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Validation of a modified method for Bxb1 mycobacteriophage integrase-mediated recombination in Plasmodium falciparum by localization of the H-protein of the glycine cleavage complex to the mitochondrion.

ABSTRACT: The glycine cleavage complex (GCV) is a potential source of the one carbon donor 5,10-methylene-tetrahydrofolate (5,10-CH(2)-THF) in the malaria parasite Plasmodium falciparum. One carbon (C1) donor units are necessary for amino acid and nucleotide biosynthesis, and for the initiation of mitochondrial and plastid translation. In other organisms, GCV activity is closely coordinated with the activity of serine hydroxymethyltransferase (SHMT) enzymes. P. falciparum contains cytosolic and mitochondrial SHMT isoforms, and thus, the subcellular location of the GCV is an important indicator of its role in malaria metabolism. To determine the subcellular localization of the GCV, we used a modified version of the published method for mycobacteriophage integrase-mediated recombination in P. falciparum to generate cell lines containing one of the component proteins of the GCV, the H-protein, fused to GFP. Here, we demonstrate that this modification results in rapid generation of chromosomally integrated transgenic parasites, and we show that the H-protein localizes to the mitochondrion.

SUBMITTER: Spalding MD

PROVIDER: S-EPMC2875341 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Validation of a modified method for Bxb1 mycobacteriophage integrase-mediated recombination in Plasmodium falciparum by localization of the H-protein of the glycine cleavage complex to the mitochondrion.

Spalding Maroya D MD Allary Marina M Gallagher John R JR Prigge Sean T ST

Molecular and biochemical parasitology 20100418 2

The glycine cleavage complex (GCV) is a potential source of the one carbon donor 5,10-methylene-tetrahydrofolate (5,10-CH(2)-THF) in the malaria parasite Plasmodium falciparum. One carbon (C1) donor units are necessary for amino acid and nucleotide biosynthesis, and for the initiation of mitochondrial and plastid translation. In other organisms, GCV activity is closely coordinated with the activity of serine hydroxymethyltransferase (SHMT) enzymes. P. falciparum contains cytosolic and mitochondr ...[more]

PMID: 20403390

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Project description:BackgroundMalaria remains a global health problem and the majority of deaths are caused by Plasmodium falciparum parasites. Due to the rapid emergence of drug-resistant strains, novel avenues of research on the biology of the parasite are needed. The massive proliferation of asexual, intra-erythrocytic parasites every 48 h could kill the human host prior to transmission of slow-developing gametocytes to the mosquito vector. A self-induced P. falciparum programmed cell death mechanism has been hypothesized to maintain this balance between the parasite and its two hosts, but molecular participants of the cell death pathway in P. falciparum have not been characterized. Proteins with SWIB/MDM2 domains play a key role in metazoan programmed cell death and this study provides the first evaluation of two parasite SWIB/MDM2 homologues, PF3D7_0518200 (PfMDM2) and PF3D7_0611400 (PfSWIB).MethodsThe function of these proteins was assessed by predicting their structural topology with the aid of bioinformatics and determining their location within live transgenic parasites, expressing green fluorescent protein-tagged PfMDM2 and PfSWIB under normal and elevated temperatures, which mimic fever and which are known to induce a programmed cell death response. Additionally, P. falciparum phage display library technology was used to identify binding partners for the two parasite SWIB/MDM2 domains.ResultsStructural features of the SWIB/MDM2 domains of PfMDM2 and PfSWIB, suggested that they are chromatin remodelling factors. The N-terminal signal sequence of PfMDM2 directed the protein to the mitochondrion under both normal and heat stress conditions. Plasmodium falciparum phage display library technology revealed that the C-terminal SWIB/MDM2 domain of PfMDM2 interacted with a conserved protein containing a LisH domain. PfSWIB localized to the cytoplasm under normal growth conditions, while approximately 10% of the heat-stressed trophozoite-stage parasites presented a rapid but short-lived nuclear localization pattern. Two PfSWIB binding partners, a putative Aurora-related kinase and a member of the inner membrane complex, were identified.ConclusionThese novel data provide insight into the function of two parasite SWIB/MDM2 homologues and suggest that PfMDM2 plays a role within the mitochondrion and that PfSWIB is involved in a stage-specific, heat-stress, response pathway.

Dataset Information

Validation of a modified method for Bxb1 mycobacteriophage integrase-mediated recombination in Plasmodium falciparum by localization of the H-protein of the glycine cleavage complex to the mitochondrion.

Publications

Validation of a modified method for Bxb1 mycobacteriophage integrase-mediated recombination in Plasmodium falciparum by localization of the H-protein of the glycine cleavage complex to the mitochondrion.

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