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.2000 Jan 4;97(1):240-4.
doi: 10.1073/pnas.97.1.240.

Guanidine hydrochloride blocks a critical step in the propagation of the prion-like determinant [PSI(+)] of Saccharomyces cerevisiae

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Guanidine hydrochloride blocks a critical step in the propagation of the prion-like determinant [PSI(+)] of Saccharomyces cerevisiae

S S Eaglestone et al. Proc Natl Acad Sci U S A..

Abstract

The cytoplasmic heritable determinant [PSI(+)] of the yeast Saccharomyces cerevisiae reflects the prion-like properties of the chromosome-encoded protein Sup35p. This protein is known to be an essential eukaryote polypeptide release factor, namely eRF3. In a [PSI(+)] background, the prion conformer of Sup35p forms large oligomers, which results in the intracellular depletion of functional release factor and hence inefficient translation termination. We have investigated the process by which the [PSI(+)] determinant can be efficiently eliminated from strains, by growth in the presence of the protein denaturant guanidine hydrochloride (GuHCl). Strains are "cured" of [PSI(+)] by millimolar concentrations of GuHCl, well below that normally required for protein denaturation. Here we provide evidence indicating that the elimination of the [PSI(+)] determinant is not derived from the direct dissolution of self-replicating [PSI(+)] seeds by GuHCl. Although GuHCl does elicit a moderate stress response, the elimination of [PSI(+)] is not enhanced by stress, and furthermore, exhibits an absolute requirement for continued cell division. We propose that GuHCl inhibits a critical event in the propagation of the prion conformer and demonstrate that the kinetics of curing by GuHCl fit a random segregation model whereby the heritable [PSI(+)] element is diluted from a culture, after the total inhibition of prion replication by GuHCl.

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Figures

Figure 1
Figure 1
Linear plot of the number of generations against growth time for one set of curing data in the presence of 3 mM GuHCl. The number of generations was obtained by comparing those values of % [PSI+] determined experimentally at each time point, with those calculated from an iterative model for curing by random segregation, in which the initial number of prions (n) was set at 32. Only values of % [PSI+] between 98 and 2 were considered. The line of best fit gives values for the gradient of 0.373 and the intercept of −0.934, withR2 = 0.982. The gradient represents 1/generation time, yielding a value for calculated generation time of 2.68 hr. This calculation compares well with the experimentally determined generation time of 2.5 hr. The intercept gives a measure of the generation shift from the real number of prion seeds to that used in the iterative model. The best-fit number of prion seeds can be calculated by 32*(2-generation shift), yielding a value of 61. Similar linear plots for other curing curves in each case yielded generation times within 10% of the experimentally determined doubling time and an average number of prion seeds of 62 ± 10.
Figure 2
Figure 2
Elimination of the [PSI+] determinant by GuHCl requires cell division. Two identical cultures of the [PSI+] strain BSC783/4a were grown at 30°C in YEPD supplemented with 3 mM GuHCl. One culture was maintained in exponential growth (●), while the other was allowed to enter stationary phase (○), after 10 hr (approximately four generations) of exponential growth. The [PSI+] fraction of the cultures were determined, as a function of time, on the basis of the red/white colony ratio of culture aliquots, grown on solid ¼YEPD medium. Each data point represents an average of three culture samples. The dashed line represents the best fit to a simple segregational model, with variables pre-existing prion seeds and generation time (as described inMaterials and Methods).
Figure 3
Figure 3
Stress response does not enhance the rate of prion elimination by GuHCl. BSC783/4a [PSI+] was grown in exponential phase at 30°C in YEPD containing 3 mM GuHCl (○) and further supplemented with 3% (vol/vol) EtOH (●). The [PSI+] fraction of the cultures was determined, as a function of time, on the basis of the red/white colony ratio of culture samples. Each data point represents an average of three culture samples. The lines represent the best fit to a simple segregational model, with variables pre-existing prion seeds and generation time.
Figure 4
Figure 4
The propagation of [PSI+] is stable upon removal of cells from GuHCl. After 12.5 hr exponential growth in the presence of 3 mM GuHCl (●), two culture aliquots of BSC783/4a were transferred to fresh medium containing 3 mM GuHCl (○) or to YEPD medium alone (▵). The cultures then were allowed to continue exponential growth. The [PSI+] fraction of the cultures were determined, as a function of time, on the basis of the red/white colony ratio of culture samples. Each data point represents an average of three culture samples. The line overlaying the data for growth in the presence of GuHCl represents the best fit to a simple segregational model, with variables pre-existing prion seeds and generation time.
Figure 5
Figure 5
Curing fits to a simple segregation model. The data of Fig. 3 are replotted with respect to generations, as calculated by the model and adjusted to account for a brief cessation of growth, during which the cells adapted to the presence of 3% (vol/vol) EtOH. BSC783/4a [PSI+] was grown in exponential phase, at 30°C in YEPD containing 3 mM GuHCl (○) and further supplemented with 3% (vol/vol) EtOH (●). The curve is a theoretical plot, based on the segregational hypothesis, where the calculated number of pre-existing prion seeds within a cell is 62. The calculated generation times for both sets of data from the model were within 10% of the experimentally determined generation times.
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