Utilizing Knockdowns, Overexpression and Tagging to Study the SUMO Proteases Ulp1 and Ulp2 in Tetrahymena thermophila

Abstract

SUMO, small ubiquitin like modifier, plays a role in post‐translational modification of proteins, altering protein‐protein interactions and regulating cellular processes. Much of what is known about the SUMO pathway and SUMOylation comes from studies conducted in yeast and vertebrates. As a unicellular ciliated eukaryote, Tetrahymena thermophila exhibit complexity and diversity of cellular processes that are not present in yeast, and they possess the advantages of other single celled model organisms, such as short cell division times and ease of growth and genetic manipulation. We aimed to develop a better understanding of the function of the two SUMO proteases, Ulp1 and Ulp2, by knocking out the genes, ULP1 and ULP2, that encode these proteins. We designed knockout plasmid constructs by cloning the 5′ and 3′ flanking sequences of each gene into a vector with a neo resistance marker. The plasmid construct was then introduced into Tetrahymena, and cells were passaged into increasingly higher concentrations of paromomycin to promote phenotypic assortment. Quantitative RT‐PCR suggests that we were able to knockdown ULP2 but not ULP1, despite high paromomycin drug resistance, suggesting that ULP1 is an essential gene in Tetrahymena. We have also created plasmid constructs using a Gateway cloning method that enable us to both overexpress and YFP tag ULP1 and ULP2. Preliminary results show that Ulp1 localizes to the nuclear envelope, and Ulp2 localizes to the nucleus. Future work will focus on characterizing the overexpression phenotypes of ULP1 and ULP2 and the knockdown phenotype of ULP2 in Tetrahymena.Support or Funding InformationThis research was supported by the St. Olaf College Office of Collaborative Undergraduate Research, St. Olaf TRIO McNair Scholars Program (L.T.) and the Beta Beta Beta National Biological Honor Society (J.L.).