Abstract
Trypanosoma brucei, the infectious agent of a deadly disease known as African trypanosomiasis, undergoes various stresses during its digenetic life cycle. We previously showed that downregulation of T. brucei mitochondrial inner membrane protein translocase 50 (TbTim50), an aspartate-based protein phosphatase and a component of the translocase of the mitochondrial inner membrane (TIM), increased the tolerance of procyclic cells to oxidative stress. Using comparative proteomics analysis and further validating the proteomics results by immunoblotting, here we discovered that TbTim50 downregulation caused an approximately 5-fold increase in the levels of PIP39, which is also an aspartate-based protein phosphatase and is primarily localized in glycosomes. A moderate upregulation of a number of glycosomal enzymes was also noticed due to TbTim50 knockdown. We found that the rate of mitochondrial ATP production by oxidative phosphorylation decreased and that substrate-level phosphorylation increased due to depletion of TbTim50. These results were correlated with relative increases in the levels of trypanosome alternative oxidase and hexokinase and a reduced-growth phenotype in low-glucose medium. The levels and activity of the mitochondrial superoxide dismutase and glutaredoxin levels were increased due to TbTim50 knockdown. Furthermore, we show that TbTim50 downregulation increased the cellular AMP/ATP ratio, and as a consequence, phosphorylation of AMP-activated protein kinase (AMPK) was increased. Knocking down both TbTim50 and TbPIP39 reduced PIP39 levels as well as AMPK phosphorylation and reduced T. brucei tolerance to oxidative stress. These results suggest that TbTim50 and PIP39, two protein phosphatases in mitochondria and glycosomes, respectively, cross talk via the AMPK pathway to maintain cellular homeostasis in the procyclic form of T. bruceiIMPORTANCETrypanosoma brucei, the infectious agent of African trypanosomiasis, must adapt to strikingly different host environments during its digenetic life cycle. Developmental regulation of mitochondrial activities is an essential part of these processes. We have shown previously that mitochondrial inner membrane protein translocase 50 in T. brucei (TbTim50) possesses a dually specific phosphatase activity and plays a role in the cellular stress response pathway. Using proteomics analysis, here we have elucidated a novel connection between TbTim50 and a protein phosphatase of the same family, PIP39, which is also a differentiation-related protein localized in glycosomes. We found that these two protein phosphatases cross talk via the AMPK pathway and modulate cellular metabolic activities under stress. Together, our results indicate the importance of a TbTim50 and PIP39 cascade for communication between mitochondria and other cellular parts in regulation of cell homeostasis in T. brucei.
Highlights
Trypanosoma brucei, the infectious agent of a deadly disease known as African trypanosomiasis, undergoes various stresses during its digenetic life cycle
We found a number of chaperone proteins, like Hsp70, DNAj, glucose-regulated protein 78 (GRP78), cyclophilin, and calreticulin (Table 2 and Data Set S1)
In spite of a large amount of evidence showing that Tim50 plays additional roles besides mitochondrial protein import, the mechanism of its action for these cellular functions is not clearly understood in any system
Summary
Trypanosoma brucei, the infectious agent of a deadly disease known as African trypanosomiasis, undergoes various stresses during its digenetic life cycle. We show that TbTim downregulation increased the cellular AMP/ATP ratio, and as a consequence, phosphorylation of AMP-activated protein kinase (AMPK) was increased Knocking down both TbTim and TbPIP39 reduced PIP39 levels as well as AMPK phosphorylation and reduced T. brucei tolerance to oxidative stress. We have shown previously that mitochondrial inner membrane protein translocase 50 in T. brucei (TbTim50) possesses a dually specific phosphatase activity and plays a role in the cellular stress response pathway. Here we have elucidated a novel connection between TbTim and a protein phosphatase of the same family, PIP39, which is a differentiation-related protein localized in glycosomes We found that these two protein phosphatases cross talk via the AMPK pathway and modulate cellular metabolic activities under stress. Stage differentiation in T. brucei is associated with changes in morphology, the metabolic pattern, surface protein expression, and mitochondrial activities [22, 23] that require a coordinated regulation of gene expression, which in trypanosomatids primarily occurs posttranscriptionally [24]
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