We identify new proteins whose altered abundance affects [PSI+] propagation and implies that amyloid propagation in vivo is impaired by alterations that promote conversion to, or stabilize, the substrate-bound state of Hsp70. Figure depicts the Hsp70 reaction cycle and where these proteins act on it.
FIG. 7. Regulation of Hsp70 (Ssa1p) reaction cycle by cochaperones. Hsp70 function is finely tuned by ATP hydrolysis and nucleotide exchange, which regulate substrate binding. It is known that Hsp40 (Ydj1p, Sis1p) and Sti1p stimulate ATP hydrolysis, promoting (more ...)
Our earlier work showed that second-site mutations in Ssa1-21p that overcome the [PSI+
]-impairing effect of the L483W substitution weaken substrate binding, which indicates that L483W enhances substrate binding of Ssa1p (27
). Consistent with this interpretation, overproducing Sti1p, which promotes substrate binding by activating Ssa1p ATPase, also impaired [PSI+
] propagation. Conversely, deletion of Sti1p restored [PSI+
] propagation in SSA1-21
cells. As predicted and in contrast to the case with Sti1p, overproduction of Fes1p, which facilitates substrate release (30
), counteracted the impairment of [PSI+
] by Ssa1-21p, while depletion of Fes1p, which should prolong the substrate-bound state of Ssa1p, impaired [PSI+
] in both wild-type and SSA1-21
cells. Thus, in addition to mutations in Hsp70 that alter its function directly, an imbalance of Hsp70 cochaperones can produce a similar [PSI+
]-impairing effect by disrupting regulation of the Hsp70 reaction cycle.
Our results agree with previous data showing that excess Sti1p destabilizes some variants of [PSI+
) and suggest that this destabilization is mediated through effects on Ssa1p activity rather than altered expression of other Hsps as previously suggested. Hsp40s also stimulate Hsp70 ATPase, and in particular Ydj1p and Sis1p stimulate Ssa1p ATPase (38
). Consistent with our conclusion, overproduction of the Hsp40 homologs Ydj1p, Sis1p, and Apj1p impairs propagation of yeast prions (33
), and a suppressing mutation in Ssa1-21p described earlier is in a residue expected to disrupt Hsp40 interaction (27
). Additionally, weakening of [PSI+
] by Ydj1p overproduction is increased when Ssa1p is simultaneously elevated. Our data suggest that the previously described effects of Sti1p and Hsp40 overproduction on yeast prions can be attributed to stimulated Ssa1p ATPase.
Data presented here support our earlier interpretation that other mutations in Ssa1p that inhibit [PSI+
] propagation also increase ATPase activity of Ssa1p. Aside from L483W, all of eight previously described Ssa1p mutations that impair [PSI+
] are in the ATPase domain and cannot directly affect interaction with the substrate (27
). The L483W substitution is within the substrate-binding domain but distant from the substrate-binding pocket, so its effects should also be indirect, perhaps altering interdomain communication. Together, effects of the Hsp70 and cochaperone mutations on [PSI+
] suggest that the increase in ATPase is reflected in the reduced generation of amyloid seeds from preexisting material. Since cells expressing Ssa1-21p as the only essential Ssap are unable to maintain [PSI+
] but grow as well as wild-type cells, an implication of our results is that it might be possible to therapeutically modulate ATPase activity of Hsp70 in other systems, directly or through cochaperones, in a way that would impair cytosolic amyloid propagation with minimal side effects.
How does promoting or prolonging substrate binding by Hsp70 impair [PSI+
] propagation? In addition to the continued growth of polymers, propagation of yeast prions (infectious amyloid) requires the generation of new self-replicating seeds from preexisting material and efficient distribution of seeds between mother and daughter cells. Generation of yeast prion seeds requires Hsp104 protein disaggregating function, which may break the polymers into smaller, more numerous pieces that continue to propagate the structure and are more readily transmitted to daughter cells (4
). Since SSA1-21
mutants have severalfold fewer seeds per cell, altered Hsp70 function can interfere with amyloid seeding. Because Ssa1-21p does not affect Hsp104's ability to provide thermotolerance (28
) or to reactivate heat-denatured luciferase in vivo (G. Jung, unpublished data), its effect on [PSI+
] might be direct. The simplest way to explain our data is that Ssa1-21p reduces generation of [PSI+
] seeds by binding too avidly to Sup35p aggregates, which sterically restricts access to Hsp104 (27
). If this explanation were true, then this altered function must affect disassembly or breakage of the presumed highly ordered amyloid fibers of yeast prions differently than disaggregation of amorphous, thermally denatured substrates.
A way to explain this difference is that amyloid is recognized differently as a substrate by Hsp70 and Hsp104. Since Hsp40 is thought to be able to present a substrate to Hsp70, and its substrate recognition overlaps that of Hsp70, Hsp40 also may contribute to the apparent substrate-specific effects on amyloid. In line with this idea, properties of a specific Hsp40 (Sis1p) have been suggested to enhance interaction of the yeast prion [PIN+
] determinant Rnq1p with Hsp70 (36
Another way that enhanced substrate binding by Hsp70 might impair [PSI+
] is that Hsp70's ability to assist Hsp104 in resolubilizing protein from aggregates is altered. Although the mechanism of protein disaggregation by Hsp104 is unknown, Hsp70 and Hsp40 cooperate in this process, and together these three chaperones can resolubilize large protein aggregates in vitro (20
). The altered function of the mutant Hsp70 may affect an aspect of a transient physical interaction between Hsp70 and Hsp104 or of their ability to act sequentially or simultaneously on a substrate. In the homologous bichaperone system of Escherichia coli
, the Hsp104 homolog ClpB binds the substrate first, increasing exposure of surfaces for subsequent Hsp70 interaction (21
). If the reaction is similar in yeast, a substrate produced from Hsp104 interaction with amyloid may interact with Hsp70 in a way that allows it to retain the ability to propagate the self-replicating structure. By binding too avidly with this substrate, Ssa1-21p may interfere with the ability of the self-replicating conformation to be maintained. Such a scenario may explain why Ssa1-21p has a detrimental effect on amyloid without greatly affecting other cellular processes that require Hsp70 function.
Our data suggest that Cpr7p, whose effect on Hsp70's enzymatic function is unknown, may also act to enhance substrate binding by Ssa1p. Since more Cpr7p was associated with Ssa1-21p than with Ssa1p, and more Ssa1-21p is expected to be in the closed ADP-bound state, Cpr7p may preferentially bind the ADP-bound form of Hsp70. If so, then Cpr7p may be acting to stabilize the closed conformation rather than to induce conversion to this state. The additive effect of depleting both Cpr7p and Sti1p is consistent with this interpretation.
, other yeast cyclophilin and Hop1 homologs, respectively, had no effect on [PSI+
] propagation in wild-type or SSA1-21
cells, showing that the effect of TPR cochaperones on [PSI+
] was not a general one. Thus, with respect to [PSI+
] there are clear functional differences in TPR cochaperone interactions with Ssa1p, which are likely rooted in differences in specificity or affinity. Such differences also may underlie functional distinctions between Ssa1p and Ssa2p regarding [PSI+
] propagation (this study), the ability of excess Ssa1p but not Ssa2p to cure cells of the yeast [URE3
] prion (50
), or the specific requirement of Ssa2p for transport of certain proteins into prevacuolar (Vid) vesicles (6
Our finding that deleting the TPR interaction motif (MEEVD) of Hsp90 had no effect on [PSI+
] suggests that Hsp90 is not involved in Sti1p and Cpr7p effects on [PSI+
] propagation. Despite wide interest in yeast prions and Hsp90 and the availability of many Hsp90 mutants, the only other experiments describing effects of Hsp90 on yeast prions were also negative, showing a lack of effect of overproduced Hsp90 on [PSI+
). Whether Hsp90 is required for [PSI+
] propagation cannot be tested directly because it is essential for viability. However, we also found that [PSI+
] propagation was unaffected in both wild-type and SSA1-21
cells by deletion of only HSC82
, which reduces the overall abundance of Hsp90 to roughly 10% of wild-type levels (5
), or when cells were treated with a wide range of concentrations of the Hsp90 inhibitors geldanamycin and radicicol (G. Jones, unpublished data). Together these observations suggest that Hsp90 function is not important for [PSI+
] propagation. Our data therefore provide evidence suggesting that Sti1p and Cpr7p can functionally interact with Ssa1p independently of Hsp90. Cpr7 and CNS1
have been found together in complexes without Hsp90 (58
), and our results may reveal effects of perturbation of such a complex.
Our findings illustrate the utility of yeast prions as a system for studying both amyloid propagation and protein chaperone function. In addition to advancing the understanding of how chaperones affect amyloid propagation in vivo, continued study with this unique system will provide new insights into the functions of the chaperone machinery in general.