The Stability and Localization of Tetrahymena thermophila UBC9 Mutants

Abstract

UBC9 is the sole E‐2 conjugating enzyme in the SUMOylation pathway, which is analogous to the ubiquitin pathway. SUMOylation can affect the localization of proteins or their ability to interact with other substrates. UBC9 is essential in most eukaryotes including the ciliate Tetrahymena thermophila. Tetrahymena thermophila are protozoa with two nuclei, a somatic macronucleus (MAC) and germline micronucleus (MIC). Transcription occurs in the macronucleus, but not in the micronucleus. Tetrahymena have a large increase in SUMOylation within the developing macronucleus during conjugation (sexual reproduction). In an effort to create UBC9 mutants with altered activity or cellular location, we designed site‐directed changes to the UBC9 gene. Some of these changes were analogous to mutations identified in yeast UBC9, others were directed at the N‐terminus that is conserved between Tetrahymena thermophila and other ciliates, but is substantially different from other eukaryotes. Plasmids containing YFP fusions of UBC9 with small deletions in the N‐ or C‐ terminus or single amino acid changes were transformed into Tetrahymena and viewed via fluorescence microscopy to determine localization. Constructs with the same mutations but N‐terminal FLAG tags were analyzed via Western blots to determine stability of the mutant proteins. Versions of UBC9‐YFP fusions with 13 amino acids removed at the N‐terminus were not detectable, or barely detectable by microscopy as compared with wild type UBC9‐YFP fusions. A YFP fusion with only 4 amino acids removed from the N‐terminus was visible with a similar intensity as wild type (full length) controls. In contrast, removal of 4 amino from the C‐terminus eliminated all signal from expression of a YFP‐fusion. The tagged versions of UBC9 with 13 and 4 amino acids of the protein removed at the N‐terminus produced a signal on a Western blot; while a UBC9 construct with 4 amino acids removed from the C‐terminus was not able to produce a signal on a Western blot. To our surprise, removing amino acids from the C‐terminus had a greater effect on protein stability than removing those from the N‐terminus. Analysis of two other mutations (R20A, R24D) predicted to create altered cellular location or stability of the protein showed small, if any differences from wild type UBC9 in the distribution between cytoplasm and nucleus. Additional mutations are being constructed in the C‐terminal region in an attempt to generated UBC9 with marginal stability that could be used for in vivo analysis of function.Support or Funding InformationDepartment of Biochemistry, Purdue University, West Lafayette, INCollege of Agriculture (Molecular Agriculture Summer Institute), Purdue University, West Lafayette, IN