In vivo propagation of [PSI؉ ], an aggregation-prone prion isoform of the yeast release factor Sup35 (eRF3), has previously been shown to require intermediate levels of the chaperone protein Hsp104. Here we perform a detailed study on the mechanism of prion loss after Hsp104 inactivation. Complete or partial inactivation of Hsp104 was achieved by the following approaches: deleting the HSP104 gene; modifying the HSP104 promoter that results in low level of its expression; and overexpressing the dominant-negative ATPase-inactive mutant HSP104 allele. In contrast to guanidine-HCl, an agent blocking prion proliferation, Hsp104 inactivation induced relatively rapid loss of [PSI ؉ ] and another candidate yeast prion, [PIN ؉ ]. Thus, the previously hypothesized mechanism of prion dilution in cell divisions due to the blocking of prion proliferation is not sufficient to explain the effect of Hsp104 inactivation. The [PSI ؉ ] response to increased levels of another chaperone, Hsp70-Ssa, depends on whether the Hsp104 activity is increased or decreased. A decrease of Hsp104 levels or activity is accompanied by a decrease in the number of Sup35 PSI؉ aggregates and an increase in their size. This eventually leads to accumulation of huge agglomerates, apparently possessing reduced prion forming capability and representing dead ends of the prion replication cycle. Thus, our data confirm that the primary function of Hsp104 in prion propagation is to disassemble prion aggregates and generate the small prion seeds that initiate new rounds of prion propagation (possibly assisted by Hsp70-Ssa).Prions (37) are protein isoforms that are capable of reproducing themselves by converting normal proteins of the same primary structure into a prion state. In mammals, including humans, the prion protein PrP Sc is associated with infectious neurodegenerative diseases, such as mad cow disease (see reference 38 for a review). In yeast and fungi, prions serve as protein-based genetic elements, inherited via cytoplasm in a non-Mendelian fashion (see references 5, 46, and 52 for reviews). Prions form insoluble proteinase-resistant aggregates in vivo, in contrast to their normal (nonprion) counterparts, which are usually soluble. In vitro, prion proteins form amyloid-like polymers. It has been suggested that in vivo replication of prion conformation occurs by a nucleated polymerization mechanism (25,26). This relates prion phenomena to other amyloidoses and neural inclusion disorders (see reference 21 for a review). An alternative model explains replication of prion conformation via a monomer-directed or template-assisted conformational switch in the heterodimer, suggesting that aggregate formation occurs as a consequence of the conformational switch (see reference 17 for a review). Recent data indicate that in vitro propagation of the yeast prion amyloids may combine features of both models and therefore could be termed a nucleated conformational conversion (45).Since prion propagation apparently operates at the level of protein folding and assemb...

Self-perpetuating protein aggregates transmit prion diseases in mammals and heritable traits in yeast. De novo prion formation can be induced by transient overproduction of the corresponding prion-forming protein or its prion domain. Here, we demonstrate that the yeast prion protein Sup35 interacts with various proteins of the actin cortical cytoskeleton that are involved in endocytosis. Sup35-derived aggregates, generated in the process of prion induction, are associated with the components of the endocytic/vacuolar pathway. Mutational alterations of the cortical actin cytoskeleton decrease aggregation of overproduced Sup35 and de novo prion induction and increase prion-related toxicity in yeast. Deletion of the gene coding for the actin assembly protein Sla2 is lethal in cells containing the prion isoforms of both Sup35 and Rnq1 proteins simultaneously. Our data are consistent with a model in which cytoskeletal structures provide a scaffold for generation of large aggregates, resembling mammalian aggresomes. These aggregates promote prion formation. Moreover, it appears that the actin cytoskeleton also plays a certain role in counteracting the toxicity of the overproduced potentially aggregating proteins.Prions are protein isoforms that cause transmissible neurodegenerative diseases in mammals (for review, see reference 50) and control heritable traits in fungi (for review, see references 10 to 12). Most known prions are self-perpetuating amyloid-like ordered fibrous protein aggregates which propagate the prion state by immobilizing the soluble protein molecules of the same amino acid sequence. Saccharomyces cerevisiae prion [PSI ϩ ] is an aggregate of the translation termination factor Sup35. The prion domain of Sup35 is rich in glutamine (Q) and asparagine (N) residues, resembling poly-Q proteins, such as huntingtin, which is involved in Huntington's disease (for review, see reference 53). While recent data shed light on the major steps of propagation of the preexisting [PSI ϩ ] aggregates in yeast cells (for review, see references 12 and 47), the mechanism of initial prion formation from nonprion protein remains a mystery. It has been shown that de novo formation of the [PSI ϩ ] prion is induced by transient overproduction of the Sup35 protein or its prion domain (14,19). This process is usually efficient only in cells containing other QNrich protein aggregates, such as [PIN ϩ ], a prion form of Rnq1 (20, 22). Likewise, preexisting QN-rich prions promote aggregation and aggregation-related toxicity of heterologous poly-Q proteins expressed in yeast cells (33,38). Possibly, preexisting QN-rich aggregates either provide initial nucleation centers for aggregation of other QN-rich proteins or sequester unknown antiaggregation factors.Assembly of amyloid fibers resembles the assembly of cytoskeletal structures such as actin filaments. The QN-rich domain of Sup35 was shown to interact with the actin assembly protein Sla1 in the two-hybrid assay (4). Deletion of SLA1 decreases de novo induction of [PSI ϩ ] ...

The maintenance of [PSI], a prion-like form of the yeast release factor Sup35, requires a specific concentration of the chaperone protein Hsp104: either deletion or overexpression of Hsp104 will cure cells of [PSI]. A major puzzle of these studies was that overexpression of Hsp104 alone, from a heterologous promoter, cures cells of [PSI] very efficiently, yet the natural induction of Hsp104 with heat shock, stationary-phase growth, or sporulation does not. These observations pointed to a mechanism for protecting the genetic information carried by the [PSI] element from vicissitudes of the environment. Here, we show that simultaneous overexpression of Ssa1, a protein of the Hsp70 family, protects [PSI] from curing by overexpression of Hsp104. Ssa1 protein belongs to the Ssa subfamily, members of which are normally induced with Hsp104 during heat shock, stationary-phase growth, and sporulation. At the molecular level, excess Ssa1 prevents a shift of Sup35 protein from the insoluble (prion) to the soluble (cellular) state in the presence of excess Hsp104. Overexpression of Ssa1 also increases nonsense suppression by [PSI] when Hsp104 is expressed at its normal level. In contrast, hsp104 deletion strains lose [PSI] even in the presence of overproduced Ssa1. Overproduction of the unrelated chaperone protein Hsp82 (Hsp90) neither cured [PSI] nor antagonized the [PSI]-curing effect of overproduced Hsp104. Our results suggest it is the interplay between Hsp104 and Hsp70 that allows the maintenance of [PSI] under natural growth conditions.

In Escherichia coli, the ribosome-associated chaperone Trigger Factor (TF) promotes the folding of newly synthesized cytosolic proteins. TF is composed of three domains: an N-terminal domain (N), which mediates ribosome binding; a central domain (P), which has peptidyl-prolyl cis/trans isomerase activity and is involved in substrate binding in vitro; and a C-terminal domain (C) with unknown function. We investigated the contributions of individual domains (N, P, and C) or domain combinations (NP, PC, and NC) to the chaperone activity of TF in vivo and in vitro. All fragments comprising the N domain (N, NP, NC) complemented the synthetic lethality of ⌬tig ⌬dnaK in cells lacking TF and DnaK, prevented protein aggregation in these cells, and cross-linked to nascent polypeptides in vitro. However, ⌬tig⌬dnaK cells expressing the N domain alone grew more slowly and showed less viability than ⌬tig⌬dnaK cells synthesizing either NP, NC, or full-length TF, indicating beneficial contributions of the P and C domains to TF's chaperone activity. In an in vitro system with purified components, none of the TF fragments assisted the refolding of denatured D-glyceraldehyde-3-phosphate dehydrogenase in a manner comparable to that of wild-type TF, suggesting that the observed chaperone activity of TF fragments in vivo is dependent on their localization at the ribosome. These results indicate that the N domain, in addition to its function to promote binding to the ribosome, has a chaperone activity per se and is sufficient to substitute for TF in vivo.

In eukaryotes, newly synthesized proteins interact co-translationally with a multitude of different ribosome-bound factors and chaperones including the conserved heterodimeric nascent polypeptide-associated complex (NAC) and a Hsp40/70-based chaperone system. These factors are thought to play an important role in protein folding and targeting, yet their specific ribosomal localizations, which are prerequisite for their functions, remain elusive. This study describes the ribosomal localization of NAC and the molecular details by which NAC is able to contact the ribosome and gain access to nascent polypeptides. We identified a conserved RRK(X) n KK ribosome binding motif within the ␤-subunit of NAC that is essential for the entire NAC complex to attach to ribosomes and allow for its interaction with nascent polypeptide chains. The motif localizes within a potential loop region between two predicted ␣-helices in the N terminus of ␤NAC. This N-terminal ␤NAC ribosome-binding domain was completely portable and sufficient to target an otherwise cytosolic protein to the ribosome. NAC modified with a UV-activatable cross-linker within its ribosome binding motif specifically cross-linked to L23 ribosomal protein family members at the exit site of the ribosome, providing the first evidence of NAC-L23 interaction in the context of the ribosome. Mutations of L23 reduced NAC ribosome binding in vivo and in vitro, whereas other eukaryotic ribosome-associated factors such as the Hsp70/40 chaperones Ssb or Zuotin were unaffected. We conclude that NAC employs a conserved ribosome binding domain to position itself on the L23 ribosomal protein adjacent to the nascent polypeptide exit site.