The cytosolic 70-kDa heat shock proteins (Hsp70s) Ssa and Ssb of are functionally unique. Ssb1 and Ssb2 which differ from each other by only four amino acids and from your members of the Ssa AEE788 family by ≈37% seem to have a more specialized function. Ssb binds to translating ribosomes and can be crosslinked to the nascent chain (18 19 This association in addition to the fact that strains lacking Ssb are hypersensitive to certain inhibitors of protein synthesis suggests that this class of Hsp70s may be involved in translation and/or very early folding events around the ribosome. In addition to the antibiotic sensitivity strains lacking Ssbs are cold-sensitive for growth. Genetic results using chimeric genes have shown that these two phenotypes are separable (20). For example rescue of the cold-sensitive phenotype requires the 44-kDa ATPase domain name from Ssb. Any two of the three (44- 18 and 10-kDa) Ssb domains are sufficient for rescue of the antibiotic sensitivity and result in chimera association with ribosomes. For example the expression of a chimera made up of the Ssa1 ATPase domain name and the 18-kDa and 10-kDa domains of Ssb1 allows for polysome association as well as growth in the presence of 70 μg/ml hygromycin B a concentration that inhibits the growth of cells lacking Ssb. Ssa1 has an ATPase activity very similar to that of other Hsp70s that have AEE788 been analyzed with a and TZ236: test (H. J. Motulsky GraphPad San Diego). Peptides A7 (RRLIEDAETAARG; catalog number A7433) and A5 (APRLRFTSL; catalog number A5308) and reduced CMLA used in the ATPase assays were obtained from Sigma and were used as 5 mg/ml and 10 mg/ml stock solutions respectively. CMLA was boiled to remove contaminating ATPase activity. For each 40-μl ATPase assay the following concentrations of each peptide unfolded protein or DnaJ-homologue were added: A5 (15 μg) A7 (15 μg) S32 (15 μg) CLMA (20 μg) Sis1 (12.9 μg) and Ydj1 (8.6 μg). RESULTS Kinetic Parameters of ATP Hydrolysis by Ssb. To begin a kinetic characterization of Ssb we compared the ATPase activity of Ssb to that of another yeast cytosolic Hsp70 Ssa1. By using a standard ATPase assay the were performed under the optimal concentrations of KOAc and AEE788 ATP for each given Hsp70. As shown in Table ?Table2 2 Ssb was not stimulated by CMLA or any of the peptides tested which are clearly capable of stimulating one or more other Hsp70 subfamily users. Furthermore Ssb ATPase activity was not stimulated by either yeast cytosolic DnaJ homolog Ydj1 or Sis1 even when these proteins were added in excess to Ssb. However both Ydj1 and Sis1 were able to stimulate two or more yeast Hsp70 subfamily users. AEE788 These data suggest that purified Ssb ATPase activity is not affected by the addition of peptide or DnaJs and that indeed Ssb may differ from other Hsp70s in this respect. However it is also possible that none of the peptides or DnaJs used in these assays interact with Ssb. Table 2 Activation factor of yeast Hsp70s ATPase activity by peptide CMLA and yeast DnaJ? homologs Ssb ATPase Activity Is usually Relatively Indie of Added Potassium. It has been shown that Ssa1 ATPase activity like that of other Hsp70s analyzed is highly K+-dependent (21). Ssa1 is nearly inactive at low concentrations of potassium and its affinity for ATP increases ≈20-fold when the potassium concentration is raised from 2.5 to 200 mM. To compare Ssa1 and Ssb we decided the K+ dependence of Ssb ATPase activity. There was little variance in ATPase activity of Ssb over a wide range of K+ concentrations (Fig. ?(Fig.11mutant strain chilly sensitivity and hypersensitivity to certain translation inhibiting drugs (20). Here we show that there are both fundamental differences between the intrinsic ATPase activities of the Ssa and Ssb 44-kDa ATPase domains and the intrinsic ability of the two C-terminal domains to AEE788 modulate the activity of an ATPase domain name. However whether these differences are critical for biological function will require more RYBP study because the results of the analysis carried out to date is usually complex. The fusion BAA rescues the cold-sensitive phenotype of a disruption strain. Here we demonstrate that this fusion BAA has biochemical properties more like Ssa1. This biochemical analysis is usually of particular interest because it suggests that it is not the B-like activity of the Ssb ATPase domain name that confers rescue of the cold-sensitive phenotype. However because wild-type Ssa1 cannot rescue Ssb function there must be some feature of the Ssb ATPase domain name that gives it Ssb-specific.