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The krebs cycle enzyme ?-ketoglutarate decarboxylase is an essential glycosomal protein in bloodstream African trypanosomes.

ABSTRACT: ?-Ketoglutarate decarboxylase (?-KDE1) is a Krebs cycle enzyme found in the mitochondrion of the procyclic form (PF) of Trypanosoma brucei. The bloodstream form (BF) of T. brucei lacks a functional Krebs cycle and relies exclusively on glycolysis for ATP production. Despite the lack of a functional Krebs cycle, ?-KDE1 was expressed in BF T. brucei and RNA interference knockdown of ?-KDE1 mRNA resulted in rapid growth arrest and killing. Cell death was preceded by progressive swelling of the flagellar pocket as a consequence of recruitment of both flagellar and plasma membranes into the pocket. BF T. brucei expressing an epitope-tagged copy of ?-KDE1 showed localization to glycosomes and not the mitochondrion. We used a cell line transfected with a reporter construct containing the N-terminal sequence of ?-KDE1 fused to green fluorescent protein to examine the requirements for glycosome targeting. We found that the N-terminal 18 amino acids of ?-KDE1 contain overlapping mitochondrion- and peroxisome-targeting sequences and are sufficient to direct localization to the glycosome in BF T. brucei. These results suggest that ?-KDE1 has a novel moonlighting function outside the mitochondrion in BF T. brucei.

SUBMITTER: Sykes S

PROVIDER: S-EPMC4346564 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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The krebs cycle enzyme α-ketoglutarate decarboxylase is an essential glycosomal protein in bloodstream African trypanosomes.

Sykes Steven S Szempruch Anthony A Hajduk Stephen S

Eukaryotic cell 20141121 3

α-Ketoglutarate decarboxylase (α-KDE1) is a Krebs cycle enzyme found in the mitochondrion of the procyclic form (PF) of Trypanosoma brucei. The bloodstream form (BF) of T. brucei lacks a functional Krebs cycle and relies exclusively on glycolysis for ATP production. Despite the lack of a functional Krebs cycle, α-KDE1 was expressed in BF T. brucei and RNA interference knockdown of α-KDE1 mRNA resulted in rapid growth arrest and killing. Cell death was preceded by progressive swelling of the flag ...[more]

PMID: 25416237

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Project description:African trypanosomes express three virtually identical non-selenium glutathione peroxidase (Px)-type enzymes which preferably detoxify lipid-derived hydroperoxides. As shown previously, bloodstream Trypanosoma brucei lacking the mitochondrial Px III display only a weak and transient proliferation defect whereas parasites that lack the cytosolic Px I and Px II undergo extremely fast lipid peroxidation and cell lysis. The phenotype can completely be rescued by supplementing the medium with the ?-tocopherol derivative Trolox. The mechanism underlying the rapid cell death remained however elusive. Here we show that the lysosome is the origin of the cellular injury. Feeding the px I-II knockout parasites with Alexa Fluor-conjugated dextran or LysoTracker in the presence of Trolox yielded a discrete lysosomal staining. Yet upon withdrawal of the antioxidant, the signal became progressively spread over the whole cell body and was completely lost, respectively. T. brucei acquire iron by endocytosis of host transferrin. Supplementing the medium with iron or transferrin induced, whereas the iron chelator deferoxamine and apo-transferrin attenuated lysis of the px I-II knockout cells. Immunofluorescence microscopy with MitoTracker and antibodies against the lysosomal marker protein p67 revealed that disintegration of the lysosome precedes mitochondrial damage. In vivo experiments confirmed the negligible role of the mitochondrial peroxidase: Mice infected with px III knockout cells displayed only a slightly delayed disease development compared to wild-type parasites. Our data demonstrate that in bloodstream African trypanosomes, the lysosome, not the mitochondrion, is the primary site of oxidative damage and cytosolic trypanothione/tryparedoxin-dependent peroxidases protect the lysosome from iron-induced membrane peroxidation. This process appears to be closely linked to the high endocytic rate and distinct iron acquisition mechanisms of the infective stage of T. brucei. The respective knockout of the cytosolic px I-II in the procyclic insect form resulted in cells that were fully viable in Trolox-free medium.

Dataset Information

The krebs cycle enzyme ?-ketoglutarate decarboxylase is an essential glycosomal protein in bloodstream African trypanosomes.

Publications

The krebs cycle enzyme α-ketoglutarate decarboxylase is an essential glycosomal protein in bloodstream African trypanosomes.

Similar Datasets

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