Search PubMed⌕ Search

Biomedical subjects

S Lindquist

Publications and source records attributed to S Lindquist.

At least 37 records · Page 2Linked to original sources
PubMed1999

Oligopeptide-repeat expansions modulate 'protein-only' inheritance in yeast.

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.

Fungal Proteins↗
PubMed1999

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

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.

Cell Division↗
PubMed1999

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

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, p60(v-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.

Amino Acid Substitution↗
PubMed1999

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

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.

Aspartic Acid↗
PubMed1999

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

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.

Animals↗
PubMed1999

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

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.

Cell Division↗
PubMed1999

Genetic analysis of viable Hsp90 alleles reveals a critical role in Drosophila spermatogenesis.

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.

Alleles↗
PubMed1998

Hsp90 as a capacitor for morphological evolution.

The heat-shock protein Hsp90 supports diverse but specific signal transducers and lies at the interface of several developmental pathways. We report here that when Drosophila Hsp90 is mutant or pharmacologically impaired, phenotypic variation affecting nearly any adult structure is produced, with specific variants depending on the genetic background and occurring both in laboratory strains and in wild populations. Multiple, previously silent, genetic determinants produced these variants and, when enriched by selection, they rapidly became independent of the Hsp90 mutation. Therefore, widespread variation affecting morphogenic pathways exists in nature, but is usually silent; Hsp90 buffers this variation, allowing it to accumulate under neutral conditions. When Hsp90 buffering is compromised, for example by temperature, cryptic variants are expressed and selection can lead to the continued expression of these traits, even when Hsp90 function is restored. This provides a plausible mechanism for promoting evolutionary change in otherwise entrenched developmental processes.

Animals↗
PubMed1998

Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins.

Hsp104 is a stress tolerance factor that promotes the reactivation of heat-damaged proteins in yeast by an unknown mechanism. Herein, we demonstrate that Hsp104 functions in this process directly. Unlike other chaperones, Hsp104 does not prevent the aggregation of denatured proteins. However, in concert with Hsp40 and Hsp70, Hsp104 can reactivate proteins that have been denatured and allowed to aggregate, substrates refractory to the action of other chaperones. Hsp104 cooperates with the chaperones present in reticulocyte lysates but not with DnaK of E. coli. We conclude that Hsp104 has a protein remodeling activity that acts on trapped, aggregated proteins and requires specific interactions with conventional chaperones to promote refolding of the intermediates it produces.

Adenosine Triphosphatases↗
PubMed1998

The ATPase activity of Hsp104, effects of environmental conditions and mutations.

Hsp104 is crucial for stress tolerance in Saccharomyces cerevisiae, and both of its nucleotide-binding domains (NBD1 and NBD2) are required. Here, we characterize the ATPase activity and oligomerization properties of wild-type (WT) Hsp104 and of NBD mutants. In physiological ionic strength buffers (pH 7.5, 37 degreesC) WT Hsp104 exhibits Michaelis-Menten kinetics between 0.5 and 25 mM ATP (Km approximately 5 mM, Vmax approximately 2 nmol min-1 microg-1). ATPase activity is strongly influenced by factors that vary with cell stress (e.g. temperature, pH, and ADP). Mutations in the P-loop of NBD1 (G217V or K218T) severely reduce ATP hydrolysis but have little effect on oligomerization. Analogous mutations in NBD2 (G619V or K620T) have smaller effects on ATPase activity but impair oligomerization. The opposite relationship was reported for another member of the HSP100 protein family, the Escherichia coli ClpA protein, in studies employing lower ionic strength buffers. In such buffers, the Km of WT Hsp104 for ATP hydrolysis decreased 10-fold and its stability under stress conditions increased, but the effects of the NBD mutations on ATPase activity and oligomerization remained opposite to those of ClpA. Either the functions of the two NBDs in ClpA and Hsp104 have been reversed or both contribute to ATP hydrolysis and oligomerization in a complex manner that can be idiosyncratically affected by such mutations.

Adenosine Triphosphatases↗
PubMed1998

The peptidyl-prolyl isomerase domain of the CyP-40 cyclophilin homolog Cpr7 is not required to support growth or glucocorticoid receptor activity in Saccharomyces cerevisiae.

CyP-40 cyclophilins are found in association with molecular chaperone Hsp90.steroid receptor complexes. The amino-terminal portion of these cyclophilins harbors the characteristic peptidyl-prolyl isomerase (PPIase) domain, whereas three copies of the tetratricopeptide (TPR) motif, a structure shown to be involved in protein-protein interactions, and a putative calmodulin-binding domain are located in the carboxyl-terminal half of the protein. The TPR domains mediate binding to Hsp90, but a requirement for the PPIase domain has not been established. To address this, we have investigated the effects of mutations that alter the PPIase domain of the Saccharomyces cerevisiae CyP-40 homolog, Cpr7. Because Cpr7 is required for rapid growth and full Hsp90 activity, a functional assessment of the PPIase domain could be performed in vivo. A mutation in the catalytic domain altering a conserved site predicted to be essential for isomerase activity did not compromise Cpr7 function. Furthermore, deletion of the entire PPIase domain did not significantly affect growth or Hsp90-mediated steroid receptor activity. These results indicate that the TPR-containing carboxyl terminus of Cpr7 is sufficient for fundamental Cpr7-dependent activity.

Carrier Proteins↗
PubMed1998

Thermotolerance in Saccharomyces cerevisiae: the Yin and Yang of trehalose.

Trehalose, a sugar produced by a wide variety of organisms, has long been known for its role in protecting certain organisms from desiccation. Recent work in yeast indicates that trehalose also promotes survival under conditions of extreme heat, by enabling proteins to retain their native conformation at elevated temperatures and suppressing the aggregation of denatured proteins. The latter property, however, seems to impair the recovery of cells from heat shock if they fail to degrade trehalose after the stress has passed. These multiple effects of trehalose on protein stability and folding suggest a host of promising applications.

Animals↗
PubMed1998

Multiple effects of trehalose on protein folding in vitro and in vivo.

The disaccharide trehalose is produced in large quantities by diverse organisms during a variety of stresses. Trehalose prevents proteins from denaturing at high temperatures in vitro, but its function in stress tolerance in vivo is controversial. We report that trehalose stabilizes proteins in yeast cells during heat shock. Surprisingly, trehalose also suppresses the aggregation of denatured proteins, maintaining them in a partially-folded state from which they can be activated by molecular chaperones. The continued presence of trehalose, however, interferes with refolding, suggesting why it is rapidly hydrolyzed following heat shock. These findings reconcile conflicting reports on the role of trehalose in stress tolerance, provide a novel tool for accessing protein folding intermediates, and define new parameters for modulating stress tolerance and protein aggregation.

Animals↗
PubMed1998

Developmentally regulated nuclear transport of transcription factors in Drosophila embryos enable the heat shock response.

Hsp70 is a broadly conserved thermotolerance factor, but inhibits growth at normal temperatures and cannot be induced in early embryos. We report that in Drosophila embryos the temporal and spatial patterns of Hsp70 inducibility were unexpectedly complex, with striking differences between the soma and the germline. In both, regulation occurred at the level of transcription. During the refractory period for Hsp70 induction, HSF (heat-shock transcription factor) exhibited specific DNA-binding activity characteristic of activation in extracts of heated embryos. Remarkably, however, HSF was restricted to the cytoplasm in intact embryos even after heat shock. HSF moved from the cytoplasm to the nucleus in the absence of heat precisely when the capacity to induce Hsp70 was acquired (cycle 12 of the germline, cycle 13 in the soma). During oogenesis, Hsp70 inducibility was lost in nurse cells around stage 10, in a posterior-to-anterior gradient and HSF redistributed from nucleus to cytoplasm in the same spatiotemporal pattern. In a highly inbred derivative of the Samarkind strain, HSF moved into embryonic nuclei earlier than in our standard wild-type strain. Correspondingly, Hsp70 was inducible earlier, confirming that nuclear transport of HSF controls the inducibility of Hsp70 in early embryos. We also report for the first time the nuclear import patterns of two general transcription factors, RNA polymerase subunit Ilc and TATA binding protein (TBP). Both enter nuclei in a highly synchronous manner, independently of each other and of HSF. The import of TBP coincides with the first reported appearance of transcripts in the embryo. We suggest that the potentiation of general and heat shock-specific transcription in Drosophila embryos is controlled by the developmentally programmed relocalization of general and heat shock-specific transcription factors. Restricted nuclear entry of HSF represents a newly described mechanism for regulating the heat-shock response.

Animals↗
PubMed1997

Interactions of the chaperone Hsp104 with yeast Sup35 and mammalian PrP.

[PSI+] is a genetic element in yeast for which a heritable change in phenotype appears to be caused by a heritable change in the conformational state of the Sup35 protein. The inheritance of [PSI+] and the physical state of Sup35 in vivo depend on the protein chaperone Hsp104 (heat shock protein 104). Although these observations provide a strong genetic argument in support of the "protein-only" or "prion" hypothesis for [PSI+], there is, as yet, no direct evidence of an interaction between the two proteins. We report that when purified Sup35 and Hsp104 are mixed, the circular dichroism (CD) spectrum differs from that predicted by the addition of the proteins' individual spectra, and the ATPase activity of Hsp104 is inhibited. Similar results are obtained with two other amyloidogenic substrates, mammalian PrP and beta-amyloid 1-42 peptide, but not with several control proteins. With a group of peptides that span the PrP protein sequence, those that produced the largest changes in CD spectra also caused the strongest inhibition of ATPase activity in Hsp104. Our observations suggest that (i) previously described genetic interactions between Hsp104 and [PSI+] are caused by direct interaction between Hsp104 and Sup35; (ii) Sup35 and PrP, the determinants of the yeast and mammalian prions, respectively, share structural features that lead to a specific interaction with Hsp104; and (iii) these interactions couple a change in structure to the ATPase activity of Hsp104.

Adenosine Triphosphatases↗