Developmentally regulated SUMOylation in the ciliate Tetrahymena thermophila

<p>The Small Ubiquitin-like MOdifier (SUMO) protein regulates numerous nuclear events such as transcription, mitosis and meiosis and DNA repair. These processes are critical to the programmed nuclear events of conjugation in ciliates and provide the potential to investigate developmentally regulated SUMOylation. We predicted a developmental increase in SUMOylation during late conjugation based on the extensive genome remodeling in the developing macronucleus (anlagen) of<em>Tetrahymena thermophila.</em> Immunoblotting of cell lysates from vegetative and mating cells using anti-SUMO antibodies revealed distinct developmental differences and an increased signal correlated with formation of the anlagen. Immunofluorescence of mating <em>Tetrahymena</em> cells with the same antibody revealed an increase in staining of the parental macronucleus until the signal shifts to the anlagen at 7 hours post-mixing. This along with the finding that GFP-Uba2 fusion proteins localize to the anlagen is consistent with a major nuclear role for SUMOylation during conjugation. Germ-line knockout mutants of <em>SUMO (SMT3)</em> and <em>UBA2 </em>are vegetative lethal and conditional mutants dependent on a cadmium-inducible metallothionein promoter exhibit reduced cell growth and increased sensitivity to DNA damaging agents upon cadmium withdrawal. Interestingly, mating of Uba2p conditional lines leads to a cadmium-dependent delay after meiosis but prior to macronuclear development predicting a SUMOylation-dependent event. Additionally, in an effort to provide further insight into the various processes affected by SUMO modification, we utilized a proteomics-based approach to generate an unbiased spectrum of SUMO protein substrates from vegetative<em>Tetrahymena. </em>We used a two-step affinity purification scheme to isolate SUMO substrates from a tagged <em>Tetrahymena</em> strain. Proteins from the purification were then identified by subsequent LC-MS.MS analysis using a QuadTOF mass spectrometer. We identified 110 candidate proteins that were identified by 2 or more peptides. The nature of the protein substrates that were identified is consistent with roles of SUMOylation in modulating diverse cellular processes including transcriptional regulation, protein folding and translation, metabolism and crosstalk with other post-translational modifications. These results support multiple roles of SUMOylation in regulating various cellular processes in eukaryotic cells. Taken together, our findings provide the foundation for additional studies of SUMOylation during conjugation in <em>Tetrahymena</em></p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Year

2015

Author dc:creator

• Nasir, Amjad M

Contributors dc:contributor

• Amjad M. Nasir
• Ourania M. Andrisani
• Scott D. Briggs
• Mark C. Hall

• Biochemistry

Repository record dc:identifier

https://docs.lib.purdue.edu/open_access_dissertations/524

OAI identifier oai:identifier

oai:docs.lib.purdue.edu:open_access_dissertations-1454