Showing papers by "Susan Lindquist published in 1996"

Support for the prion hypothesis for inheritance of a phenotypic trait in yeast.

Abstract: A cytoplasmically inherited genetic element in yeast, [PSI+], was confirmed to be a prionlike aggregate of the cellular protein Sup35 by differential centrifugation analysis and microscopic localization of a Sup35-green fluorescent protein fusion. Aggregation depended on the intracellular concentration and functional state of the chaperone protein Hsp104 in the same manner as did [PSI+] inheritance. The amino-terminal and carboxy-terminal domains of Sup35 contributed to the unusual behavior of [PSI+]. [PSI+] altered the conformational state of newly synthesized prion proteins, inducing them to aggregate as well, thus fulfilling a major tenet of the prion hypothesis.

A cyclophilin function in Hsp90-dependent signal transduction.

Abstract: Cpr6 and Cpr7, the Saccharomyces cerevisiae homologs of cyclophilin-40 (CyP-40), were shown to form complexes with Hsp90, a protein chaperone that functions in several signal transduction pathways. Deletion of CPR7 caused severe growth defects when combined with mutations that decrease the amount of Hsp90 or Sti1, another component of the Hsp90 chaperone machinery. The activities of two heterologous Hsp90-dependent signal transducers expressed in yeast, glucocorticoid receptor and pp60v−src kinase, were adversely affected by cpr7 null mutations. These results suggest that CyP-40 cyclophilins play a general role in Hsp90-dependent signal transduction pathways under normal growth conditions.

Heat-shock protein 104 expression is sufficient for thermotolerance in yeast.

Abstract: In all organisms, mild heat pretreatments induce tolerance to high temperatures. In the yeast Saccharomyces cerevisiae, such pretreatments strongly induce heat-shock protein (Hsp) 104, and hsp104 mutations greatly reduce high-temperature survival, indicating Hsp1O4 plays a critical role in induced thermotolerance. Surprisingly, however, a heat-shock transcription factor mutation (hsf1-m3) that blocks the induction of Hsps does not block induced thermotolerance. To resolve these apparent contradictions, we reexamined Hsp expression in hsf1-m3 cells. HsplO4 was expressed at a higher basal level in this strain than in other S. cerevisiae strains. Moreover, whereas the hsf1-m3 mutation completely blocked the induction of Hsp26 by heat, it did not block the induction of Hsp1O4. HSP104 could not be deleted in hsf1-m3 cells because the expression of heat-shock factor (and the viability of the strain) requires nonsense suppression mediated by the yeast prion [PSI+], which in turn depends upon Hsp1O4. To determine whether the level of Hsp1O4 expressed in hsf1-m3 cells is sufficient for thermotolerance, we used heterologous promoters to regulate Hsp1O4 expression in other strains. In the presence of other inducible factors (with a conditioning pretreatment), low levels of Hsp1O4 are sufficient to provide full thermotolerance. More remarkably, in the absence of other inducible factors (without a pretreatment), high levels of Hsp1O4 are sufficient. We conclude that Hsp1O4 plays a central role in ameliorating heat toxicity. Because Hsp1O4 is nontoxic and highly conserved, manipulating the expression of Hsp1OO proteins provides an excellent prospect for manipulating thermotolerance in other species.

Effect of engineering Hsp70 copy number on Hsp70 expression and tolerance of ecologically relevant heat shock in larvae and pupae of Drosophila melanogaster.

Abstract: To determine how the accumulation of the major Drosophila melanogaster heat-shock protein, Hsp70, affects inducible thermotolerance in larvae and pupae, we have compared two sister strains generated by site-specific homologus recombination. One strain carried 12 extra copies of the Hsp70 gene at a single insertion site (extra-copy strain) and the other carried remnants of the transgene construct but lacked the extra copies of Hsp70 (excision strain). Hsp70 levels in whole-body lysates of larvae and pupae were measured by ELISA with an Hsp70-specific antibody. In both extra-copy and excision strains, Hsp70 was undetectable prior to heat shock. Hsp70 concentrations were higher in the extra-copy strain than in the excision strain at most time points during and after heat shock. Pretreatment (i.e. exposure to 36 degrees C before heat shock) significantly improved thermotolerance, and this improvement was greater and more rapid in larvae and pupae of the extra-copy strain than in those of the excision strain. The experimental conditions resemble thermal regimes actually experienced by Drosophila in the field. Thus, these findings represent the best evidence to date that the amount of a heat-shock protein affects the fitness of a complex animal in the wild.