Isolation of a Dosage Dependent Lethal Mutation in Ubiquitin Gene of Saccharomyces Cerevisiae

Abstract

AbstractSummary: Ubiquitin is a small protein with a highly conserved sequence, playing a pivotal role in ubiquitin proteasome system (UPS). Considering the central role UPS has in cellular homeostasis, several drugs have been developed to target UPS to remove cells responsible for cancer and other neurodegenerative diseases. As an alternative to the above approach, in the present study we have isolated dose dependent lethal form of ubiquitin gene by in vitro evolution. In vitro evolution is a powerful tool for developing proteins with novel and desirable properties. The ubiquitin gene of Saccharomyces cerevisiae was subjected to in vitro evolution and lethal mutations were selected. The ubiquitin of S. cerevisiae differs only by three residues from human ubiquitin. The mutants were selected by expressing the protein in temperature sensitive ubi4 deletion mutants of ubiquitin. Most of the mutations in ubiquitin gene failed to complement UBI4 phenotype under heat shock. Only one of the mutants caused cell lysis, even at permissive temperature. Interestingly, expression of the same protein in wild type S. cerevisiae cells left them unaffected, establishing the mutant protein as a competitive inhibitor for UPS. Sequencing of the mutant gene showed four completely novel amino acid substitutions. They are namely, Ser20 to Phe, Ala46 to Ser, Leu50 to Pro and Ile61 to Thr. Construction of the mutant ubiquitin gene and characterization of the mutant phenotype along with the nature and location of the mutations are presented.