Effects of altered expression of the sumo conjugating enzyme, UBC9 on mitosis, meiosis and conjugation in Tetrahymena thermophila
SUMOylation is a critical posttranslational modification in eukaryotic species. Ubc9p is the E2-conjugating enzyme for SUMOylation and consequently it influences multiple cellular pathways. Nuclear proteins are common targets of SUMOylation and regulate nuclear events such as transcription, DNA repair and mitosis. The segregation of the Tetrahymena thermophila genome into two different nuclear compartments provides an unusual context for the analysis of SUMOylation. Each cell contains a transcriptionally silent, diploid germ line micronucleus (MIC) that divides by mitosis and a polyploid transcriptionally active somatic macronucleus (MAC) that divides by an amitotic mechanism. With the long-term goal to exploit these opportunities we initiated studies of Ubc9p and therefore indirectly SUMOylation, on the functionally distinct nuclei in T. thermophila using genetic analysis combined with proteomics study. We found that complete deletion of the UBC9 gene is lethal. Rescue of the lethal phenotype with a GFP-UBC9 fusion gene driven by a metallothionein promoter generated a cell line with a slow growth phenotype in the absence of CdCl 2-dependent expression of GFP-Ubc9p. Altered expression of Ubc9p resulted in differential effects in MICs and MACs. MICs were lost from cells during vegetative growth but MACs were capable of division. Interestingly, cells expressing a catalytically inactive dominant negative Ubc9p (DN-Ubc9p) accumulated multiple MICs. Ubc9p depleted cells were hypersensitive to DNA damaging agents that promote double-strand DNA breaks. Additional studies point to critical roles for Ubc9p during the sexual life cycle of Tetrahymena. Crosses between cell lines expressing the dominant negative Ubc9p were delayed in meiosis and produced fewer exconjugant progeny who successfully completed genetic exchange and conjugation than from wild-type controls. In contrast, cell lines that were depleted for Ubc9p did not form pairs and therefore could not complete any of the subsequent stages of conjugation including meiosis and macronuclear development. The results are consistent with roles for Ubc9p in mitosis, meiosis and double strand break repair. A proteomics-based approach generated an unbiased spectrum of Ubc9p interacting proteins during Tetrahymena vegetative growth and conjugation. We identified 128 high-confidence Ubc9p interacting proteins during Tetrahymena vegetative growth and 106 proteins during conjugation, among which 58 are conjugation-specific. Seven proteins with homologs in other species have been reported previously as SUMO substrates, or Ubc9p interacting proteins. The Ubc9p interactome covers a wide range of cellular processes, including chromatin remodeling, cell cycle progression, stress response, gene transcription and Tetrahymena macronuclear development, which further support our observations from phenotypic analysis. The findings provide evidence for distinct roles for SUMOylation in ciliate nuclei and provide opportunities for future studies of SUMOylated substrates in a context specific for gene expression (MAC) or mitotic and meiotic division (MIC).
Forney, Purdue University.
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