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 Saccharomyces cerevisiae as a small halo-forming mutant of secreted mouse alpha-amylase. The mutation site was identified as a point mutation at t1337 to c1337 resulting in the Ile-404Thr mutation of mature Kar2-404p, located at the most NH2-terminal first beta-sheet structure (beta 1) 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 MF alpha 1-prepro-signal-mouse-alpha-amylase between the wild type and the kar2-404 mutant. However, only about half the amount of secreted heterologous protein, mouse alpha-amylase, was detected in the mutant culture medium compared with wild type. A smaller amount of homologous protein, alpha-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.
Kawamura-Watabe
A
A
Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
Tokunaga
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003470:Culture Media; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D015202:Protein Engineering / Q000379:methods; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism
D003470:Culture Media; D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; C060153:KAR2 protein, yeast; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D000516:alpha-Amylases