Biology subjects

Chaudhuri, M.

Publications and source records attributed to Chaudhuri, M..

3 recordsLinked to original sources

bioRxiv2023

Distinct structural motifs are necessary for targeting and import of Tim17 in Trypanosoma brucei mitochondrion

Nuclear-encoded mitochondrial proteins are correctly translocated to their proper sub-mitochondrial destination using location specific mitochondrial targeting signals (MTSs) and via multi-protein import machineries (translocases) in the outer and inner mitochondrial membranes (TOM and TIMs, respectively). However, MTSs of multi-pass Tims are less defined. Here we report the characterization of the MTSs of Trypanosoma brucei Tim17 (TbTim17), an essential component of the most divergent TIM complex. TbTim17 possesses a characteristic secondary structure including four predicted transmembrane (TM) domains in the center with hydrophilic N- and C-termini. After examining mitochondrial localization of various deletion and site-directed mutants of TbTim17 in T. brucei using subcellular fractionation and confocal microscopy we located at least two internal signals, 1) within TM1 (31-50 AAs) and 2) TM4 + Loop 3 (120-136 AAs). Both signals are required for proper targeting and integration of TbTim17 in the membrane. Furthermore, a positively charged residue (K122) is critical for mitochondrial localization of TbTim17. This is the first report of characterizing the internal mitochondrial targeting signals (ITS) for a multipass inner membrane protein in a divergent eukaryote, like T. brucei. SummaryInternal targeting signals within the TM1, TM4 with Loop 3, and residue K122 are required collectively for import and integration of TbTim17 in the T. brucei mitochondrion. This information could be utilized to block parasite growth.

cell biology↗

bioRxiv2023

Unique interactions and functions of the mitochondrial small Tims in Trypanosoma brucei

Trypanosoma brucei is an early divergent parasitic protozoan that causes a fatal disease, African trypanosomiasis. T. brucei possesses a unique and essential translocase of the mitochondrial inner membrane, the TbTIM17 complex. TbTim17 associates with 6 small TbTims, (TbTim9, TbTim10, TbTim11, TbTim12, TbTim13, and TbTim8/13). However, the interaction pattern of the small TbTims with each other and TbTim17 are not clear. Here, we demonstrated by yeast two-hybrid (Y2H) analysis that all six small TbTims interact with each other, but stronger interactions were found among TbTim8/13, TbTim9, and TbTim10. Each of the small TbTims also interact directly with the C-terminal region of TbTim17. RNAi studies indicated that among all small TbTims, TbTim13 is most crucial to maintain the steady-state levels of the TbTIM17 complex. Co-immunoprecipitation analyses from T. brucei mitochondrial extracts also showed that TbTim10 has a stronger association with TbTim9 and TbTim8/13, but a weaker association with TbTim13, whereas TbTim13 has a stronger connection with TbTim17. Analysis of the small TbTim complexes by size exclusion chromatography revealed that each small TbTim, except TbTim13, is present in [~]70 kDa complexes, which could be heterohexameric forms of the small TbTims. However, TbTim13 is primarily present in the larger complex (>800 kDa) and co-fractionated with TbTim17. Altogether, our results demonstrated that TbTim13 is a part of the TbTIM complex and the smaller complexes of the small TbTims likely interact with the larger complex dynamically. Therefore, relative to other eukaryotes, the architecture and function of the small TbTim complexes are specific in T. brucei.

cell biology↗

bioRxiv2021

Trypanosoma brucei Tim50 Plays a Critical Role in Cell Cycle Regulation and Parasite Infectivity

Tim50 is a receptor subunit of the preprotein-translocase of the mitochondrial inner membrane, TIM23. Trypanosoma brucei, the infective agent for African trypanosomiasis, possesses a homologue of Tim50 (TbTim50) with a pair of characteristic DXDX(T/V) phosphatase signature motifs. Here, we demonstrated that besides its protein phosphatase activity, the recombinant TbTim50 binds and hydrolyzes phosphatidic acid in a concentration-dependent manner. In silico structural homology models identify the putative binding interfaces that may accommodate different phospho-substrates. Interestingly, TbTim50 depletion in the bloodstream form (BF) of T. brucei reduced cardiolipin (CL) levels and decreased mitochondrial membrane potential ({Delta}{Psi}). TbTim50 knockdown (KD) also reduced the population of G2 phase and increased G1 phase; thus, BF cell growth was reduced. Confocal and electron microscopy revealed a defect in regulation of kinetoplast (kDNA) replication due to TbTim50 KD. Depletion of TbTim50 increased the levels of AMPK phosphorylation, and parasite morphology was changed to stumpy-like with upregulation of few stumpy marker gene expressions. Importantly, we observed that TbTim50-depleted parasites were unable to establish infection in mice and rats. Proteomics analysis showed reductions of the translation factors, flagellar transport proteins, and many proteasomal subunits, including the mitochondrial HslVU that is known to play a role in kDNA replication. Reduction of the level of HslV in TbTim50 KD cells was further validated by immunoblot analysis. Altogether, our results showed that TbTim50 is essential for mitochondrial function, regulation of kDNA replication, and cell cycle in the BF. Therefore, TbTim50 is an important target for structure-based drug design to combat African trypanosomiasis. ImportanceAfrican trypanosomiasis, a neglected tropical disease caused by parasitic protozoan Trypanosoma brucei, is transmitted by the tsetse fly prevalent in sub-Saharan Africa. During its digenetic life cycle, T. brucei undergoes multiple developmental changes to adapt in different environments. T. brucei BF, dwelling in mammalian blood, generates ATP from glycolysis and hydrolyzes ATP in mitochondria for inner membrane potential. We found that TbTim50, a HAD-family phosphatase, is critical for T. brucei BF survival in vitro and in vivo. Depletion of TbTim50 in BF reduced CL levels and mitochondrial {Delta}{Psi} and caused a detrimental effect on many cellular functions. Cells accumulated in G1-S phase, and kinetoplast was over-replicated due to depletion of mitochondrial proteasomes, HslVU, a master-regulator of kDNA replication. Cell growth inhibition was accompanied by changes in morphology, AMPK phosphorylation, and upregulation of stumpy-specific gene expression. TbTim50 is essential for T. brucei survival and an important T. brucei therapeutic target.

cell biology↗