Investigating the Plasmodium falciparum mitochondrial proteome for functional analysis of conserved essential proteins

Ian  Michael Lamb

doi:10.17918/00001439

Mitochondrial functions are essential throughout the life cycle of malaria parasites and are validated as targets of antimalarial drugs such as atovaquone. Using a mitochondrially targeted proximity biotinylation approach, here, we explore the possibility that identification of novel, conserved, and essential proteins encoding mitochondrial functions may provide additional targets for the discovery of antimalarial drugs in P. falciparum. To initiate this investigation, we generated parasites that express a protein where the Hsp60 leader sequence (for mitochondrial targeting) is fused to the promiscuous biotin ligase TurboID from an ectopic locus. Initial experiments suggested inefficient targeting to the mitochondrion, as many cytosolic proteins were identified in the mass spectrometry analysis, presumably due to labeling by Hsp60L-TurboID proteins in transit to the mitochondrion. To overcome this challenge, we employed hypotonic lysis and centrifugation to fractionate parasite pellets into "soluble" and "membrane" fractions after labeling. After three biological replicates of this experiment and subsequent mass spectrometry analysis, we identified 121 proteins that we considered to be "putative mitochondrial" proteins. Of these, 26 were annotated as conserved Plasmodium proteins of unknown function. We characterized five of these 122 for their sub-cellular localization and found that four were localized to the mitochondrion. Of these, PF3D7_0707400 attracted our attention as a candidate for phenotypic characterization. This protein is abundantly expressed and is an ortholog of mammalian ATAD3A (ATPase family AAA+ domain-containing protein 3), a mitochondrial membrane localized protein. In mammals, ATAD3A spans the matrix, the inner and outer mitochondrial membranes, and communicates with the endoplasmic reticulum, playing a central role in mitochondrial biogenesis. ATAD3A orthologues are conserved in all apicomplexans, including Cryptosporidium, suggesting it to have a critical role in mitochondrial physiology of all of these parasites. To initiate its characterization in malaria parasites, we generated parasites in which this protein was endogenously tagged and subjected to conditional expression. These parasites grew normally in the presence of aTc but failed to thrive in the absence of aTc within 72 hours. Mitochondrial membrane potential in PfATAD3A knockdown parasites was collapsed by 48 hours. Native gel electrophoresis suggested PfATAD3A to be a part of a complex estimated to be >5 million Dalton. Characterization of this complex will likely reveal hitherto unknown essential features of mitochondrial physiology in malaria parasites.

Investigating the Plasmodium falciparum mitochondrial proteome for functional analysis of conserved essential proteins

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Investigating the Plasmodium falciparum mitochondrial proteome for functional analysis of conserved essential proteins

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