Characterization of deletion mutations in the carboxy-terminal peptide-binding domain of the Kar2 protein inSaccharomyces cerevisiae

✍ Scribed by Tokunaga, Masao; Kato, Shinya; Kawamura-Watabe, Akiko; Tanaka, Ryoichi; Tokunaga, Hiroko

• Publisher: John Wiley and Sons
• Year: 1998
• Tongue: English
• Weight: 287 KB
• Volume: 14
• Category: Article
• ISSN: 0749-503X
• DOI: 10.1002/(sici)1097-0061(1998100)14:14<1285::aid-yea329>3.0.co;2-0

✦ Synopsis

Hsp70 is structurally composed of three domains, an amino-terminal ATPase domain, a proximal 18 kDa peptide-binding domain and a distal 10 kDa carboxy-terminal (C-terminal) domain. To dissect the functional significance of the distal 10 kDa domain, and the boundary region between the proximal and distal C-terminal domains of Kar2p in vivo in Saccharomyces cerevisiae, we constructed a series of plasmids which were truncated or had internal deletion mutations in this region. We found that all these mutations are recessive, and that the distal 10 kDa C-terminal domain, including the HDEL ER-retention sequence, is not essential for cell growth, although the major role of this 10 kDa C-terminal domain is due to the function of the HDEL ER-retention signal. We also found that the Kar2p region (Thr 492 -Thr 512 ), corresponding to the 8-sheet in the peptide-binding domain, which constitutes the bottom plate of the binding pocket in E. coli DnaK, is essential for cell viability, and that the following Kar2p region (Glu 513 -Lys 542 ), corresponding to -helices A and B of E. coli DnaK, which was proposed to compose the lid of the binding pocket, is critical but not essential for yeast cell growth. This was further supported by the fact that the latter deletion showed a fully reversible ts phenotype in its growth and only a slight inhibitory effect on the secretion of -amylase at non-permissive temperature.