Background:The trypanosome mitochondrion imports ϳ1000 nucleus-encoded proteins. However, its protein translocation machinery remains elusive. Results: We identified several trypanosome-specific members of the translocase of mitochondrial inner membrane (TIM) in T. brucei. Conclusion:The TIM complex in T. brucei is significantly divergent from those of other eukaryotes. Significance: This TIM complex could be a potential drug target in trypanosomatids.

bTrypanosoma brucei, a parasitic protozoan that causes African trypanosomiasis, possesses a single member of the presequence and amino acid transporter (PRAT) protein family, which is referred to as TbTim17. In contrast, three homologous proteins, ScTim23, ScTim17, and ScTim22, are found in Saccharomyces cerevisiae and higher eukaryotes. Here, we show that TbTim17 cannot rescue Tim17, Tim23, or Tim22 mutants of S. cerevisiae. We expressed S. cerevisiae Tim23, Tim17, and Tim22 in T. brucei. These heterologous proteins were properly imported into mitochondria in the parasite. Further analysis revealed that although ScTim23 and ScTim17 were integrated into the mitochondrial inner membrane and assembled into a protein complex similar in size to TbTim17, only ScTim17 was stably associated with TbTim17. In contrast, ScTim22 existed as a protease-sensitive soluble protein in the T. brucei mitochondrion. In addition, the growth defect caused by TbTim17 knockdown in T. brucei was partially restored by the expression of ScTim17 but not by the expression of either ScTim23 or ScTim22, whereas the expression of TbTim17 fully complemented the growth defect caused by TbTim17 knockdown, as anticipated. Similar to the findings for cell growth, the defect in the import of mitochondrial proteins due to depletion of TbTim17 was in part 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 T. brucei mitochondrion and thus failed to complement the function of TbTim17.A majority of proteins in the mitochondria are encoded by nuclear DNA. These proteins are 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. For translocation of proteins to the mitochondrial matrix, the ATP-dependent action of the import motor of the TIM23 complex is additionally required (4-6). The TIM22 complex, on the other hand, is involved in the translocation and insertion of a special class of mitochondrial inner membrane proteins. These pr...

Recognition of mitochondrial targeting signals (MTS) by receptor translocases of outer and inner membranes of mitochondria is one of the prerequisites for import of nucleus-encoded proteins into this organelle. The MTS for a majority of trypanosomatid mitochondrial proteins have not been well defined. Here we analyzed the targeting signal for trypanosome alternative oxidase (TAO), which functions as the sole terminal oxidase in the infective form of Trypanosoma brucei. Deleting the first 10 of 24 amino acids predicted to be the classical N-terminal MTS of TAO did not affect its import into mitochondria in vitro. Furthermore, ectopically expressed TAO was targeted to mitochondria in both forms of the parasite even after deletion of first 40 amino acid residues. However, deletion of more than 20 amino acid residues from the N terminus reduced the efficiency of import. These data suggest that besides an N-terminal MTS, TAO possesses an internal mitochondrial targeting signal. In addition, both the N-terminal MTS and the mature TAO protein were able to target a cytosolic protein, dihydrofolate reductase (DHFR), to a T. brucei mitochondrion. Further analysis identified a cryptic internal MTS of TAO, located within amino acid residues 115 to 146, which was fully capable of targeting DHFR to mitochondria. The internal signal was more efficient than the N-terminal MTS for import of this heterologous protein. Together, these results show that TAO possesses a cleavable N-terminal MTS as well as an internal MTS and that these signals act together for efficient import of TAO into mitochondria.

Trypanosoma brucei Tim17 (TbTim17), the single member of the Tim17/23/22 protein family, is an essential component of the translocase of the mitochondrial inner membrane (TIM). In spite of the conserved secondary structure, the primary sequence of TbTim17, particularly the N-terminal hydrophilic region, is significantly divergent. In order to understand the function of this region we expressed two N-terminal deletion mutants (Δ20 and Δ30) of TbTim17 in T. brucei. Both of these mutants of TbTim17 were targeted to mitochondria, however, they failed to complement the growth defect of TbTim17 RNAi cells. In addition, the import defect of other nuclear encoded proteins into TbTim17 knockdown mitochondria were not restored by expression of the N-terminal deletion mutants but complemented by knock-in of the full-length protein. Further analysis revealed that Δ20-TbTim17 and Δ30-TbTim17 mutants were not localized in the mitochondrial inner membrane. Analysis of the protein complexes in the wild type and mutant mitochondria by two-dimensional Blue-native/SDS-PAGE revealed that none of these mutants are assembled into the TbTim17 protein complex. However, FL-TbTim17 was integrated into mitochondrial inner membrane and assembled into TbTim17 complex. Co-immunoprecipitation analysis showed that unlike the FL-TbTim17, mutant proteins are not associated with the endogenous TbTim17 as well as its interacting partner TbTim62, a novel trypanosome specific Tim. Together, these results show that the N-terminal domain of TbTim17 plays unique and essential roles for its sorting and assembly into the TbTim17 protein complex.

The coronavirus disease 2019 (COVID-19) pandemic has led to a reimagining of many aspects of higher education, including how instructors interact with their students and how they encourage student participation. Text-based chatting during synchronous remote instruction is a simple form of student-student and student-instructor interaction. The importance of student participation has been documented, as have clear disparities in participation between those well-represented and those under-represented in science disciplines. Thus, we conducted an investigation into who is texting, what students are texting, and how these texts align with course content. We focused on two sections of a large-enrollment, introductory biology class offered remotely during Fall 2020. Using an analysis of in-class chatting, in combination with student survey responses, we find that text-based chatting suggests not only a high level of student engagement, but a type of participation that is disproportionately favored by women. Given the multiple lines of evidence indicating that women typically under-participate in their science courses, any vehicle that counters this trend merits further exploration. We conclude with suggestions for further research, and ideas for carrying forward text-based chatting in the post-COVID-19, in-person classroom.