Beyond the Dinoflagellate Transcriptome: Validation of Protein Production via Biochemical Analysis and Mass Spectrometry
AdvisorPlace, Allen R.
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AbstractDinoflagellates are members of the Alveolata (meaning “with cavities”), a monophyletic group of single cell protists which includes apicomplexans and ciliates that exhibit a diverse mode of nutrition, ranging from predation to photo autotrophy to intracellular parasitism. Dinoflagellates are both primary producers and consumers in the food web, sometimes at the same time, and best known for their dominant role in causing harmful algal blooms. In keeping with their unusual nuclear chromatin architecture (condensed throughout the cell cycle) the degree to which dinoflagellates use transcriptional responses to alter mRNA expression levels appears limited. In addition, it appears dinoflagellates have extensive gene duplications for most biological processes. For these reasons, the goal for this dissertation was to measure protein levels directly for important cellular phenotypes. We began with characterizing a proton selective channel (Hv1) that was hypothesized to be involved in bioluminescence in Lingulodinium polyedrum with goals of elucidating function, location, and production of this protein. Next, we focused on acetyl CoA carboxylases (ACC) which are responsible for the rate limiting step in fatty acid (FAS) and polyketide synthases (PKS). We find the presence of these proteins in thirteen dinoflagellates surveyed and show there is distribution in the plastid and cytosol. In Amphidinium carterae we verify protein production with high performance liquid chromatography-coupled to tandem mass spectrometry (LC-MS/MS), otherwise known as shotgun proteomics as well as compare transcript and protein levels across a diel cycle. Lastly, we focused on a multi-modular polyketide protein in A. carterae with implications in toxin and fatty acid production due to the domain arrangement which is indicative of a noniterative trans-AT PKS process. We show successful inhibition of 14C acetate incorporation into toxins and fatty acids with the addition of the ketosynthase specific (KS) fatty acid inhibitor cerulenin. Partial protein production is verified by western blotting and LC-MS/MS analysis of KS peptides reveals a strong covalent bond with cerulenin addition. Taken together the work discussed in this thesis has resulted in a better understanding of three different cellular processes in dinoflagellates using a shotgun proteomic approach. Future work continuing this trend of protein characterization in dinoflagellates will help elucidate many uncharacterized pathways in dinoflagellate biology.
University of Maryland, Baltimore