Characterization of the Bar Proteinase, An Extracellular Enzyme from the Yeast Saccharomyces Cerevisiae
Haploid S. cerevisiae cells of the a mating type constitutively secrete an extracellular proteinase that cleaves the peptide mating pheromone (α-factor) secreted by mating-type α cells. DNA sequence analysis of the BAR1 gene that encodes Bar proteinase demonstrated that the primary translation product of 587 amino acids has strong homology to two-domain aspartic proteinases such as pepsin, chymosin, and others, but contains a unique third domain that is not homologous to these enzymes. When produced by wild-type yeast cells, the Bar enzyme exists as a heterogeneous, heavily glycosylated protein with apparent molecular weight >200,000 Da. By producing the proteinase in mutant yeast strains that are defective in glycosylation, we have been able to purify and characterize a homogeneous species. In this paper, we will describe some of the enzyme’s physical properties and substrate requirements, as well as present data indicating that the third domain is required for secretion of the proteinase to the culture medium.
KeywordsTriose Phosphate Isomerase Aspartyl Proteinase Putative Active Site Scissile Bond Mutant Yeast Strain
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