, formed by the aggregated states of the cytosolic proteins Sup35, Rnq1, and Ure2, respectively (1). Sup35 is a translation termination factor; Ure2 is a regulator that acts to repress transcription of a set of genes involved in nitrogen catabolism; the function of Rnq1 is unknown. Prion proteins can form different conformational states resulting in prion strains having different heritable traits. For propagation in the cell population, physical transmission of the prion template, often referred to as the propagon or seed, is required to allow conversion of newly synthesized protein to the prion conformation (2).Somewhat paradoxically, the propagation of yeast prions appears to be inexorably reliant on the function of molecular chaperones, proteins that normally function to prevent protein misfolding (2). Two chaperone systems have been linked to prion propagation: the hexameric AAAϩ ATPase Hsp104 and the J-protein (Hsp40):Hsp70 chaperone machinery, with its associated nucleotide exchange factors (3). Hsp104, like its ortholog ClpB, functions in protein remodeling by threading partially folded proteins through its central pore and is stringently required for the propagation of all identified yeast prions (1,4,5).Hsp70s function with their obligate cochaperones, J-proteins, which act to stimulate Hsp70 ATPase activity and stabilize their interaction with client proteins (6). Although J-proteins are very diverse in sequence and structure, they possess a highly conserved J-domain that is responsible for the stimulation of the ATPase activity of Hsp70s. One cytosolic J-protein, Sis1, is required for propagation of [RNQ ϩ ] (7). In addition, multiple individual amino acid substitutions in the cytosolic Hsp70s Ssa1/2 that impair propagation of [PSI ϩ propagation implies an involvement of an unidentified J-protein as well.The currently favored model for prion propagation posits chaperone-mediated fragmentation of prion complexes to produce sufficient prion seeds to assure consistent transmission of seeds to daughter cells, thus maintaining the prion in the cell population (2,(10)(11)(12)(13)(14). Supporting this model, inhibition of Hsp104 activity results in an increase in the size of Sup35 and Rnq1 prion complexes and subsequent prion loss, which has been shown in the case of [PSI ϩ ] to be dependent on cell division (10,11,15,16). Additional support for this idea comes from reports of fragmentation of prion fibers in vitro by Hsp104 (17) and the apparent decrease in the number of [PSI ϩ ] prion seeds in cells expressing a dominant mutation in the Hsp70 SSA1 gene (8). An increase in the size of Rnq1 polymers, followed by [RNQ ϩ ] loss, also occurs upon depletion of Sis1, a partner of Ssa1 (15). Together these data suggest cooperation between the 2 chaperone systems. Such cooperation has precedent, as Hsp104 is known to function in disaggregation of amorphous protein aggregates in conjunction with J-protein:Hsp70 chaperone machinery, with J-protein/Hsp70 and Hsp104 machineries acting sequentially (4).The yeast cytos...

The yeast prion [SWI+], formed of heritable amyloid aggregates of the Swi1 protein, results in a partial loss of function of the SWI/SNF chromatin-remodeling complex, required for the regulation of a diverse set of genes. Our genetic analysis revealed that [SWI+] propagation is highly dependent upon the action of members of the Hsp70 molecular chaperone system, specifically the Hsp70 Ssa, two of its J-protein co-chaperones, Sis1 and Ydj1, and the nucleotide exchange factors of the Hsp110 family (Sse1/2). Notably, while all yeast prions tested thus far require Sis1, [SWI+] is the only one known to require the activity of Ydj1, the most abundant J-protein in yeast. The C-terminal region of Ydj1, which contains the client protein interaction domain, is required for [SWI+] propagation. However, Ydj1 is not unique in this regard, as another, closely related J-protein, Apj1, can substitute for it when expressed at a level approaching that of Ydj1. While dependent upon Ydj1 and Sis1 for propagation, [SWI+] is also highly sensitive to overexpression of both J-proteins. However, this increased prion-loss requires only the highly conserved 70 amino acid J-domain, which serves to stimulate the ATPase activity of Hsp70 and thus to stabilize its interaction with client protein. Overexpression of the J-domain from Sis1, Ydj1, or Apj1 is sufficient to destabilize [SWI+]. In addition, [SWI+] is lost upon overexpression of Sse nucleotide exchange factors, which act to destabilize Hsp70's interaction with client proteins. Given the plethora of genes affected by the activity of the SWI/SNF chromatin-remodeling complex, it is possible that this sensitivity of [SWI+] to the activity of Hsp70 chaperone machinery may serve a regulatory role, keeping this prion in an easily-lost, meta-stable state. Such sensitivity may provide a means to reach an optimal balance of phenotypic diversity within a cell population to better adapt to stressful environments.

Yeast prions are heritable amyloid aggregates of functional yeast proteins; their propagation to subsequent cell generations is dependent upon fragmentation of prion protein aggregates by molecular chaperone proteins. Mounting evidence indicates the J-protein Sis1 may act as an amyloid specificity factor, recognizing prion and other amyloid aggregates and enabling Ssa and Hsp104 to act in prion fragmentation. Chaperone interactions with prions, however, can be affected by variations in amyloid-core structure resulting in distinct prion variants or ‘strains’. Our genetic analysis revealed that Sis1 domain requirements by distinct variants of [PSI +] are strongly dependent upon overall variant stability. Notably, multiple strong [PSI +] variants can be maintained by a minimal construct of Sis1 consisting of only the J-domain and glycine/phenylalanine-rich (G/F) region that was previously shown to be sufficient for cell viability and [RNQ +] prion propagation. In contrast, weak [PSI +] variants are lost under the same conditions but maintained by the expression of an Sis1 construct that lacks only the G/F region and cannot support [RNQ +] propagation, revealing mutually exclusive requirements for Sis1 function between these two prions. Prion loss is not due to [PSI +]-dependent toxicity or dependent upon a particular yeast genetic background. These observations necessitate that Sis1 must have at least two distinct functional roles that individual prions differentially require for propagation and which are localized to the glycine-rich domains of the Sis1. Based on these distinctions, Sis1 plasmid-shuffling in a [PSI +]/[RNQ +] strain permitted J-protein-dependent prion selection for either prion. We also found that, despite an initial report to the contrary, the human homolog of Sis1, Hdj1, is capable of [PSI +] prion propagation in place of Sis1. This conservation of function is also prion-variant dependent, indicating that only one of the two Sis1-prion functions may have been maintained in eukaryotic chaperone evolution.

Prions of budding yeast serve as a tractable model of amyloid behavior. Here we address the issue of the effect of yeast strain variation on prion stability, focusing also on the effect of amyloid conformation and the involvement of the co-chaperone Sis1, an essential J-protein partner of Hsp70. We found, despite an initial report to the contrary, that yeast strain background has little effect on the requirement for particular Sis1 domains for stable propagation of the prion [RNQ+], if the level of Sis1 expression is controlled. On the other hand, some variation in prion behavior was observed between yeast strains, in particular, the stability of certain [PSI+] variants. Future examination of such yeast strain-specific phenomena may provide useful insights into the basis of prion/chaperone dynamics.

Fructose-1,6-bisphosphatase (FBPase) governs a key step in gluconeogenesis, the conversion of fructose 1,6-bisphosphate into fructose 6-phosphate. In mammals, the enzyme is subject to metabolic regulation, but regulatory mechanisms of bacterial FBPases are not well understood. Presented here is the crystal structure (resolution, 1.45 Å ) of recombinant FBPase from Escherichia coli, the first structure of a prokaryotic Type I FBPase. The E. coli enzyme is a homotetramer, but in a quaternary state between the canonical Rand T-states of porcine FBPase. Phe 15 and residues at the C-terminal side of the first ␣-helix (helix H1) occupy the AMP binding pocket. Residues at the N-terminal side of helix H1 hydrogen bond with sulfate ions buried at a subunit interface, which in porcine FBPase undergoes significant conformational change in response to allosteric effectors. Phosphoenolpyruvate and sulfate activate E. coli FBPase by at least 300%. Key residues that bind sulfate anions are conserved among many heterotrophic bacteria, but are absent in FBPases of organisms that employ fructose 2,6-bisphosphate as a regulator. These observations suggest a new mechanism of regulation in the FBPase enzyme family: anionic ligands, most likely phosphoenolpyruvate, bind to allosteric activator sites, which in turn stabilize a tetramer and a polypeptide fold that obstructs AMP binding.Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3. 1.3.11; FBPase) 2 is a key enzyme of the gluconeogenic pathway, hydrolyzing fructose 1,6-bisphosphate (Fru-1,6-P 2 ) to fructose 6-phosphate (Fru-6-P) and inorganic phosphate (P i ) (1, 2). In Escherichia coli, the most likely role of FBPase is the generation of fivecarbon precursors for nucleotide and polysaccharide production (3, 4). Microbial strains without FBPase (gene deletion mutants) cannot grow on gluconeogenic substrates, but thrive on glucose or other hexoses (3).FBPase and fructose-6-phosphate-1-kinase are constitutive enzymes in E. coli, and in the absence of some kind of coordinate regulation, define a futile cycle (5). Futile cycling, however, is minimal under glycolytic (6) and gluconeogenic (7) conditions, and hence FBPase must be under metabolic control. In mammals, AMP and fructose 2,6-bisphosphate (Fru-2,6-P 2 ) synergistically inhibit FBPase (8). Fru-2,6-P 2 , the concentration of which is subject to hormonal control, directly inhibits FBPase by binding to its active site, whereas AMP binds to an allosteric site (5, 9, 10) and induces a conformational change, converting the enzyme from an active quaternary conformation (R-state) to an inactive form (T-state) (11-13). In contrast, little is known regarding the regulation of FBPase in E. coli. AMP is a potent inhibitor of the E. coli enzyme (4, 14 -16), but Fru-2,6-P 2 is not present. (Fru-2,6-P 2 , however, does inhibit E. coli FBPase in assays (15, 16).) Dynamic regulation by AMP alone is unlikely, because AMP concentrations in vivo remain relatively constant under gluconeogenic and glycolytic condit...

SummaryYeast prions require a core set of chaperone proteins including Sis1, Hsp70 and Hsp104 to generate new amyloid templates for stable propagation, yet emerging studies indicate that propagation of some prions requires additional chaperone activities, demonstrating chaperone specificity beyond the common amyloid requirements. To comprehensively assess such prion‐specific requirements for the propagation of the [URE 3] prion variant [URE 3‐1], we screened 12 yeast cytosolic J‐proteins, and here we report a novel role for the J‐protein Swa2/Aux1. Swa2 is the sole yeast homolog of the mammalian protein auxilin, which, like Swa2, functions in vesicle‐mediated endocytosis by disassembling the structural lattice formed by the protein clathrin. We found that, in addition to Sis1, [URE 3‐1] is specifically dependent upon Swa2, but not on any of the 11 other J‐proteins. Further, we show that [URE 3‐1] propagation requires both a functional J‐domain and the tetratricopeptide repeat (TPR) domain, but surprisingly does not require Swa2‐clathrin binding. Because the J‐domain of Swa2 can be replaced with the J‐domains of other proteins, our data strongly suggest that prion‐chaperone specificity arises from the Swa2 TPR domain and supports a model where Swa2 acts through Hsp70, most likely to provide additional access points for Hsp104 to promote prion template generation.

Prions of budding yeast serve as a tractable model of amyloid behavior. Here we address the issue of the effect of yeast strain variation on prion stability, focusing also on the effect of amyloid conformation and the involvement of the co-chaperone Sis1, an essential J-protein partner of Hsp70. We found, despite an initial report to the contrary, that yeast strain background has little effect on the requirement for particular Sis1 domains for stable propagation of the prion [RNQ+], if the level of Sis1 expression is controlled. On the other hand, some variation in prion behavior was observed between yeast strains, in particular, the stability of certain [PSI+] variants. Future examination of such yeast strain-specific phenomena may provide useful insights into the basis of prion/chaperone dynamics.

Yeast prions are heritable protein-based elements, most of which are formed of amyloid aggregates that rely on the action of molecular chaperones for transmission to progeny. Prions can form distinct amyloid structures, known as ‘strains’ in mammalian systems, that dictate both pathological progression and cross-species infection barriers. In yeast these same amyloid structural polymorphisms, called ‘variants’, dictate the intensity of prion-associated phenotypes and stability in mitosis. We recently reported that [PSI+] prion variants differ in the fundamental domain requirements for one chaperone, the Hsp40/J-protein Sis1, which are mutually exclusive between 2 different yeast prions, demonstrating a functional plurality for Sis1.1 Here we extend that analysis to incorporate additional data that collectively support the hypothesis that Sis1 has multiple functional roles that can be accomplished by distinct sets of domains. These functions are differentially required by distinct prions and prion variants. We also present new data regarding Hsp104-mediated prion elimination and show that some Sis1 functions, but not all, are conserved in the human homolog Hdj1/DNAJB1. Importantly, of the 10 amyloid-based prions indentified to date in Saccharomyces cerevisiae, the chaperone requirements of only 4 are known, leaving a great diversity of amyloid structures, and likely modes of amyloid-chaperone interaction, largely unexplored.