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Showing papers by "Susan Lindquist published in 2001"


Journal ArticleDOI
TL;DR: It is demonstrated that PrP can accumulate in the cytoplasm and is likely to enter this compartment through normal protein quality-control pathways, which has implications for pathogenesis.
Abstract: The cytoplasm seems to provide an environment that favors conversion of the prion protein (PrP) to a form with the physical characteristics of the PrPSc conformation, which is associated with transmissible spongiform encephalopathies. However, it is not clear whether PrP would ever exist in the cytoplasm under normal circumstances. We report that PrP accumulates in the cytoplasm when proteasome activity is compromised. The accumulated PrP seems to have been subjected to the normal proteolytic cleavage events associated with N- and C-terminal processing in the endoplasmic reticulum, suggesting that it arrives in the cytoplasm through retrograde transport. In the cytoplasm, PrP forms aggregates, often in association with Hsc70. With prolonged incubation, these aggregates accumulate in an “aggresome”-like state, surrounding the centrosome. A mutant (D177N), which is associated with a heritable and transmissible form of the spongiform encephalopathies, is less efficiently trafficked to the surface than wild-type PrP and accumulates in the cytoplasm even without proteasome inhibition. These results demonstrate that PrP can accumulate in the cytoplasm and is likely to enter this compartment through normal protein quality-control pathways. Its potential to accumulate in the cytoplasm has implications for pathogenesis.

290 citations


Journal ArticleDOI
TL;DR: Using a prion chimera, it is demonstrated that the prion‐determinant domain of Rnq1 is genetically sufficient for control by Sis1, indicating that multiple physical states are compatible with prion maintenance and that changes in chaperone activity can create prion variants.
Abstract: Yeast prions are inherited through proteins that exist in alternate, self-perpetuating conformational states. The mechanisms by which these states arise and are maintained are still poorly defined. Here we demonstrate for the first time that Sis1, a member of the Hsp40 chaperone family, plays a critical role in the maintenance of a prion. The prion [RNQ+] is formed by Rnq1, which is present in the same physical complex as Sis1, but only when Rnq1 is in the prion state. The G/F domain of Sis1 is dispensable for rapid growth on rich medium, but is required for [RNQ+] maintenance, distinguishing essential regions of Sis1 from those needed for prion interaction. A specific Sis1 deletion mutant altered the physical aggregation pattern of Rnq1 without curing the prion. This variant state propagated in a heritable fashion after wild-type Sis1 function was restored, indicating that multiple physical states are compatible with prion maintenance and that changes in chaperone activity can create prion variants. Using a prion chimera we demonstrate that the prion-determinant domain of Rnq1 is genetically sufficient for control by Sis1.

199 citations


Journal ArticleDOI
TL;DR: It is found that [PSI+] variants contain different ratios of Sup35 in the prion and non‐prion state that correlate with different translation termination efficiencies, and that the partially purified prion form of Sup 35 from a strong [PSi+] variant converted purified NM much more efficiently than that of several weak variants.
Abstract: Yeast prions are protein-based genetic elements that produce phenotypes through self-perpetuating changes in protein conformation. For the prion [PSI+] this protein is Sup35, which is comprised of a prion-determining region (NM) fused to a translational termination region. [PSI+] strains (variants) with different heritable translational termination defects (weak or strong) can exist in the same genetic background. [PSI+] variants are reminiscent of mammalian prion strains, which can be passaged in the same mouse strain yet have different disease latencies and brain pathologies. We found that [PSI+] variants contain different ratios of Sup35 in the prion and non-prion state that correlate with different translation termination efficiencies. Indeed, the partially purified prion form of Sup35 from a strong [PSI+] variant converted purified NM much more efficiently than that of several weak variants. However, this difference was lost in a second round of conversion in vitro. Thus, [PSI+] variants result from differences in the efficiency of prion-mediated conversion, and the maintenance of [PSI+] variants involves more than nucleated conformational conversion (templating) to NM alone.

138 citations


Journal ArticleDOI
TL;DR: It is proposed that the structural flexibility of NM is particularly suited to allowing heritable protein-based changes in cellular behavior.
Abstract: The [PSI(+)] factor of Saccharomyces cerevisiae is a protein-based genetic element (prion) comprised of a heritable altered conformation of the cytosolic translation termination factor Sup35p. In vitro, the prion-determining region (NM) of Sup35p undergoes conformational conversion from a highly flexible soluble state to structured amyloid fibers, with a rate that is greatly accelerated by preformed NM fiber nuclei. Nucleated conformational conversion is the molecular basis of the genetic inheritance of [PSI(+)] and provides a new model for studying amyloidogenesis. Here we investigate the importance of structure and structural flexibility in soluble NM. Elevated temperatures, chemical chaperones and certain mutations in NM increase or change its structural content and inhibit or enhance nucleated conformational conversion. We propose that the structural flexibility of NM is particularly suited to allowing heritable protein-based changes in cellular behavior.

105 citations


Journal ArticleDOI
TL;DR: Interactions between subunits influence the ATPase activity of Hsp104, play a vital role in its biological functions, and provide a mechanism for conditionally inactivating Hsp 104 function in vivo.
Abstract: ‡Point mutations in either of the two nucleotide-binding domains (NBD) of Hsp104 (NBD1 and NBD2) eliminate its thermotolerance function in vivo. In vitro, NBD1 mutations virtually eliminate ATP hydrolysis with little effect on hexamerization; analogous NBD2 mutations reduce ATPase activity and severely impair hexamerization. We report that high protein concentrations overcome the assembly defects of NBD2 mutants and increase ATP hydrolysis severalfold, changing Vmax with little effect on Km. In a complementary fashion, the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate inhibits hexamerization of wildtype (WT) Hsp104, lowering Vmax with little effect on Km. ATP hydrolysis exhibits a Hill coefficient between 1.5 and 2, indicating that it is influenced by cooperative subunit interactions. To further analyze the effects of subunit interactions on Hsp104, we assessed the effects of mutant Hsp104 proteins on WT Hsp104 activities. An NBD1 mutant that hexamerizes but does not hydrolyze ATP reduces the ATPase activity of WT Hsp104 in vitro. In vivo, this mutant is not toxic but specifically inhibits the thermotolerance function of WT Hsp104. Thus, interactions between subunits influence the ATPase activity of Hsp104, play a vital role in its biological functions, and provide a mechanism for conditionally inactivating Hsp104 function in vivo.

88 citations


Journal ArticleDOI
TL;DR: This work genetically engineered a mutant of NM so that it contained an accessible cysteine residue that was easily labeled after fiber formation and propagated the heritable genetic trait [PSI(+)] with the same fidelity, indicating that NM fiber growth is bidirectional.

85 citations


Journal ArticleDOI
01 Oct 2001-Genetics
TL;DR: The N domain of Sup35p is highly conserved in amino acid sequence and is highly biased in codon usage toward preferred codons, which concludes that Amino acid changes are under weak purifying selection based on a quantitative analysis of polymorphism and divergence.
Abstract: The prion-like behavior of Sup35p, the eRF3 homolog in the yeast Saccharomyces cerevisiae, mediates the activity of the cytoplasmic nonsense suppressor known as [PSI(+)]. Sup35p is divided into three regions of distinct function. The N-terminal and middle (M) regions are required for the induction and propagation of [PSI(+)] but are not necessary for translation termination or cell viability. The C-terminal region encompasses the termination function. The existence of the N-terminal region in SUP35 homologs of other fungi has led some to suggest that this region has an adaptive function separate from translation termination. To examine this hypothesis, we sequenced portions of SUP35 in 21 strains of S. cerevisiae, including 13 clinical isolates. We analyzed nucleotide polymorphism within this species and compared it to sequence divergence from a sister species, S. paradoxus. The N domain of Sup35p is highly conserved in amino acid sequence and is highly biased in codon usage toward preferred codons. Amino acid changes are under weak purifying selection based on a quantitative analysis of polymorphism and divergence. We also conclude that the clinical strains of S. cerevisiae are not recently derived and that outcrossing between strains in S. cerevisiae may be relatively rare in nature.

54 citations


Journal ArticleDOI
TL;DR: This change in translation is produced by a self-perpetuating change in the conformation of the translationtermination factor, Sup35 as mentioned in this paper, a dominant cytoplasmically inherited factor that alters translational fidelity.
Abstract: Our work supports the hypothesis that a protein can serve as an element of genetic inheritance. This protein–only mechanism of inheritance is propagated in much the same way as hypothesized for the transmission of the protein–only infectious agent in the spongiform encephalopathies; hence these protein factors have been called yeast prions. Our work has focused on [ PSI + ], a dominant cytoplasmically inherited factor that alters translational fidelity.This change in translation is produced by a self–perpetuating change in the conformation of the translation–termination factor, Sup35. Most recently, we have determined that new elements of genetic inheritance can be created by deliberate genetic engineering, opening prospects for new methods of manipulating heredity. We have also uncovered evidence that other previously unknown elements of protein–based inheritance are encoded in the yeast genome. Finally, we have begun to use yeast as a model system for studying human protein folding diseases, such as Huntington9s disease. Proteins responsible for some of these diseases have properties uncannily similar to those that produce protein–based mechanisms of inheritance.

36 citations


Book ChapterDOI
TL;DR: Future work especially aimed at understanding the molecular role of chaperone proteins in regulating conversion as well as the early steps in de novo formation of the prion state in yeast will likely provide invaluable lessons that may be more broadly applicable to related processes in higher eukaryotes.
Abstract: Biochemical characterization of the yeast prions has revealed many similarities with the mammalian amyloidogenic proteins. The ease of generating in vivo mutations in yeast and the developing in vitro models for [PSI+] and [URE3] circumvent many of the difficulties of studying the proteins linked to the mammalian amyloidoses. Future work especially aimed at understanding the molecular role of chaperone proteins in regulating conversion as well as the early steps in de novo formation of the prion state in yeast will likely provide invaluable lessons that may be more broadly applicable to related processes in higher eukaryotes. It is important to remember, however, that there are clear distinctions between disease states associated with amyloidogenesis and the epigenetic modulation of protein function by self-perpetuating conformational conversions. Amyloid formation is detrimental to mammals and is likely selected against, providing a possible explanation for the late onset of these disorders ( Lansbury, 1999 ). In contrast, the known yeast prions are compatible with normal growth and, if beneficial to the organism, may be subject to evolutionary pressures that ultimately maximize transmission. In the prion proteins examined to date, distinct domains are responsible for normal function and for the conformational switches producing a prion conversion of that function. Recent work has demonstrated that the prion domains are both modular and transferable to other proteins on which they can confer a heritable epigenetic alteration of function (Edskes et al. 1999; Li and Lindquist 2000; Patino et al. 1996; Santoso et al. 2000; Sondheimer and Lindquist 2000). That is, prion domains need not coevolve with particular functional domains but might be moved from one protein to another during evolution. Such processes may be widely used in biology. Mechanistic studies of [PSI+] and [URE3] replication are sure to lay a foundation of knowledge for understanding a host of nonconventional genetic elements that currently remain elusive.

23 citations


Book ChapterDOI
TL;DR: This chapter focuses on recent advances in the understanding of the molecular processes underlying the inheritance and functional consequences of the Saccharomyces cerevisiaeprion [PSI+], and a fundamentalUnderstanding of the regulation of the [ PSI + ] prion cycle in vivo remains elusive.
Abstract: Publisher Summary This chapter focuses on recent advances in the understanding of the molecular processes underlying the inheritance and functional consequences of the Saccharomyces cerevisiaeprion [PSI+]. Prions are unique proteins that can exist in more than one stable conformation, and at least one of these states can be transmitted to newly synthesized protein as a form of templated replication. Because each physical state is associated with a distinct phenotypic state, the trait becomes heritable. A single prion protein can adopt alternate conformations, and hence functional states, expanding the phenotypic breadth of a single genome. Because prion inheritance is reversible (i.e., epigenetic; associated phenotypes can be sampled by the organism prior to fixation. In strains classified as [PSI + ], nonsense mutations were suppressed (or read-through) at low efficiency, while in [psi - ] strains, translation terminated faithfully at all stop codons. Strong [PSI + ] is inherited by nearly 100% of progeny, while weak [PSI + ] can be transmitted to less than 70% of progeny. Nevertheless, a fundamental understanding of the regulation of the [PSI + ] prion cycle in vivo remains elusive.

21 citations


Journal ArticleDOI
TL;DR: Evidence for an interdependence between HSP100 and A2, which are both expressed predominantly in the amastigote stage of Leishmania donovani, shows that Mutant strains lacking either of these proteins display very similar phenotypes.
Abstract: HSP100 protein in Leishmania spp. plays an important role for the survival and integrity of intracellular amastigotes. The A2 proteins of L. donovani are functionally linked to HSP100. There is evidence for an interdependence between these two proteins, which are both expressed predominantly in the amastigote stage of Leishmania donovani. Mutant strains lacking either of these proteins display very similar phenotypes, i.e. loss of virulence both in vivo and in vitro. Also, both proteins colocalise specifically to small foci within the cytoplasm of amastigotes.

Patent
20 Mar 2001
TL;DR: A transgenic plant having increased stress tolerance, such as thermotolerance, comprises a Hsp 100 family nucleic acid sequence as mentioned in this paper, which is also directed to methods of producing products from transgenic HSP 100 plants.
Abstract: A transgenic plant having increased stress tolerance, such as thermotolerance, comprises a Hsp 100 family nucleic acid sequence. The invention is also directed to methods of producing products from transgenic Hsp 100 plants.

Journal ArticleDOI
TL;DR: A mechanism exists to ensure replication of the conformational information that underlies protein-only inheritance, and is characterized by which the alternative conformation of Sup35 is adopted by an unstructured oilgomeric intermediate at the time of assembly.
Abstract: Recently, a novel mode of inheritance has been described in the yeast Saccharomyces cerevisiae. The mechanism is based on the prion hypothesis, which posits that self-perpetuating changes in the conformation of single protein, PrP, underlie the severe neurodegeneration associated with the transmissible spongiform enchephalopathies in mammals. In yeast, two prions, [URE3] and [PSI+], have been identified, but these factors confer unique phenotypes rather than disease to the organism. In each case, the prion-associated phenotype has been linked to alternative conformations of the Ure2 and Sup35 proteins. Remarkably, Ure2 and Sup35 proteins existing in the alternative conformations have the unique capacity to transmit this physical state to the newly synthesized protein in vivo. Thus, a mechanism exists to ensure replication of the conformational information that underlies protein-only inheritance. We have characterized the mechanism by which Sup35 conformational information is replicated in vitro. The assembly of amyloid fibres by a region of Sup35 encompassing the N-terminal 254 amino acids faithfully recapitulates the in vivo propagation of [PSI+]. Mutations that alter [PSI+] inheritance in vivo change the kinetics of amyloid assembly in vitro in a complementary fashion, and lysates from [PSI+] cells, but not [psi-] cells, accelerate assembly in vitro. Using this system we propose a mechanism by which the alternative conformation of Sup35 is adopted by an unstructured oilgomeric intermediate at the time of assembly.