Biogenesis of cytochromescandc₁in the electron transport chain of malaria parasites

Abstract

Plasmodiummalaria parasites retain an essential mitochondrional electron transport chain (ETC) that is critical for growth within humans and mosquitoes and a key antimalarial drug target. ETC function requires cytrochromes c and c1that are unusual among heme proteins due to their covalent binding to heme via conserved CXXCH sequence motifs. Heme attachment to these proteins requires the mitochondrial enzyme holocytochrome c synthase (HCCS) that binds heme and the apo cytochrome to facilitate biogenesis of the mature cytochrome c or c1. Although humans encode a single bifunctional HCCS that attaches heme to both proteins,Plasmodiumparasites are like yeast and encode two separate HCCS homologs thought to be specific for heme attachment to cyt c (HCCS) or cyt c1(HCC1S). To test the function and specificity ofP. falciparumHCCS and HCC1S, we used CRISPR/Cas9 to tag both genes for conditional expression. HCC1S knockdown selectively impaired cyt c1biogenesis and caused lethal ETC dysfunction that was not reversed by over-expression of HCCS. Knockdown of HCCS caused a more modest growth defect but strongly sensitized parasites to mitochondrial depolarization by proguanil, revealing key defects in ETC function. These results and prior heterologous studies inE. coliof cyt c hemylation byP. falciparumHCCS and HCC1S strongly suggest that both homologs are essential for mitochondrial ETC function and have distinct specificities for biogenesis of cyt c and c1, respectively, in parasites. This study lays a foundation to develop novel strategies to selectively block ETC function in malaria parasites.

Identification of a divalent metal transporter required for cellular iron metabolism in malaria parasites

“…2A). This rapid sensitivity differs from our prior knockdown studies of proteins localized to the apicoplast and mitochondrion, in which growth phenotypes were not observed until at least the second cycle of RBC infection 12, 48, 49, 75 . The unusual sensitivity of parasites to PfDMT1 knockdown may reflect the need to fully synthesize this protein de novo in each life cycle, since the FV is not inherited by daughter parasites (unlike the apicoplast and mitochondrion).…”

Malaria parasites require a divergent heme oxygenase for apicoplast gene expression and biogenesis

“…Critically, we observed that growth in -aTC conditions reduced PfHO levels by >85% (Figure 3C), indicating substantial downregulation of PfHO protein expression. PfHO mRNA levels were also selectively decreased by ~75% upon aTC washout, consistent with prior reports that TetR-DOZI association with transcripts targets mRNA to P-bodies for degradation (Figure 3 -figure supplement 6) [48][49][50] . Because of the consistency and stringency of knockdown achieved by the PfHO-aptamer/TetR-DOZI system, all subsequent knockdown experiments were performed in this line.…”