Isolation and characterization of kar2-404 mutation in saccharomyces cerevisiae

Abstract

We have devised a direct screening method to isolate mutations in the KAR2 gene, and have isolated a BiP/KAR2 mutant, kar2-404, from Saccharomyces cerevisiae as a small halo-forming mutant of secreted mouse α-amylase. The mutation site was identified as a point mutation at tl337 to cl337 resulting in the Ile404Thr mutation of mature Kar2-404p, located at the most NH2-terminal first β-sheet structure (β1) of the putative peptide-binding domain. This isoleucine is highly conserved in the Hsp70 family. By pulse-chase experiments, no obvious difference was detected in the intracellular secretion rate of MFα1-prepro-signal-mouse-α-aniylase between the wild type and the kar2-404 mutant. However, only about half the amount of secreted heterologous protein, mouse α-amylase, was detected in the mutant culture medium compared with wild type. A smaller amount of homologous protein, α-factor, was also detected and decreased faster in the mutant culture medium than in wild type. Kar2-404p was expressed about 3-fold more than wild type Kar2p, probably to cover its defective functions, and the turnover rates of Kar2p and Kar2-404p were about the same in vivo. The purified Kar2-404p was slightly more sensitive to chymotryptic digestion than Kar2p in vitro. © 1997, Taylor & Francis Group, LLC. All rights reserved.