The translationally controlled tumor protein (TCTP; also known as TPT1 in mammals) is highly conserved and ubiquitously expressed in eukaryotes. It is involved in growth and development, cell cycle progression, protection against cellular stresses and apoptosis, indicating the multifunctional role of the protein. Here, for the first time, we characterize the expression and function of TCTP in the human and animal pathogen, Trypanosoma brucei. We identified two paralogs (TCTP1 and TCTP2) that are differentially expressed in the life cycle of the parasite. The genes have identical 5′ untranslated regions (UTRs) and almost identical open-reading frames. The 3′UTRs differ substantially in sequence and length, and are sufficient for the exclusive expression of TCTP1 in procyclic- and TCTP2 in bloodstream-form parasites. Furthermore, we characterize which parts of the 3′UTR are needed for TCTP2 mRNA stability. RNAi experiments demonstrate that TCTP1 and TCTP2 expression is essential for normal cell growth in procyclic- and bloodstream-form parasites, respectively. Depletion of TCTP1 in the procyclic form cells leads to aberrant cell and mitochondrial organelle morphology, as well as enlarged, and a reduced number of, acidocalcisomes.
Mitochondrial DNA replication is an essential process in most eukaryotes. Similar to the diversity in mitochondrial genome size and organization in the different eukaryotic supergroups, there is considerable diversity in the replication process of the mitochondrial DNA. In this review, we summarize the current knowledge of mitochondrial DNA replication and the associated factors in trypanosomes with a focus on Trypanosoma brucei, and provide a new model of minicircle replication for this protozoan parasite. The model assumes the mitochondrial DNA (kinetoplast DNA, kDNA) of T. brucei to be loosely diploid in nature and the replication of the genome to occur at two replication centers at the opposing ends of the kDNA disc (also known as antipodal sites, APS). The new model is consistent with the localization of most replication factors and in contrast to the current model, it does not require the assumption of an unknown sorting and transport complex moving freshly replicated DNA to the antipodal sites. In combination with the previously proposed sexual stages of the parasite in the insect vector, the new model provides a mechanism for maintenance of the mitochondrial genetic diversity.
Trypanosoma brucei is a single celled eukaryotic parasite in the group of the Excavates. T. brucei cells harbor a single mitochondrion with a singular mitochondrial genome, that consists of a unique network of thousands of interwoven circular DNA molecule copies and is termed the kinetoplast DNA (kDNA). To ensure proper inheritance of the kDNA to the daughter cells the genome is linked to the basal body, the master organizer of the cell cycle in trypanosomes. The structure connecting the basal body and kDNA is termed the tripartite attachment complex (TAC). Using a combination of proteomics and RNAi (depletomics) we test the current model of hierarchical TAC assembly and identify TbmtHMG44 and Tb927.11.16120 as novel candidates of a structure that connects the TAC to the kDNA. Both proteins localize in the region of the unilateral filaments between TAC102 and the kDNA and depletion of each leads to a strong kDNA loss phenotype. TbmtHMG44 and Tb927.11.16120 stably associate with extracted flagella, even after DNase treatment however they do require the kDNA for initial assembly. Furthermore we demonstrate that recombinant Tb927.11.16120 is a DNA binding protein and thus a promising candidate to link the TAC to the kDNA.
In contrast to many eukaryotic organisms, trypanosomes only contain a single mitochondrion per cell. Within that singular mitochondrion, the protist carries a single mitochondrial genome that consists of a complex DNA network, the kinetoplast DNA (kDNA). Segregation of the replicated kDNA is coordinated by the basal body of the cell′s single flagellum. The tripartite attachment complex (TAC) forms a physical connection between the proximal end of the basal body and the kDNA. This allows anchoring of the kDNA throughout the cell cycle and couples kDNA segregation with the separation of the basal bodies prior to cell division. Over the past years, several components of the TAC have been identified. To shed light on the structure of the cytoplasmic part of the TAC (known as the exclusion zone), we performed cryo-electron tomography on whole cells. This allowed us to acquire three-dimensional high-resolution images of the exclusion zone in situ. We observed that the exclusion zone filaments offer great mechanical flexibility for basal body movement. We measured the dimensions of the individual structural elements of the area, as well as the overall orientation and positioning of the basal bodies towards the mitochondrial kDNA pocket. Using a combination of experimental data and modelling, we generated a structural model of the exclusion zone protein p197. Our findings suggest that the majority of p197 consists of a string of spectrin-like repeats. We propose that these structural units provide the architecture of a molecular spring and that they are required in the TAC to withstand the mechanical forces generated through basal body repositioning events during kDNA segregation and motility of the organism.
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