To assess whether the regulation of ATP hydrolysis by Sse1p is similar to that of Ssa1p, we first purified hexahistidine-tagged forms of both proteins and compared their ATPase activities in the presence and absence of select Hsp40 co-chaperones. As shown in , we found that the activity of the Ssa1p but not Sse1p was significantly enhanced by two cytosolic yeast Hsp40s, Hlj1p and Sis1p, which are respectively ER- and ribosome-associated Hsp70 co-chaperones that are known to interact with Ssa1p [19
]. A mammalian Hsp40, Csp, which activates ATP hydrolysis by mammalian Hsc70 [24
] also selectively stimulated ATP hydrolysis by Ssa1p. Notably, the unstimulated rates of ATP hydrolysis by Ssa1p and Sse1p were very similar; the turnover numbers were 0.33 per minute for Ssa1p and 0.34 per minute for Sse1p. This is consistent with the range of published values for the unstimulated rates of ATP hydrolysis by Hsp70 chaperones [25
The ATPase activity of yeast Hsp70 but not Hsp110 is stimulated by select Hsp40 co-chaperones
Next, we employed a method that was previously used to isolate peptides that bound to the E. coli
Hsp70, DnaK [28
] and Hsp40, DnaJ [29
]. In brief, nitrocellulose blots containing partially overlapping 14 amino acid peptides derived from firefly luciferase, an Sse1p substrate [5
], were probed with purified Sse1p. Peptides corresponding to select “hits” from this analysis were synthesized and one peptide, designated “LIC”, was conjugated to 6-carboxyfluorescein (6CF) so that binding could be assessed in solution. We found that 6CF-LIC associated with Sse1p with a KD
of 2.3 nM (, open circles). This is higher than the affinity of Hsp70 for other peptides, such as p5 and APPY, measured in the presence of ADP or in the absence of nucleotide (~60 nM;[31
]), but the binding of 6CF-LIC to Ssa1p under these conditions (KD
= 27 nM; , closed circles) was quite similar to the published values for peptide binding. Furthermore, addition of unlabeled LIC peptide to a final concentration of 1 μM displaced ~25% of the bound 6CF-LIC under steady-state conditions; in contrast, incubation of Sse1p with 6CF-LIC in the presence of ATP or ADP had no statistically significant effect on peptide binding (data not shown), consistent with the conjecture that a tight coupling between Sse1p’s ATPase cycle and peptide binding may not be vital (see Introduction). Based on these data, we conclude that Sse1p has a significantly higher binding affinity for the LIC peptide than Ssa1p.
Sse1p exhibits high-affinity peptide binding
To determine whether Sse1p could also bind to an Hsp70 peptide substrate, we made use of a FITC-conjugate of ala-p5 (see Materials and methods). Although Ssa1p bound FITC-ala-p5 with a KD
of ~100 nM, no interaction was observed between this peptide and Sse1p (, compare closed to open circles). Of note, we recently reported that the binding of FITC-ala-p5 to Hsp70 is ATP-dependent [21
], indicating the validity of using this modified substrate to measure the peptide binding affinity for the chaperone. Combined with the data presented in , these results indicate that Sse1p exhibits peptide binding specificity and that the peptide preferences for Sse1p and Ssa1p are not entirely overlapping. A complete analysis of the peptide preferences of Sse1p will require continued analysis using peptide-display technologies, but based on our data we suggest that these efforts should prove informative.
Having identified 6CF-LIC as a high-affinity peptide substrate for Sse1p, we then examined whether the peptide stimulates the chaperone’s ATPase activity. Experiments using FITC-ala-p5 were performed in parallel and the combined data are summarized in . As anticipated, we found that a 30-fold molar excess of ala-p5 significantly enhanced the ATPase activity of Ssa1p. In contrast, neither FITC-ala-p5 nor the binding peptide 6CF-LIC affected the ATP hydrolytic rate of Sse1p ().
The ATPase activity of Sse1p is not enhanced by the addition of a peptide substrate
Based on the sensitivity of the fluorescence methods employed in this study, our data also establish a means to screen for small molecule compounds that might interfere with peptide binding to Hsp110s. Because defects in Sse1p function enhance the degradation of an atherosclerotic-inducing apolipoprotein (apoB) [33
], we suggest that inhibitors that block Hsp110-substrate binding in hepatic cells might provide a means to lower circulating cholesterol levels. Furthermore, mammalian Hsp110 and an ER-localized Hsp110 homolog, Grp170, bind tumor-derived peptides and protein substrates, and these chaperones have been used to trigger innate and antigen-associated immunity [34
]. Based on our data, one reason Hsp110/Grp170 may be highly effective in anti-cancer vaccines is that they have such a high affinity for peptide substrates. Further studies can now be directed to test this hypothesis.