprotein complexes whose structure and function have been extensively characterized in fungi and recently in humans and plants. The TOM complex serves as the entry gate for virtually all mitochondrial proteins (3). There are two TIM complexes, TIM23 and TIM22, in the majority of eukaryotes analyzed so far. Unlike TOM, the TIM complexes have substrate specificities. The TIM23 complex imports proteins that contain an N-terminal targeting signal (MTS) into the mitochondrial matrix and, if they contain an additional sorting signal, into the inner membrane (4, 5). Tim23 and Tim17, together with the receptor Tim50, form the core of the TIM23 complex. This core complex seems to be sufficient for transport of proteins into the mitochondrial inner membrane by a stop-transfer pathway. Sildenafil Mesylate For translocation of proteins to the mitochondrial matrix, the ATP-dependent action of the import motor of the TIM23 complex is additionally required (4,C6). The TIM22 complex, on the other hand, is involved in the translocation and insertion of a special class of mitochondrial inner membrane proteins. These proteins have multiple internal targeting signals, such as mitochondrial metabolite carrier proteins (MCPs) (7, 8). The membrane-embedded part of TIM22 consists of Tim22, Tim54, and Tim18. Tim12 is an additional component of this complex and aids with the docking of MCPs carried by the small Tim proteins (Tim9 and Tim10) in the intermembrane space (IMS) (9). Tim17, Tim23, and Tim22 all belong to the presequence and amino acid transporter (PRAT) protein family (10, 11). These proteins have 4 transmembrane domains located at the center and possess a PRAT signature motif [(G/A)X2(F/Y)X10RX3DX6(G/A/S)GX3G]. In spite of the similarity of their secondary structure, these proteins perform distinct functions. Tim23 (ScTim23) and ScTim22 form a protein import channel in the TIM23 and TIM22 complexes, respectively (12, 13). The function of ScTim17 is less clear; it seems to act as the structural component that is involved in gating the Tim23 channel (14). Homologs of these Tim proteins are also present in human and plants (15,C18). belongs to a group of ancient and divergent eukaryotes. It possesses a single mitochondrion with many unique and essential activities (19,C21). Similar to other eukaryotes, hundreds of nuclear DNA-encoded proteins are imported into the mitochondrion. However, possesses relict import machinery for mitochondrial proteins (22). There is no canonical TOM complex in this parasite. Instead, an archaic protein termed ATOM imports proteins across the outer mitochondrial membrane (23, 24). possesses a single PRAT family protein named Tim17 (TbTim17) (25, 26) and also has a homolog of Tim50 (27). Three small Tim proteins have been identified in mitochondrion (29). Although possesses a large set of mitochondrial metabolite carrier proteins, the homologs of any of the components of the mitochondrial carrier translocase TIM22 have not been detected in trypanosome genome databases. However, it is speculated that TbTim17 Sildenafil Mesylate along with small TbTim proteins perform this function (22). TbTim17 possesses 4 predicted transmembrane domains and has an 20 to 30% primary sequence similarity with fungal Tim23, Tim17, and Tim22 (25). However, the functional equivalent of TbTim17 among these three proteins has not been determined. Here, we performed a functional Sildenafil Mesylate complementation analysis of TbTim17 with Tim17, Tim23, and Tim22 and vice versa. We show that the function of TbTim17 is partially complemented by the expression of ScTim17 but not by the expression of ScTim23 or ScTim22. All three proteins Sildenafil Mesylate were targeted to the mitochondrion in strains, media, and cell Sildenafil Mesylate growth. The procyclic form of cells of the 427 double-resistant 29-13 cell collection expressing a tetracycline repressor gene and a T7 RNA polymerase was cultivated in Schneider’s Drosophila medium supplemented with 10% fetal bovine serum and antibiotics (50 g/ml hygromycin and 15 g/ml G418) (30). To measure cell growth, cells were seeded at a denseness of 2 106 cells/ml in new medium containing.

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