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Dynamic localization of Trypanosoma brucei mitochondrial DNA polymerase ID.

ABSTRACT: Trypanosomes contain a unique form of mitochondrial DNA called kinetoplast DNA (kDNA) that is a catenated network composed of minicircles and maxicircles. Several proteins are essential for network replication, and most of these localize to the antipodal sites or the kinetoflagellar zone. Essential components for kDNA synthesis include three mitochondrial DNA polymerases TbPOLIB, TbPOLIC, and TbPOLID). In contrast to other kDNA replication proteins, TbPOLID was previously reported to localize throughout the mitochondrial matrix. This spatial distribution suggests that TbPOLID requires redistribution to engage in kDNA replication. Here, we characterize the subcellular distribution of TbPOLID with respect to the Trypanosoma brucei cell cycle using immunofluorescence microscopy. Our analyses demonstrate that in addition to the previously reported matrix localization, TbPOLID was detected as discrete foci near the kDNA. TbPOLID foci colocalized with replicating minicircles at antipodal sites in a specific subset of the cells during stages II and III of kDNA replication. Additionally, the TbPOLID foci were stable following the inhibition of protein synthesis, detergent extraction, and DNase treatment. Taken together, these data demonstrate that TbPOLID has a dynamic localization that allows it to be spatially and temporally available to perform its role in kDNA replication.

SUBMITTER: Concepcion-Acevedo J

PROVIDER: S-EPMC3416495 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Dynamic localization of Trypanosoma brucei mitochondrial DNA polymerase ID.

Concepción-Acevedo Jeniffer J Luo Juemin J Klingbeil Michele M MM

Eukaryotic cell 20120127 7

Trypanosomes contain a unique form of mitochondrial DNA called kinetoplast DNA (kDNA) that is a catenated network composed of minicircles and maxicircles. Several proteins are essential for network replication, and most of these localize to the antipodal sites or the kinetoflagellar zone. Essential components for kDNA synthesis include three mitochondrial DNA polymerases TbPOLIB, TbPOLIC, and TbPOLID). In contrast to other kDNA replication proteins, TbPOLID was previously reported to localize th ...[more]

PMID: 22286095

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Project description:The sleeping sickness parasite Trypanosoma brucei has a complex life cycle, alternating between a mammalian host and the tsetse fly vector. A tightly controlled developmental programme ensures parasite transmission between hosts as well as survival within them and involves strict regulation of mitochondrial activities. In the glucose-rich bloodstream, the replicative 'slender' stage is thought to produce ATP exclusively via glycolysis and uses the mitochondrial F1FO-ATP synthase as an ATP hydrolysis-driven proton pump to generate the mitochondrial membrane potential (??m). The 'procyclic' stage in the glucose-poor tsetse midgut depends on mitochondrial catabolism of amino acids for energy production, which involves oxidative phosphorylation with ATP production via the F1FO-ATP synthase. Both modes of the F1FO enzyme critically depend on FO subunit a, which is encoded in the parasite's mitochondrial DNA (kinetoplast or kDNA). Comparatively little is known about mitochondrial function and the role of kDNA in non-replicative 'stumpy' bloodstream forms, a developmental stage essential for disease transmission. Here we show that the L262P mutation in the nuclear-encoded F1 subunit ? that permits survival of 'slender' bloodstream forms lacking kDNA ('akinetoplastic' forms), via FO-independent generation of ??m, also permits their differentiation into stumpy forms. However, these akinetoplastic stumpy cells lack a ??m and have a reduced lifespan in vitro and in mice, which significantly alters the within-host dynamics of the parasite. We further show that generation of ??m in stumpy parasites and their ability to use ?-ketoglutarate to sustain viability depend on F1-ATPase activity. Surprisingly, however, loss of ??m does not reduce stumpy life span. We conclude that the L262P ? subunit mutation does not enable FO-independent generation of ??m in stumpy cells, most likely as a consequence of mitochondrial ATP production in these cells. In addition, kDNA-encoded genes other than FO subunit a are important for stumpy form viability.

Dataset Information

Dynamic localization of Trypanosoma brucei mitochondrial DNA polymerase ID.

Publications

Dynamic localization of Trypanosoma brucei mitochondrial DNA polymerase ID.

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