Characterization of deletion mutations in the carboxy-terminal peptide-binding domain of the Kar2 protein in Saccharomyces cerevisiae.
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 (Thr492-Thr512), corresponding to the beta 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 (Glu513-Lys542), corresponding to alpha-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 alpha-amylase at non-permissive temperature.
Tokunaga
M
M
Laboratory of Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, Japan.
Kato
S
S
Kawamura-Watabe
A
A
Tanaka
R
R
Tokunaga
H
H
D001665:Binding Sites / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017384:Sequence Deletion; D000516:alpha-Amylases / Q000378:metabolism
D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; C060153:KAR2 protein, yeast; D000516:alpha-Amylases