ISOLATION AND CHARACTERIZATION OF KAR2-404 MUTATION IN SACCHAROMYCES-CEREVISIAE

We have devised a direct screening method to isolate mutations in the KAR2 gene, and have isolated a BiP/KAR2 mutant, kar2-404, from Sacchar omyces cerevisiae as a small halo-forming mutant of secreted mouse alp ha-amylase. The mutation site was identified as a point mutation at t1 337 to c1337 resulting in the Ile404Thr mutation of mature Kar2-404p, located at the most NH2-terminal first beta-sheet structure (beta 1) o f the putative peptide-binding domain. This isoleucine is highly conse rved in the HSP 70 family. By pulse-chase experiments, no obvious diff erence was detected in the intracellular secretion rate of MF alpha 1- prepro-signal-mouse-alpha-amylase between the wild type and the kar2-4 04 mutant. However, only about half the amount of secreted heterologou s protein, mouse alpha-amylase, was detected in the mutant culture med ium compared with wild type. A smaller amount of homologous protein, a lpha-factor, was also detected and decreased faster in the mutant cult ure medium thean in wild type. Kar2-404p was expressed about 3-fold mo re than wild type Kar2p, probably to cover its defective functions, an d the turnover rates of Kar2p and Kar2-404p were about the same in viv o. The pruified Kar2-404p was slightly more sensitive to chymotryptic digestion than Kar2p in vitro.