Despite significant progress in understanding propagation of preexisting prions, the mechanism of initial prion formation remains a mystery. In vitro experiments suggest that prion-forming domains tend to aggregate spontaneously (27
). However, this aggregation potential is apparently suppressed in vivo, since in the absence of a prion seed, the potential prion-forming proteins remain soluble and frequency of spontaneous aggregate formation is low. This suggests that spontaneous prion formation could be caused by dysfunction or imbalance of proteins, which influence the normal process of folding and suppress the aggregation potential of the prion-forming domains. Here, we demonstrate for the first time that inactivation of the chaperone protein (Ssb) may increase the frequency of spontaneous prionization of another protein (Sup35). If prion conformers are considered analogous to the mutants, this is equivalent to discovery of the mutator genes.
Quite remarkably, representatives of two major cytosolic Hsp70 subfamilies, Ssa and Ssb, exhibit opposite effects on [PSI
] prion. While Ssa protein assists [PSI
] and protects it from curing by Hsp104, Ssb protein antagonizes [PSI
] formation and assists Hsp104 in [PSI
] curing. Functional divergence between Ssa and Ssb has been noted previously in other assays (29
). While the Ssa subfamily is essential for vegetative growth, inducible by high temperature and some other stresses, and involved in stress defense, Ssb protein is dispensable for viability and stress response and does not appear to be heat inducible. Genetic and biochemical data demonstrate an Ssb association with translating ribosomes (38
), in particular with nascent polypeptide (47
). It has been suggested that Ssb is involved in cotranslational protein folding (38
). However, Ssb is certainly not required for this process, since its inactivation causes only slight growth deficiencies. Phenotypes associated with the double ssb1/2
deletion, i.e., cold sensitivity and sensitivity to translational inhibitors, do not contradict the role of Ssb in cotranslational folding but cannot be easily explained as a simple consequence of the protein folding defects.
Another cellular function attributed to the Ssb protein is stimulation of proteasome-dependent protein turnover (41
). The prokaryotic Hsp70 homolog, DnaK, is also involved in protein degradation mediated by proteases La and ClpA/B (52
). It is worth noting that ClpB, a subunit of the protease complex which converts multiprotein substrates to a form accessible for protease, is a prokaryotic homolog of the yeast Hsp104 protein (51
). It has been suggested that yeast Ssb protein is responsible for proofreading of the newly synthesized and folded polypeptides and for targeting the incorrectly folded products for proteasome-mediated degradation (41
). This does not rule out the possibility of Ssb being involved in the actual process of folding, since coupling of the processes of synthesis and breakdown is frequently observed in biological systems. The proofreading role for Ssb protein would be consistent with our data.
We propose that Ssb either prevents formation of the misfolded intermediates which serve as the raw material for the initial formation of prion aggregates, stimulates degradation of such intermediates, or both. This is why spontaneous [PSI] formation, and especially [PSI] formation induced by Sup35 (or Sup35N) overproduction is increased in Ssb− cells. Ssb could also assist Hsp104 in curing yeast cells of [PSI] by two mechanisms: (i) decreasing the amount of the misfolded newly synthesized Sup35 protein and therefore eliminating the substrate for generation of the new prion polymers and (ii) refolding the misfolded Sup35 molecules, generated due to Hsp104-mediated breakdown of preexisting prion polymers, or targeting these molecules for proteasome-mediated degradation and therefore preventing them from reverting to prion form. Since we detected no significant effect of Ssb levels on prion propagation under the normal circumstances (that is, when Hsp104 levels are low), the latter mechanism seems more likely. This would mean that the proofreading function of Ssb is not restricted to the newly synthesized proteins. However, one has to remember that Sup35 is a ribosome-binding protein, and thus at least a fraction of the Ssb and Sup35 molecules are located close to each other. This might enable Ssb to monitor conformation of some preexisting Sup35 molecules.
Other phenotypes of the double ssb1/2
Δ deletion are also consistent with the proofreading function of the Ssb protein. Both translational inhibitors, such as paromomycin and hygromycin (42
), and growth at low temperature (28
) increase translational misreading, resulting in production of the erroneous and potentially misfolded proteins. If such proteins are not corrected or removed by the Ssb-mediated proofreading system, they may cause deleterious effects. Interestingly, we have observed for the first time that Ssb−
strains are extremely sensitive to the protein-denaturing agent GuHCl. Growth in the presence of 1 to 5 mM GuHCl has been shown to efficiently cure yeast cells of prions [PSI
) and [URE3
) and of the non-Mendelian element [PIN
), even though such low concentrations of GuHCl are not sufficient to cause significant protein denaturing and solubilize protein aggregates in vitro. GuHCl was shown to induce expression of some heat shock proteins including Hsp104 (36
), leading to the hypothesis that the GuHCl effect is mediated by Hsp104 induction (11
). However, Hsp104 overproduction does not appear to cure yeast cells of [URE3
) and [PIN
). Extreme sensitivity of the Ssb−
strains to the millimolar concentrations of GuHCl indicates that low concentrations of GuHCl can specifically target cellular processes involving Ssb, in particular, cotranslational protein folding. It is possible that growth in the presence of GuHCl results in synthesis of the amorphous unfolded polypeptide products, which are unable both to perform their normal function and convert to the prion form. If these are not corrected or targeted for degradation due to Ssb action, they are toxic for the yeast cells. Dynamics of [PSI
] loss in the presence of GuHCl confirm that GuHCl prevents formation of new prions rather than acts on preexisting ones (16
Interestingly, toxicity of the overproduced Sup35 appears to decrease in the Ssb−
background, even though translational readthrough and [PSI
] induction increase. This finding suggests that growth defects caused by the overproduced Sup35 protein may not necessarily result directly from translational readthrough and aggregate formation. One possibility is that prion protein incorporated in the huge aggregates is harmless, so that aggregate formation might in fact protect cells by localizing and compartmentalizing prions. Proteins like Ssb, which interfere with prion formation and/or promote interactions between prion conformer and cellular metabolic systems (e.g., proteolysis machinery), may inadvertently increase toxicity, since wrongly shaped prion conformers or misfolded intermediates inhibit processes in which they become involved. Recent data indicate that in some human neural inclusion diseases, such as Huntington disease, aggregate formation may indeed protect cells by localizing and inactivating the misfolded protein rather than contribute to toxicity (50
). This may also explain why the presence of [PSI
] prion slightly increases resistance of the Ssb−
strains to GuHCl (Fig. ). This raises the possibility of yeast prions being by-products of the cellular processes aimed at protecting cells from the toxic effects of misfolded and mislocalized proteins. By analogy with mutagenic DNA repair systems, protein repair pathways, which are supposed to correct and/or remove damaged proteins, can generate protein “mutations” becoming reproducible in a prion-like fashion. Further experiments to test this hypothesis are under way.
Protein-based transmission of the phenotypic traits mediated by prion-like elements constitutes a new mechanism of inheritance. The very fact that several proteins of different functions and origins exhibit this phenomenon suggests a wide distribution of the protein-based systems of structural (as opposed to sequential) coding in nature. Our research uncovers the enzymatic machinery for protein-based inheritance, playing a role comparable to that of the DNA repair machinery in DNA-based inheritance. This machinery is composed of evolutionarily conserved proteins of the Hsp100 and Hsp70 families, suggesting that we are dealing with ancient phenomenon, probably having implications for organisms other than yeast.