Showing papers by "Susan Lindquist published in 1999"

Antagonistic Interactions between Yeast Chaperones Hsp104 and Hsp70 in Prion Curing

Abstract: 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.

Oligopeptide-repeat expansions modulate ‘protein-only’ inheritance in yeast

Abstract: The yeast [PSI+] element represents a new type of genetic inheritance, in which changes in phenotype are transmitted by a 'protein only' mechanism reminiscent of the 'protein-only' transmission of mammalian prion diseases. The underlying molecular mechanisms for both are poorly understood and it is not clear how similar they might be. Sup35, the [PSI+] protein determinant, and PrP, the mammalian prion determinant, have different functions, different cellular locations and no sequence similarity; however, each contains five imperfect oligopeptide repeats-PQGGYQQYN in Sup35 and PHGGGWGQ in PrP. Repeat expansions in PrP produce spontaneous prion diseases. Here we show that replacing the wild-type SUP35 gene with a repeat-expansion mutation induces new [PSI+] elements, the first mutation of its type among these newly described elements of inheritance. In vitro, fully denatured repeat-expansion peptides can adopt conformations rich in beta-sheets and form higher-order structures much more rapidly than wild-type peptides. Our results provide insight into the nature of the conformational changes underlying protein-based mechanisms of inheritance and suggest a link between this process and those producing neurodegenerative prion diseases in mammals.

Maturation of the tyrosine kinase c-src as a kinase and as a substrate depends on the molecular chaperone Hsp90

Abstract: Although Hsp90 displays general chaperone activity in vitro, few substrates of the chaperone have been identified in vivo, and the characteristics that render these substrates dependent on Hsp90 remain elusive. To investigate this issue, we exploited a paradoxical observation: several unrelated oncogenic viral tyrosine kinases, including v-src, attain their native conformation after association with Hsp90, yet their nearly identical cellular homologs interact only weakly with the chaperone. It has been controversial whether Hsp90 is vital for normal maturation of the cellular kinases or is simply binding a misfolded subfraction of the proteins. By modulating Hsp90 levels in Saccharomyces cerevisiae, we determined that Hsp90 is indeed necessary for the maturation of c-src (the normal homolog of v-src). c-src maturation is, however, less sensitive to Hsp90 perturbations than is v-src maturation. Dependence of the two proteins on Hsp90 does not correspond to their relative efficiency in reaching their final destination (the plasma membrane); we observed that in yeast, unlike in vertebrate cells, neither c-src nor v-src concentrate in the membrane. Expression of different v/c-src chimeras in cells carrying wild-type or temperature-sensitive Hsp90 alleles revealed that the difference between the proteins instead arises from multiple, naturally occurring mutations in the C-terminal region of v-src.

Genetic Analysis of Viable Hsp90 Alleles Reveals a Critical Role in Drosophila Spermatogenesis

Abstract: The Hsp90 chaperone protein maintains the activities of a remarkable variety of signal transducers, but its most critical functions in the context of the whole organism are unknown. Point mutations of Hsp83 (the Drosophila Hsp90 gene) obtained in two different screens are lethal as homozygotes. We report that eight transheterozygous mutant combinations produce viable adults. All exhibit the same developmental defects: sterile males and sterile or weakly fertile females. We also report that scratch, a previously identified male-sterile mutation, is an allele of Hsp82 with a P-element insertion in the intron that reduces expression. Thus, it is a simple reduction in Hsp90 function, rather than possible altered functions in the point mutants, that leads to male sterility. As shown by light and electron microscopy, all stages of spermatogenesis involving microtubule function are affected, from early mitotic divisions to later stages of sperm maturation, individualization, and motility. Aberrant microtubules are prominent in yeast cells carrying mutations in HSP82 (the yeast Hsp90 gene), confirming that Hsp90 function is connected to microtubule dynamics and that this connection is highly conserved. A small fraction of Hsp90 copurifies with taxol-stabilized microtubule proteins in Drosophila embryo extracts, but Hsp90 does not remain associated with microtubules through repeated temperature-induced assembly and disassembly reactions. If the spermatogenesis phenotypes are due to defects in microtubule dynamics, we suggest these are indirect, reflecting a role for Hsp90 in maintaining critical signal transduction pathways and microtubule effectors, rather than a direct role in the assembly and disassembly of microtubules themselves.

The yeast non‐Mendelian factor [ETA+] is a variant of [PSI+], a prion‐like form of release factor eRF3

Abstract: The yeast non-Mendelian factor [ETA+] is lethal in the presence of certain mutations in the SUP35 and SUP45 genes, which code for the translational release factors eRF3 and eRF1, respectively. One such mutation, sup35-2, is now shown to contain a UAG stop codon prior to the essential region of the gene. The non-Mendelian inheritance of [ETA+] is reminiscent of the yeast [PSI+] element, which is due to a self-propagating conformation of Sup35p. Here we show that [ETA+] and [PSI+] share many characteristics. Indeed, like [PSI+], the maintenance of [ETA+] requires the N-terminal region of Sup35p and depends on an appropriate level of the chaperone protein Hsp104. Moreover, [ETA+] can be induced de novo by excess Sup35p, and [ETA+] cells have a weak nonsense suppressor phenotype characteristic of weak [PSI+]. We conclude that [ETA+] is actually a weak, unstable variant of [PSI+]. We find that although some Sup35p aggregates in [ETA+] cells, more Sup35p remains soluble in [ETA+] cells than in isogenic strong [PSI+] cells. Our data suggest that the amount of soluble Sup35p determines the strength of translational nonsense suppression associated with different [PSI+] variants.

De novo generation of a PrPSc-like conformation in living cells.

Abstract: Conformational conversion of the cellular PrPC protein to PrPSc is a central aspect of the prion diseases, but how PrP initially converts to this conformation remains a mystery. Here we show that PrP expressed in the yeast cytoplasm, instead of the endoplasmic reticulum, acquires the characteristics of PrPSc, namely detergent insolubility and a distinct pattern of protease resistance. Neuroblastoma cells cultured under reducing, glycosylation-inhibiting conditions produce PrP with the same characteristics. We therefore describe what is, to our knowledge, the first conversion of full-length PrP in a heterologous system, show the importance of reducing and deglycosylation conditions in PrP conformational transitions, and suggest a model for initiating events in sporadic and inherited prion diseases.

[PSI+]: An Epigenetic Modulator of Translation Termination Efficiency

Abstract: ▪ Abstract The [PSI+] factor of the yeast Saccharomyces cerevisiae is an epigenetic regulator of translation termination. More than three decades ago, genetic analysis of the transmission of [PSI+] revealed a complex and often contradictory series of observations. However, many of these discrepancies may now be reconciled by a revolutionary hypothesis: protein conformation-based inheritance (the prion hypothesis). This model predicts that a single protein can stably exist in at least two distinct physical states, each associated with a different phenotype. Propagation of one of these traits is achieved by a self-perpetuating change in the protein from one form to the other. Mounting genetic and biochemical evidence suggests that the determinant of [PSI+] is the nuclear encoded Sup35p, a component of the translation termination complex. Here we review the series of experiments supporting the yeast prion hypothesis and provide another look at the 30 years of work preceding this theory in light of our curren...

Identification of SSF1, CNS1, and HCH1 as multicopy suppressors of a Saccharomyces cerevisiae Hsp90 loss-of-function mutation.

Abstract: Hsp90 functions in a multicomponent chaperone system to promote the maturation and maintenance of a diverse, but specific, set of target proteins that play key roles in the regulation of cell growth and development. To identify additional components of the Hsp90 chaperone system and its targets, we searched for multicopy suppressors of various temperature-sensitive mutations in the yeast Hsp90 gene, HSP82. Three suppressors were isolated for one Hsp90 mutant (glutamate → lysine at amino acid 381). Each exhibited a unique, allele-specific pattern of suppression with other Hsp90 mutants and had unique structural and biological properties. SSF1 is a member of an essential gene family and functions in the response to mating pheromones. CNS1 is an essential gene that encodes a component of the Hsp90 chaperone machinery. The role of HCH1 is unknown; its sequence has no strong homology to any protein of known function. SSF1 and CNS1 were weak suppressors, whereas HCH1 restored wild-type growth rates at all temperatures tested to cells expressing the E381K mutant. Overexpression of CNS1 or HCH1, but not SSF1, enhanced the maturation of a heterologous Hsp90 target protein, p60v-src. These results suggest that like Cns1p, Hch1p is a general modulator of Hsp90 chaperone functions, whereas Ssf1p likely is either an Hsp90 target protein or functions in the same pathway as an Hsp90 target protein.

Yeast prion [psi +] and its determinant, Sup35p.

Abstract: Publisher Summary [PSI+] and [URE3] are two non-Mendelian genetic elements of the yeast Saccharomyces cerevisiae that appear to be inherited through an unusual mechanism—the continued propagation of an alternate protein conformation. The protein determinants of these elements, Sup35p for [PSI+] and Ure2p for [URE3], have the unique ability to exist in at least two different, stable conformations in vivo. Although the spontaneous generation of one conformer is rare, this alternate form, once acquired, becomes predominant, influencing the other conformer to change states. This self-perpetuation of protein conformation is the key to the non-Mendelian inheritance of both [PSI+] and [URE3]. In addition, the [Het-S] phenotype of Podospora anserina, another fungus, may be inherited by a similar mechanism. This chapter focuses on both in vivo and in vitro methods used to analyze [PSI+], the most extensively studied member of this group. The study of amyloidogenic proteins is complex both in vivo and in vitro. Each assay presented in this chapter provides unique information about the physical state of Sup35p, but these techniques also have inherent pitfalls.

Contribution of N- and C-terminal domains to the function of Hsp90 in Saccharomyces cerevisiae.

Abstract: The molecular chaperone Hsp90 is a regulatory component of some key signalling proteins in the cytosol of eukaryotic cells. For some of these functions, its interaction with co-chaperones is required. Limited proteolysis defined stable folded units of Hsp90. Both an N-terminal (N210) and a C-terminal (262C) fragment interact with non-native substrate proteins in vitro, but with different specificity and ATP dependence. Here, we analysed the functional properties of these Hsp90 fragments in vivo and in vitro. We determined their influence on the general viability and cell growth of Saccharomyces cerevisiae. Expression of N210 or 262C resulted in a dominant-negative phenotype in several yeast strains tested. Their expression was not toxic, but inhibited cell growth. Further, both were unable to restore viability to Hsp90-depleted cells. In addition, N210 and 262C influence the maturation of Hsp90 substrates, such as the glucocorticoid receptor and pp60v–Src kinase. Specifically, 262C forms partially active chaperone complexes, leading to an arrest of the chaperoned substrate at a certain stage of its maturation cycle. This demonstrates the requirement of a sophisticated and cofactor-regulated interplay between N- and C-terminal activities for Hsp90 function in vivo.