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Dissertation
Investigation of the unusual ATP synthase complexes in the alveolates Tetrahymena thermophila and Plasmodium falciparum
Doctor of Philosophy (Ph.D.), Drexel University
Dec 2010
DOI:
https://doi.org/10.17918/00008142
Abstract
ATP synthesis is carried out by the ATP synthase complex, which uses proton flow to drive the rotation of protein subunits to produce ATP. The enzyme has two sectors: F1 has the catalytic sites for ATP synthesis, and Fo forms channels for proton movement and functions as a bearing and stator. Because the parts of the complex have to interact with each other, critical protein subunits of the enzyme are conserved from bacteria to higher eukaryotes. We were surprised that a group of unicellular organisms called alveolates, which includes Plasmodium falciparum and Tetrahymena thermophila, lacked several critical proteins of the Fo sector, according to whole genome sequence analyses. The role of the putative mitochondria! ATP synthase in Plasmodium has been unclear, as blood stages of P. falcinarum appear to derive ATP largely through glycolysis, and the missing Fo subunits suggested a nonfunctional enzyme. However, Tetrahymena is fully capable of oxidative phosphorylation, suggesting the presence of undetected, divergent Fo subunits. We hypothesized that similar divergent Fo subunits might be shared by other alveolates, including Plasmodium. To test that, we isolated intact ATP synthase complexes from the ciliate Tetrahymena thermophila and examined their structure by electron microscopy and their protein composition by mass spectrometry. We found that the ATP synthase complex of this organism is highly divergent both in its overall structure and many of the associated protein subunits. At least 13 novel proteins are present within this complex that have no orthologues in any organism outside of the ciliates. Except for one possible protein (PF11_0262 - putative 1-) subunit), we could not identify any orthologues of the novel subunits in Plasmodium. Our investigation of Plasmodium ATP synthase has revealed the predominant form to be dimers, as seen in other eukaryotic organisms. Also, immunofluoresence microscopy of parasite lines expressing epitope-tagged versions of all the identified subunits of the ATP synthase was consistent with the localization of these subunits (with one exception) to the mitochondrion. Furthermore, our efforts to knock out key subunits (beta and gamma) of the enzyme were unsuccessful, suggesting an important role in the parasite life cycle.
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Details
- Title
- Investigation of the unusual ATP synthase complexes in the alveolates Tetrahymena thermophila and Plasmodium falciparum
- Creators
- Praveen Balabaskaran Nina
- Contributors
- Thomas D. Edlind (Advisor) - Drexel University, Drexel University (1970-)Akhil B. Vaidya (Advisor) - Drexel University, Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xi, 198 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Microbiology and Immunology; College of Medicine; Drexel University
- Other Identifier
- 991021889099204721