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

• Published in: Molecular and biochemical parasitology 2010-08, Vol.172 (2), p.156-160
• Main Authors: Spalding, Maroya D., Allary, Marina, Gallagher, John R., Prigge, Sean T.
• Format: Article
• Language: English
• Subjects: Bxb1 integrase; DNA, Mitochondrial - genetics; Genes, Reporter; Genetics, Microbial - methods; Glycine cleavage; Glycine Decarboxylase Complex - genetics; Green Fluorescent Proteins - genetics; H-protein; Integrases - genetics; Integrases - metabolism; Malaria; Mitochondrion; Mycobacteriophages - genetics; Plasmodium falciparum; Plasmodium falciparum - genetics; Recombinant Fusion Proteins - genetics; Recombination, Genetic; Viral Proteins - genetics; Viral Proteins - metabolism
• ISSN: 0166-6851; 1872-9428
• DOI: 10.1016/j.molbiopara.2010.04.005

The Bxb1 integrase system was used to localize the Plasmodium falciparum H-protein to the mitochondrion. Modifications to the transfection method resulted in decreased selection times. The glycine cleavage complex (GCV) is a potential source of the one carbon donor 5,10-methylene-tetrahydrofolate (5,10-CH2-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.