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Ubiquitin is a eukaryotic protein with 96% sequence conservation from yeast to human. Ubiquitin plays a central role in protein homeostasis and regulation of protein function. We have reported on the generation of variants of ubiquitin by in vitro evolution in Saccharomyces cerevisiae to advance our understanding of the role of the invariant amino acid residues of ubiquitin in relation to its function. One of the mutants generated, namely UbEP42, was a dosage-dependent lethal form of the ubiquitin gene, causing lethality to UBI4-deficient cells but not to ubiquitin wild-type cells. In the present study we investigated the functional reasons for the observed lethality. Expression of UbEP42 in a UBI4-deleted stress-sensitive strain resulted in an increased generation time due to a delayed S phase caused by decreased levels of Cdc28 protein kinase. Cells expressing UbEP42 displayed heightened sensitivity towards heat stress and exposure to cycloheximide. Furthermore, its expression had a negative effect on the degradation of substrates of the ubiquitin fusion degradation pathway. However, UbEP42 is incorporated into polyubiquitin chains. Collectively, our results establish that the effects seen with the mutant ubiquitin protein UbEP42 are not due to malfunction at the stage of polyubiquitination., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Ubiquitin is a globular protein with a highly conserved sequence. Sequence conservation and compact structure make it an ideal protein for structure-function studies. One of the atypical secondary structural features found in ubiquitin is a parallel G1 beta-bulge. Glutamate at 64 is the first residue of this beta-bulge and the third residue in a type II turn. However, glycine is seen in these positions in several proteins. To understand the effects of substitution of glutamate64 by glycine on the structure, stability and function of ubiquitin, mutant UbE64G has been constructed and characterized in Saccharomyces cerevisiae. The secondary and tertiary structures of UbE64G mutant protein are only marginally different from wild-type protein (UbWt) and fluorescent form of ubiquitin (UbF45W). The earlier studies have shown that the structure and stability of UbWt and UbF45W were similar. However, UbE64G has less surface hydrophobicity than UbWt. UbE64G is found to be more stable compared with UbF45W towards guanidinium chloride induced denaturation. In vivo, complementation shows substrate proteins with Pro as the N-terminal residue, which undergo ubiquitination, have extended half-lives with UbE64G. This altered preference for Pro as opposed to Met might be related to natural preference of glutamate at 64th position in ubiquitin.