Daniel C. Masison

Prion-Inducing Domain of Yeast Ure2p and Protease Resistance of Ure2p in Prion-Containing Cells

The genetic properties of the [URE3] non-Mendelian element of Saccharomyces cerevisiae suggest that it is a prion (infectious protein) form of Ure2p, a regulator of nitrogen catabolism. In extracts from [URE3] strains, Ure2p was partially resistant to proteinase K compared with Ure2p from wild-type extracts. Overexpression of Ure2p in wild-type strains induced a 20- to 200-fold increase in the frequency with which [URE3] arose. Overexpression of just the amino-terminal 65 residues of Ure2p increased the frequency of [URE3] induction 6000-fold. Without this “prion-inducing domain” the carboxyl-terminal domain performed the nitrogen regulation function of Ure2p, but could not be changed to the [URE3] prion state. Thus, this domain induced the prion state in trans, whereas in cis it conferred susceptibility of the adjoining nitrogen regulatory domain to prion infections.

Guanidine Hydrochloride Inhibits Hsp104 Activity In Vivo: A Possible Explanation for Its Effect in Curing Yeast Prions

The presence of millimolar concentrations of guanidine hydrochloride (Gdn-HCl) in growth media causes efficient loss of the normally stable [PSI+] element from yeast cells. Although it has become common practice to include 5 mm Gdn-HCl in growth media to cure [PSI+] and other prions of yeast, the biochemical mechanism by which it cures is unknown. We find that 5 mm Gdn-HCl significantly reduces Hsp104-mediated basal and acquired thermotolerance. Gdn-HCl also reduced the ability of Hsp104 to restore activity of thermally denatured luciferase in vivo. The abundance of Hsp104 was not reduced in cells grown in the presence of Gdn-HCl, ruling out negative effects on expression or stability of Hsp104. We therefore conclude that Gdn-HCl inhibits Hsp104 activity in vivo. Since replication of yeast prions is dependent on Hsp104, our results suggest that Gdn-HCl cures prions by inhibiting Hsp104 activity.

Amino acid residue 184 of yeast Hsp104 chaperone is critical for prion-curing by guanidine, prion propagation, and thermotolerance

Inactivation of Hsp104 by guanidine is contended to be the mechanism by which guanidine cures yeast prions. We now find an Hsp104 mutation (D184N) that confers resistance to guanidine-curing of the yeast [PSI+] prion. In an independent screen we isolated an HSP104 allele altered in the same residue (D184Y) that dramatically impairs [PSI+] propagation in a temperature-dependent manner. Directed mutagenesis of HSP104 produced additional alleles that conferred varying degrees of resistance to guanidine-curing or impaired [PSI+] propagation. The mutations similarly affected propagation of the [URE3] prion. Basal and induced abundance of all mutant proteins was normal. Thermotolerance of cells expressing mutant proteins was variably resistant to guanidine, and the degree of thermotolerance did not correlate with [PSI+] stability. We thus show that guanidine cures yeast prions by inactivating Hsp104 and identify a highly conserved Hsp104 residue that is critical for yeast prion propagation. Our data suggest that Hsp104 activity can be reduced substantially without affecting [PSI+] stability, and that Hsp104 interacts differently with prion aggregates than with aggregates of thermally denatured protein.

N-Terminal Domain of Yeast Hsp104 Chaperone Is Dispensable for Thermotolerance and Prion Propagation but Necessary for Curing Prions by Hsp104 Overexpression

Hsp104 is a hexameric protein chaperone that resolubilizes stress-damaged proteins from aggregates. Hsp104 promotes [PSI+] prion propagation by breaking prion aggregates, which propagate as amyloid fibers, into more numerous prion “seeds.” Inactivating Hsp104 cures cells of [PSI+] and other amyloid-like yeast prions. Overexpressing Hsp104 also eliminates [PSI+], presumably by completely resolubilizing prion aggregates. Inexplicably, however, excess Hsp104 does not cure the other prions. Here we identify missense mutations in Hsp104s amino-terminal domain (NTD), which is conserved among Hsp100 proteins but whose function is unknown, that improve [PSI+] propagation. Hsp104Δ147, engineered to lack the NTD, supported [PSI+] and functioned normally in thermotolerance and protein disaggregation. Hsp104Δ147 failed to cure [PSI+] when overexpressed, however, implying that excess Hsp104 does not eliminate [PSI+] by direct dissolution of prion aggregates. Curing of [PSI+] by overexpressing catalytically inactive Hsp104 (Hsp104KT), which interferes with endogenous Hsp104, did not require the NTD. We further found that Hsp104 mutants defective in threading peptides through the hexamer pore had reduced ability to support [PSI+] in proportion to protein resolubilization defects, suggesting that [PSI+] propagation depends on this threading and that Hsp104 “breaks” prion aggregates by extracting protein monomers from the amyloid fibers.

Species-specific collaboration of heat shock proteins (Hsp) 70 and 100 in thermotolerance and protein disaggregation

Yeast Hsp104 and its bacterial homolog, ClpB, are Clp/Hsp100 molecular chaperones and AAA+ ATPases. Hsp104 and ClpB collaborate with the Hsp70 and DnaK chaperone systems, respectively, to retrieve and reactivate stress-denatured proteins from aggregates. The action of Hsp104 and ClpB in promoting cell survival following heat stress is species-specific: Hsp104 cannot function in bacteria and ClpB cannot act in yeast. To determine the regions of Hsp104 and ClpB necessary for this specificity, we tested chimeras of Hsp104 and ClpB in vivo and in vitro. We show that the Hsp104 and ClpB middle domains dictate the species-specificity of Hsp104 and ClpB for cell survival at high temperature. In protein reactivation assays in vitro, chimeras containing the Hsp104 middle domain collaborate with Hsp70 and those with the ClpB middle domain function with DnaK. The region responsible for the specificity is within helix 2 and helix 3 of the middle domain. Additionally, several mutants containing amino acid substitutions in helix 2 of the ClpB middle domain are defective in protein disaggregation in collaboration with DnaK. In a bacterial two-hybrid assay, DnaK interacts with ClpB and with chimeras that have the ClpB middle domain, implying that species-specificity is due to an interaction between DnaK and the middle domain of ClpB. Our results suggest that the interaction between Hsp70/DnaK and helix 2 of the middle domain of Hsp104/ClpB determines the specificity required for protein disaggregation both in vivo and in vitro, as well as for cellular thermotolerance.

Propagation of Saccharomyces cerevisiae [PSI+] Prion Is Impaired by Factors That Regulate Hsp70 Substrate Binding

ABSTRACT The Saccharomyces cerevisiae [PSI+ ] prion is believed to be a self-propagating cytoplasmic amyloid. Earlier characterization of HSP70 (SSA1) mutations suggested that [PSI+ ] propagation is impaired by alterations that enhance Ssa1ps substrate binding. This impairment is overcome by second-site mutations in Ssa1ps conserved C-terminal motif (GPTVEEVD), which mediates interactions with tetratricopeptide repeat (TPR) cochaperones. Sti1p, a TPR cochaperone homolog of mammalian Hop1 (Hsp70/90 organizing protein), activates Ssa1p ATPase, which promotes substrate binding by Ssa1p. Here we find that in SSA1-21 cells depletion of Sti1p improved [PSI+] propagation, while excess Sti1p weakened it. In contrast, depletion of Fes1p, a nucleotide exchange factor for Ssa1p that facilitates substrate release, weakened [PSI +] propagation, while overproducing Fes1p improved it. Therefore, alterations of Hsp70 cochaperones that promote or prolong Hsp70 substrate binding impair [PSI +] propagation. We also find that the GPTVEEVD motif is important for physical interaction with Hsp40 (Ydj1p), another Hsp70 cochaperone that promotes substrate binding but is dispensable for viability. We further find that depleting Cpr7p, an Hsp90 TPR cochaperone and CyP-40 cyclophilin homolog, improved [PSI +] propagation in SSA1 mutants. Although Cpr7p and Sti1p are Hsp90 cochaperones, we provide evidence that Hsp90 is not involved in [PSI +] propagation, suggesting that Sti1p and Cpr7p functionally interact with Hsp70 independently of Hsp90.

Role for Hsp70 chaperone in Saccharomyces cerevisiae prion seed replication.

ABSTRACT The Saccharomyces cerevisiae [PSI+] prion is a misfolded form of Sup35p that propagates as self-replicating cytoplasmic aggregates. Replication is believed to occur through breakage of transmissible [PSI+] prion particles, or seeds, into more numerous pieces. In [PSI+] cells, large Sup35p aggregates are formed by coalescence of smaller sodium dodecyl sulfate-insoluble polymers. It is uncertain if polymers or higher-order aggregates or both act as prion seeds. A mutant Hsp70 chaperone, Ssa1-21p, reduces the number of transmissible [PSI+] seeds per cell by 10-fold but the overall amount of aggregated Sup35p by only two- to threefold. This discrepancy could be explained if, in SSA1-21 cells, [PSI+] seeds are larger or more of the aggregated Sup35p does not function as a seed. To visualize differences in aggregate size, we constructed a Sup35-green fluorescent protein (GFP) fusion (NGMC) that has normal Sup35p function and can propagate like [PSI+]. Unlike GFP fusions lacking Sup35ps essential C-terminal domain, NGMC did not form fluorescent foci in log-phase [PSI+] cells. However, using fluorescence recovery after photobleaching and size fractionation techniques, we find evidence that NGMC is aggregated in these cells. Furthermore, the aggregates were larger in SSA1-21 cells, but the size of NGMC polymers was unchanged. Possibly, NGMC aggregates are bigger in SSA1-21 cells because they contain more polymers. Our data suggest that Ssa1-21p interferes with disruption of large Sup35p aggregates, which lack or have limited capacity to function as seed, into polymers that function more efficiently as [PSI+] seeds.

Independent Regulation of Hsp70 and Hsp90 Chaperones by Hsp70/Hsp90-organizing Protein Sti1 (Hop1)

Hsp70 and Hsp90 protein chaperones cooperate in a protein-folding pathway required by many “client” proteins. The co-chaperone Sti1p coordinates functions of Hsp70 and Hsp90 in this pathway. Sti1p has three tetratricopeptide repeat (TPR) domains. TPR1 binds Hsp70, TPR2a binds Hsp90, and the ligand for TPR2b is unknown. Although Sti1p is thought to be dedicated to the client folding pathway, we earlier showed that Sti1p regulated Hsp70, independently of Hsp90, in a way that impairs yeast [PSI+] prion propagation. Using this prion system to monitor Sti1p regulation of Hsp70 and an Hsp90-inhibiting compound to monitor Hsp90 regulation, we identified Sti1p mutations that separately affect Hsp70 and Hsp90. TPR1 mutations impaired Sti1p regulation of Hsp70, but deletion of TPR2a and TPR2b did not. Conversely, TPR2a and TPR2b mutations impaired Sti1p regulation of Hsp90, but deletion of TPR1 did not. All Sti1p mutations variously impaired the client folding pathway, which requires both Hsp70 and Hsp90. Thus, Sti1p regulated Hsp70 and Hsp90 separately, Hsp90 is implicated as a TPR2b ligand, and mutations separately affecting regulation of either chaperone impair a pathway that is dependent upon both. We further demonstrate that client folding depended upon bridging of Hsp70 and Hsp90 by Sti1p and find conservation of the independent regulation of Hsp70 and Hsp90 by human Hop1.

Ure2p Function Is Enhanced by Its Prion Domain in Saccharomyces cerevisiae

The Ure2 protein of Saccharomyces cerevisiae can become a prion (infectious protein). At very low frequencies Ure2p forms an insoluble, infectious amyloid known as [URE3], which is efficiently transmitted to progeny cells or mating partners that consequently lose the normal Ure2p nitrogen regulatory function. The [URE3] prion causes yeast cells to grow slowly, has never been identified in the wild, and confers no obvious phenotypic advantage. An N-terminal asparagine-rich domain determines Ure2p prion-forming ability. Since ure2Δ strains are complemented by plasmids that overexpress truncated forms of Ure2p lacking the prion domain, the existence of the [URE3] prion and the evolutionary conservation of an N-terminal extension have remained mysteries. We find that Ure2p function is actually compromised in vivo by truncation of the prion domain. Moreover, Ure2p stability is diminished without the full-length prion domain. Mca1p, like Ure2p, has an N-terminal Q/N-rich domain whose deletion reduces its steady-state levels. Finally, we demonstrate that the prion domain may affect the interaction of Ure2p with other components of the nitrogen regulation system, specifically the negative regulator of nitrogen catabolic genes, Gzf3p.

Uncovering a Region of Heat Shock Protein 90 Important for Client Binding in E. coli and Chaperone Function in Yeast

The heat shock protein 90 (Hsp90) family of heat shock proteins is an abundantly expressed and highly conserved family of ATP-dependent molecular chaperones. Hsp90 facilitates remodeling and activation of hundreds of proteins. In this study, we developed a screen to identify Hsp90-defective mutants in E. coli. The mutations obtained define a region incorporating residues from the middle and C-terminal domains of E. coli Hsp90. The mutant proteins are defective in chaperone activity and client binding in vitro. We constructed homologous mutations in S. cerevisiae Hsp82 and identified several that caused defects in chaperone activity in vivo and in vitro. However, the Hsp82 mutant proteins were less severely defective in client binding to a model substrate than the corresponding E. coli mutant proteins. Our results identify a region in Hsp90 important for client binding in E. coli Hsp90 and suggest an evolutionary divergence in the mechanism of client interaction by bacterial and yeast Hsp90.