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.

Figure 1. Integration of pMA013 at the attB locus

(A) Schematic of primer annealing sites at the unintegrated attB locus and at the integrated locus, in which the attP site on pMA013 combines with the attB site to yield attL and attR sequences. (B) Genomic DNA was extracted from the parental Dd2^attB parasites and from the transgenic cell line H-protein^Dd2 and analyzed for the presence of the attB (no integration), attL (5′ integration), and attR (3′ integration) sites.

Figure 2. Expression and localization of the H-protein in H-protein^Dd2 cells

(A) Expression of H-protein-GFP. H-protein^Dd2 parasites were isolated from red blood cells by lysis with 0.2% saponin for 3 minutes and the reaction was quenched with ice cold PBS. The parasite pellet was washed twice in 10 ml of ice cold PBS. Parasites were resuspended in SDS-PAGE sample loading buffer and lysed by three cycles of heating at 95°C followed by vortexing. Proteins were separated by SDS-PAGE and transblotted onto nitrocellulose. H-protein-GFP was detected with monoclonal antibody specific for GFP (1:1000, Clonetech). (B) Co-localization of GFP fluorescence from H-protein^Dd2 cells with mitotracker and Hoechst dyes. Live cells from the H-protein^Dd2 cell line were incubated with 100 nM mitotracker CMX-Ros (Invitrogen) and 1 μg/ml Hoechst stain for 20 minutes at 37°C followed by 2 washout periods in complete media for five minutes. Cells were imaged on a Nikon Eclipse E800 epifluorescent microscope, and the images were processed with ImageJ software.