The genetic properties of the [URE3] non-Mendelian element Saccharomyces cerevisiae suggest that it is a prion (infectious protein) form Ure2p, regulator nitrogen catabolism. In extracts from strains, Ure2p was partially resistant to proteinase K compared with wild-type extracts. Overexpression in strains induced 20- 200-fold increase frequency which arose. just amino-terminal 65 residues increased induction 6000-fold. Without this "prion-inducing domain" carboxyl-terminal domain performed...

Abstract [PSI+] is a prion (infectious protein) of Sup35p, subunit the Saccharomyces cerevisiae translation termination factor. We isolated dominant allele, SSA1-21, gene encoding an Hsp70 chaperone that impairs mitotic stability and weakens allosuppression caused by [PSI+]. While normal in strains lacking SSA1, SSA2, or both, SSA1-21 with deletion SSA2 cannot propagate are hypersensitive to curing guanidine-hydrochloride partially cured rapid induction heat-shock response but not growth at...

CP1 is a sequence-specific DNA-binding protein of the yeast Saccharomyces cerevisiae which recognizes highly conserved DNA element I (CDEI) centromeres. We cloned and sequenced gene encoding CP1. The codes for molecular weight 39,400. When expressed in Escherichia coli, directed synthesis CDEI-binding having same gel mobility as purified have given genetic designation CEP1 (centromere 1). was mapped found to reside on chromosome X, 2.0 centimorgans from SUP4. Strains were constructed most...

Yeast Hsp104 and its bacterial homolog, ClpB, are Clp/Hsp100 molecular chaperones AAA+ ATPases. ClpB collaborate with the Hsp70 DnaK chaperone systems, respectively, to retrieve reactivate stress-denatured proteins from aggregates. The action of in promoting cell survival following heat stress is species-specific: cannot function bacteria act yeast. To determine regions necessary for this specificity, we tested chimeras vivo vitro. We show that middle domains dictate species-specificity at...

Heat shock protein 90 (Hsp90) is a highly conserved ATP-dependent molecular chaperone that essential in eukaryotes. It required for the activation and stabilization of more than 200 client proteins, including many kinases steroid hormone receptors involved cell-signaling pathways. Hsp90 activity requires collaboration with subset cochaperones, Hsp70 chaperone. In higher eukaryotes, between indirect involves Hop, cochaperone interacts both Hsp70. Here we show yeast (Hsp82) (Ssa1), directly...

ABSTRACT The yeast [ PSI + ], URE3 and PIN ] genetic elements are prion forms of Sup35p, Ure2p, Rnq1p, respectively. Overexpression or Rnq1p leads to increased de novo appearance This inducible was shown be dependent on the presence overexpression other proteins that have stretches polar residues similar prion-determining domains known proteins. In a manner, facilitate ]. contrast these positive interactions, here we find in repressed each other's propagation appearance. Elevated expression...

The genetic properties of the non-Mendelian element, [URE3], suggest that it is a prion (infectious protein) form Ure2p, mediator nitrogen regulation in Saccharomyces cerevisiae . Into ure2 Δ strain (necessarily lacking [URE3]), we introduced plasmid overproducing Ure2p. This induced frequent “spontaneous generation” with identical to original [URE3]. Altering translational frame only prion-inducing domain URE2 shows Ure2 protein (and not RNA) induces appearance proteinase K-resistance Ure2p...

Abstract Hsp104 is a hexameric protein chaperone that resolubilizes stress-damaged proteins from aggregates. promotes [PSI+] prion propagation by breaking aggregates, which propagate as amyloid fibers, into more numerous “seeds.” Inactivating cures cells of and other amyloid-like yeast prions. Overexpressing also eliminates [PSI+], presumably completely resolubilizing Inexplicably, however, excess does not cure the Here we identify missense mutations in Hsp104's amino-terminal domain (NTD),...

Inactivation of Hsp104 by guanidine is contended to be the mechanism which cures yeast prions. We now find an mutation (D184N) that confers resistance guanidine-curing [PSI(+)] prion. In independent screen we isolated HSP104 allele altered in same residue (D184Y) dramatically impairs propagation a temperature-dependent manner. Directed mutagenesis produced additional alleles conferred varying degrees or impaired propagation. The mutations similarly affected [URE3] Basal and induced abundance...

The major coat protein of the L-A double-stranded RNA virus Saccharomyces cerevisiae covalently binds m7 GMP from 5' capped mRNAs in vitro. We show that this cap binding also occurs vivo and that, while activity is required for expression viral information (killer toxin mRNA level production) a wild-type strain, requirement suppressed by deletion SKI1/XRN1/SEP1. propose creates decapped cellular to decoy 5'-->3' exoribonuclease specific cap- encoded XRN1. SKI2 antiviral gene represses copy...

AbstractThe Saccharomyces cerevisiae [PSI+] prion is believed to be a self-propagating cytoplasmic amyloid. Earlier characterization of HSP70 (SSA1) mutations suggested that propagation impaired by alterations enhance Ssa1p's substrate binding. This impairment overcome second-site in conserved C-terminal motif (GPTVEEVD), which mediates interactions with tetratricopeptide repeat (TPR) cochaperones. Sti1p, TPR cochaperone homolog mammalian Hop1 (Hsp70/90 organizing protein), activates Ssa1p...

Hsp70 and Hsp90 protein chaperones cooperate in a protein-folding pathway required by many "client" proteins. The co-chaperone Sti1p coordinates functions of this pathway. has three tetratricopeptide repeat (TPR) domains. TPR1 binds Hsp70, TPR2a Hsp90, the ligand for TPR2b is unknown. Although thought to be dedicated client folding pathway, we earlier showed that regulated independently way impairs yeast [PSI+] prion propagation. Using system monitor regulation an Hsp90-inhibiting compound...

Abstract We previously described an Hsp70 mutant (Ssa1-21p), altered in a conserved residue (L483W), that dominantly impairs yeast [PSI+] prion propagation without affecting growth. generated new SSA1 mutations impaired and second-site SSA1-21 restored normal propagation. Effects of on growth did not correlate with phenotype, revealing differences function required for suggesting interacts differently aggregates than other cellular substrates. Complementary suppression activity between...

The Saccharomyces cerevisiae [PSI+] prion is a misfolded form of Sup35p that propagates as self-replicating cytoplasmic aggregates. Replication believed to occur through breakage transmissible particles, or seeds, into more numerous pieces. In cells, large aggregates are formed by coalescence smaller sodium dodecyl sulfate-insoluble polymers. It uncertain if polymers higher-order both act seeds. A mutant Hsp70 chaperone, Ssa1-21p, reduces the number seeds per cell 10-fold but overall amount...

Although propagation of Saccharomyces cerevisiae prions requires Hsp104 protein disaggregating activity, overproducing "cures" cells [PSI(+)] prions. Earlier evidence suggests that the Hsp70 mutant Ssa1-21 impairs by a related mechanism. Here, we confirm this link finding deletion STI1 both suppresses impairment and blocks curing [PSI(+)]. Hsp104's tetratricopeptide repeat (TPR) interaction motif was dispensable for curing; however, expressing Sti1 defective in or Hsp90 cured less...

Replication of amyloid-based yeast prions [PSI(+)], [URE3], and [PIN(+)] depends on the protein disaggregation machinery that includes Hsp104, Hsp70, Hsp40 molecular chaperones. Yet, overexpressing Hsp104 cures cells [PSI(+)] prions. An Hsp70 mutant (Ssa1-21p) antagonizes propagation in a manner resembling elevated Hsp104. The major cytosolic Sis1p is only required for replication these prions, but its role curing unknown. Here we find all nonessential functional regions are dispensable...

Hsp100 family chaperones of microorganisms and plants cooperate with the Hsp70/Hsp40/NEF system to resolubilize reactivate stress-denatured proteins. In yeast this machinery also promotes propagation prions by fragmenting prion polymers. We previously showed bacterial cooperates Hsp40 Ydj1 support thermotolerance Sis1 propagate [PSI+] prions. Here we find these Hsp40s similarly directed specific activities Hsp104-based machinery. By assessing ability Ydj1-Sis1 hybrid proteins complement...

Saccharomyces cerevisiae Hsp104 and Escherichia coli ClpB are Hsp100 family AAA+ chaperones that provide stress tolerance by cooperating with Hsp70 Hsp40 to solubilize aggregated protein. also remodels amyloid in vitro promotes propagation of prions yeast, but does neither, leading a view evolved these activities. Although biochemical analyses identified disaggregation machinery components required for resolubilizing proteins, interactions among vivo functions not clearly defined. We express...

ABSTRACT The [ PSI + ] yeast prion is formed when Sup35 misfolds into amyloid aggregates. ], like other prions, dependent on the molecular chaperone Hsp104, which severs seeds so that they pass as cells divide. Surprisingly, however, overexpression of Hsp104 also cures ]. Several models have been proposed to explain this effect: inhibition severing, asymmetric segregation between mother and daughter cells, dissolution seeds. First, we found neither kinetics curing nor heterogeneity in...

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 nitrogen regulatory function. [URE3] causes yeast grow slowly, has never been identified in wild, and confers no obvious phenotypic advantage. An N-terminal asparagine-rich domain determines prion-forming ability. Since...

Why eukaryotes encode multiple Hsp70 isoforms is unclear. Saccharomyces cerevisiae Ssa1p and Ssa2p are constitutive 98% identical Hsp70's. Stress-inducible Ssa3p Ssa4p 80% to Ssa1/2p. We show Ssa1p-4p have distinct functions affecting [PSI(+)] [URE3] prions. When expressed as the only Ssa, antagonized [PSI(+)]. influenced somewhat differently but overall their effects paralleled those of Ssa2p, respectively. Additionally, suppressed a prion-inhibitory effect elevated temperature. Our...