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S Lindquist

Publications and source records attributed to S Lindquist.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1997

Chaperone-supervised conversion of prion protein to its protease-resistant form.

Transmissible spongiform encephalopathies (TSEs) are lethal, infectious disorders of the mammalian nervous system. A TSE hallmark is the conversion of the cellular protein PrPC to disease-associated PrPSc (named for scrapie, the first known TSE). PrPC is protease-sensitive, monomeric, detergent soluble, and primarily alpha-helical; PrPSc is protease-resistant, polymerized, detergent insoluble, and rich in beta-sheet. The "protein-only" hypothesis posits that PrPSc is the infectious TSE agent that directly converts host-encoded PrPC to fresh PrPSc, harming neurons and creating new agents of infection. To gain insight on the conformational transitions of PrP, we tested the ability of several protein chaperones, which supervise the conformational transitions of proteins in diverse ways, to affect conversion of PrPC to its protease-resistant state. None affected conversion in the absence of pre-existing PrPSc. In its presence, only two, GroEL and Hsp104 (heat shock protein 104), significantly affected conversion. Both promoted it, but the reaction characteristics of conversions with the two chaperones were distinct. In contrast, chemical chaperones inhibited conversion. Our findings provide new mechanistic insights into nature of PrP conversions, and provide a new set of tools for studying the process underlying TSE pathogenesis.

Adenosine Triphosphate↗
PubMed1997

In vivo functions of the Saccharomyces cerevisiae Hsp90 chaperone.

In the highly concentrated environment of the cell, polypeptide chains are prone to aggregation during synthesis (as nascent chains await the emergence of the remainder of their folding domain), translocation, assembly, and exposure to stresses that cause previously folded proteins to unfold. A large and diverse group of proteins, known as chaperones, transiently associate with such folding intermediates to prevent aggregation, but in many cases the specific functions of individual chaperones are still not clear. In vivo, Hsp90 (heat shock protein 90) plays a role in the maturation of components of signal transduction pathways but also exhibits chaperone activity with diverse proteins in vitro, suggesting a more general function. We used a unique temperature-sensitive mutant of Hsp90 in Saccharomyces cerevisiae, which rapidly and completely loses activity on shift to high temperatures, to examine the breadth of Hsp90 functions in vivo. The data suggest that Hsp90 is not required for the de novo folding of most proteins, but it is required for a specific subset of proteins that have greater difficulty reaching their native conformations. Under conditions of stress, Hsp90 does not generally protect proteins from thermal inactivation but does enhance the rate at which a heat-damaged protein is reactivated. Thus, although Hsp90 is one of the most abundant chaperones in the cell, its in vivo functions are highly restricted.

HSP90 Heat-Shock Proteins↗
PubMed1997

Cdc37 is a molecular chaperone with specific functions in signal transduction.

Cdc37 is required for cyclin-dependent kinase activation and is genetically linked with the activity of several other kinases, including oncogenic v-Src, casein kinase II, MPS-1 kinase, and sevenless. Strikingly, many pathways involving Cdc37 also involve the protein chaperone Hsp90. The identification of Cdc37 as the 50-kD protein in several Hsp90-kinase complexes, together with other data, led to the recent suggestion that Cdc37 is a kinase-targeting "subunit" of Hsp90. We directly examined the effect of Cdc37 on Hsp90 functions. Rather than simply acting as an accessory factor for Hsp90, Cdc37 is itself a protein chaperone with properties remarkably similar to those of Hsp90. In vitro, Cdc37 maintains denatured beta-galactosidase in an activation-competent state without reactivating it and stabilizes mature, but unstable, casein kinase II. In vivo, Cdc37 overexpression can compensate for decreased Hsp90 function, but the proteins are not interchangeable. Cdc37 can compensate for Hsp90 in maintaining the activity of v-Src kinase but does not maintain the activity of the glucocorticoid receptor. Thus, the very similar chaperone activities of the two proteins, uncovered through in vitro analysis, diverge in vivo in specific signal transduction pathways.

Cell Cycle Proteins↗
PubMed1997

Self-seeded fibers formed by Sup35, the protein determinant of [PSI+], a heritable prion-like factor of S. cerevisiae.

The [PSI+] factor of S. cerevisiae represents a new form of inheritance: cytosolic transmission of an altered phenotype is apparently based upon inheritance of an altered protein structure rather than an altered nucleic acid. The molecular basis of its propagation is unknown. We report that purified Sup35 and subdomains that induce [PSI+] elements in vivo form highly ordered fibers in vitro. Fibers bind Congo red and are rich in beta sheet, characteristics of amyloids found in certain human diseases, including the prion diseases. Some fibers have distinct structures and these, once initiated, are self-perpetuating. Preformed fibers greatly accelerate fiber formation by unpolymerized protein. These data support a "protein-only" seeded polymerization model for the inheritance of [PSI+].

Fungal Proteins↗