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Construction and Characterization of UBC4 Mutants with Single Residues Swapped from UBC5

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Abstract

Ubiquitination is tightly regulated to control degradation, localization and function of various proteins. Ubiquitination is catalysed by three enzymes, namely E1, E2 and E3. The specificity shown by E2s for E3s holds key to regulation of ubiquitination. Here we focussed on the E2 enzymes, UBC4 and UBC5 of Saccharomyces cerevisiae, which are almost identical differing only by 11 residues. They show functional complementation in protein degradation, especially during stress response. Existence of two almost identical proteins suggests specialized requirement of one of them under selective conditions. To understand the reasons for the residue differences between them, mutations were introduced in the UBC4 gene to generate single residue variants by swapping with codons from UBC5. Though the variants are found to be functionally active in Δubc4Δubc5 strain of yeast, they cause reduced growth under normal conditions, altered survival under heat and antibiotic stresses, when compared with UBC4. The variants indicated decrease in protein stability theoretically. Hence, the residues of UBC5 individually do not confer any structural advantage to UBC4. Interactive proteins of UBC4 are nearly three times more than those of UBC5. UBC5, therefore, is a functionally minimized version, evolved as another means of regulation to meet cell stage specific needs.

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Acknowledgements

The authors express their sincere gratitude to Prof. Stefan Jentsch of Max Planck Institute of Biochemistry, Germany, for the plasmid carrying UBC4 gene and Prof. Mark Hochstrasser of Yale University, New Haven, CT, USA, for providing the S. cerevisiae strains MHY501 and MHY508. V.R. and B.P. were supported by University Fellowship of The M. S. University of Baroda, Vadodara, India.

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  1. Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, 390002, India

    Varsha Raimalani, Brinda Panchamia & C. Ratna Prabha

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  1. Varsha Raimalani
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Correspondence to C. Ratna Prabha.

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Raimalani, V., Panchamia, B. & Prabha, C.R. Construction and Characterization of UBC4 Mutants with Single Residues Swapped from UBC5. Cell Biochem Biophys 78, 43–53 (2020). https://doi.org/10.1007/s12013-019-00894-1

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