Identification and Characterization of smt4∆ High-Copy Suppressors in Saccharomyces cerevisiae
The conjugation and de-conjugation of SUMO-l/Smt3, a pair of ubiquitin-like proteins, play an important but yet to be fully understood role in cell cycle progression and cellular checkpoint control. To better understand the SUMO-l/Smt3 system, a high-copy suppression screen was performed to isolate potential substrates of Smt3. Using the yeast Saccharomyces cerevisiae, we isolated genes which could suppress thesmt4∆ defect. Smt4 is responsible for the deconjugation of Smt3 from its substrates and so genes capable of the suppression of this defect are likely candidates for a Smt3 substrate. The screen isolated 20 clones capable of high-copy suppression. Clones were characterized by a number of tests and showed variation in their ability to suppress the growth defect and morphological characteristics associated with the smt4∆ defect under various conditions, including high-temperature and chemicals affecting multiple cell cycle checkpoints. A number of genes isolated through partial sequencing bear similarity in function to known substrates for Smt3 and/or SUMO-1 and so are promising candidates for possible Smt3/SUMO-1 substrates. Nfi 1 and YGL250W are two genes that warrant further testing. However, it still remains to be seen how many of the isolated genes are directly associated with the SUMO-l/Smt3 conjugation system. UBP3, a ubiquitin cleaving enzyme, is only capable of suppression when under high-copy control is unlikely to be directly associated with Smt3 in vivo. Further testing is needed to discover which of other genes isolated in the clones are responsible for the suppression, but it appears likely that continuation of this experiment and others like it will provide further information on the Smt3/SUMO-1-protein conjugation system and that this experiment can serve as a model for future experiments where the discovery of a substrate is desired.
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