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Dissertation
Characterization of PfCoq10 and its use in exploring the Plasmodium falciparum mitochondrial proteome
Doctor of Philosophy (Ph.D.), Drexel University
Apr 2016
DOI:
https://doi.org/10.17918/00000884
Abstract
The Plasmodium mitochondrion is essential throughout the span of the parasite lifecycle, and is a validated target for antimalarial drugs; however, several key metabolic pathways are diminished in the asexual stages, and genetic studies have demonstrated that several critical functions such as the tricarboxylic acid cycle are expendable. While some of these biosynthetic and metabolic pathways have been characterized, little is known about the mitochondria proteome as a whole, including components of import/export, maintenance, and regulation. We have characterized PfCoq10, which complements a Saccharomyces cerevisiae coq10 null allele. ScCoq10 is a mitochondrial ubiquinone-binding protein; its gene deletion results in respiration deficiency without inhibiting the synthesis of ubiquinone (CoQ), suggesting that the Coq10 protein is critical for the delivery of CoQ to the site(s) of respiration. Because CoQ is essential to the parasite and employed during mitochondrial electron transport and pyrimidine biosynthesis, we sought to determine PfCoq10's role in the mitochondria, and in delivery of antimalarials. PfCoq10 is predominantly expressed in trophozoite-stage parasites, and localizes to the parasite mitochondrion. While CoQ has some structural similarity to the antimalarial drug atovaquone, our data suggests that PfCoq10 does not functionally interact with atovaquone. To determine how PfCoq10 may be involved in essential mitochondrial pathways, we utilized the promiscuous prokaryotic biotin ligase, BirA*, which non-discriminately biotinylates proteins in its immediate vicinity. Parasites expressing a PfCoq10-BirA* fusion display a unique profile of biotinylation primarily localized within the mitochondria, and these proteins were extracted from parasites and identified by mass spectrometry. Over 300 proteins were detected at significant levels among three PfCoq10-BirA* samples, and 150 of these were detected in two or more samples. Of the 79 proteins detected in all three, over 70% are predicted or confirmed to be mitochondrial, and include components of metabolic pathways, translation machinery, chaperones, and degradation systems, some of which were previously not confirmed to be mitochondrial. We are additionally using BirA* fused to the mitochondrial targeting sequence of HSP60 to explore the mitochondrial proteome in specific stages of the parasite lifecycle to aid in the characterization of new and important elements in this essential organelle.
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Details
- Title
- Characterization of PfCoq10 and its use in exploring the Plasmodium falciparum mitochondrial proteome
- Creators
- Bethany Joy Jenkins
- Contributors
- Akhil B. Vaidya (Advisor) - Drexel University, Microbiology and Immunology
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xi, 240 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Microbiology and Immunology; College of Medicine; Drexel University
- Other Identifier
- 991016814379804721