Characterization of the chimeric protein cUBC1 engineered by substituting the linker of E2-25K into UBC1 enzyme of Saccharomyces cerevisiae

Abstract

Ubiquitination is an important posttranslational modification of proteins in eukaryotic cells, wherein ubiquitin molecules are conjugated to target proteins. Ubiquitination is catalyzed by the cascade of ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3). The number of E2s encoded in eukaryotes partly explains their contribution to the inherent specificity of the ubiquitin system. The ubiquitin conjugating enzyme UBC1 of Saccharomyces cerevisiae participates the degradation of short-lived and abnormal proteins. UBC1 consists of two well-defined domains separated by a long flexible linker. E2-25K, the human homolog of UBC1 is crucial to neurons and its failure leads to neurodegenerative disorders. The linker of UBC1 is of 22 amino acids, while that of E2-25K has 6 amino acids. To understand the importance of the linker, the chimeric protein, cUBC1 was constructed by substituting the linker of E2-25K in UBC1. cUBC1 shows minor changes in its secondary structure. cUBC1 expression in ubc1 deletion mutants showed no effect over growth, thermotolerance and resistance to antibiotic stress. However, survival under heat stress was enhanced with cUBC1. Western blot analysis of the enzymatic activity showed cUBC1 performed equally well as UBC1. Hence, cUBC1 demonstrates that the shorter linker increased the stability of UBC1.