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.
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.
Spalding
Maroya D
MD
Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA.
Allary
Marina
M
Gallagher
John R
JR
Prigge
Sean T
ST
D004272:DNA, Mitochondrial / Q000235:genetics*; D017930:Genes, Reporter; D005827:Genetics, Microbial / Q000379:methods*; D050959:Glycine Decarboxylase Complex / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D009160:Mycobacteriophages / Q000235:genetics*; D010963:Plasmodium falciparum / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D004272:DNA, Mitochondrial; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D050959:Glycine Decarboxylase Complex; D019426:Integrases
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