Q2N and E64G double mutation of ubiquitin confers a stress sensitive phenotype on Saccharomyces cerevisiae.

The eukaryotic protein, ubiquitin harbours a parallel β-bulge in its structure which is formed by residues Glu64(1), Ser65(2) and Gln2(X). Despite their low % frequency of occurrence in parallel β-bulges, the residues Gln2 and Glu64 have been totally conserved in ubiquitin. In a previous study, two single mutants UbQ2N and UbE64G were constructed by replacing the residues Gln2 and Glu64 with Asn and Gly, respectively to understand their importance. The choice of the residues for substitution was made on the basis of their high preference for existence in parallel β-bulge, so that the structure of mutants remains unaltered and any functional differences observed would highlight the importance of Gln2 and Glu64 in ubiquitin biology. The results from this study established that yeast cells expressing either UbQ2N or UbE64G, displayed functional differences with respect to survival upon exposure to cycloheximide and degradation of substrates by ubiquitin fusion degradation (UFD) pathway. It describes construction of the double mutant UbQ2N-E64G and its characterization. Our results showed expression of UbQ2N-E64G in stress hypersensitive SUB60 cells led to significant decrease in growth rate and prolonged half-life of substrates of UFD pathway, besides failure of complementation under heat and antibiotic stresses, providing the reason for conservation of Gln2 and Glu64 in ubiquitin sequence.

Construction and functional characterization of double and triple mutants of parallel -bulge of ubiquitin.

Ubiquitin, a small eukaryotic protein serving as a post-translational modification on many important proteins, plays central role in cellular homeostasis and cell cycle regulation. Ubiquitin features two -bulges, the second -bulge, located at the C-terminal region of the protein along with type II turn, holds 3 residues Glu64(1), Ser65(2) and Gln2(X). Percent frequency of occurrence of such a sequence in parallel -bulge is very low. However, the sequence and structure have been conserved in ubiquitin through out the evolution. Present study involves replacement of residues in unusual -bulge of ubiquitin by introducing mutations in combination through site directed mutagenesis, generating double and triple mutants and their functional characterization. Mutant ubiquitins cloned in yeast expression vector YEp96 tested for growth profile, viability assay and heat stress complementation study have revealed significant decrease in growth rate, loss of viability and non-complementation of heat sensitive phenotype with UbE64G-S65D and UbQ2N-E64G-S65D mutations. However, UbQ2N-S65D did not show any negative effects in the above assays. Present results show that, replacement of residues in -bulge of ubiquitin exerts severe effects on growth and viability in Saccharomyces cerevisiae due to functional failure of the mutant ubiquitins UbE64G-S65D and UbQ2N-E64G-S65D.