Cell division is essential for elimination of the yeast [PSI+] prion by guanidine hydrochloride

Abstract

Guanidine hydrochloride (Gdn.HCl) blocks the propagation of yeast prions by inhibiting Hsp104, a molecular chaperone that is absolutely required for yeast prion propagation. We had previously proposed that ongoing cell division is required for Gdn.HCl-induced loss of the [PSI+] prion. Subsequently, Wu et al.[Wu Y, Greene LE, Masison DC, Eisenberg E (2005) Proc Natl Acad Sci USA 102:12789-12794] claimed to show that Gdn.HCl can eliminate the [PSI+] prion from alpha-factor-arrested cells leading them to propose that in Gdn.HCl-treated cells the prion aggregates are degraded by an Hsp104-independent mechanism. Here we demonstrate that the results of Wu et al. can be explained by an unusually high rate of alpha-factor-induced cell death in the [PSI+] strain (780-1D) used in their studies. What appeared to be no growth in their experiments was actually no increase in total cell number in a dividing culture through a counterbalancing level of cell death. Using media-exchange experiments, we provide further support for our original proposal that elimination of the [PSI+] prion by Gdn.HCl requires ongoing cell division and that prions are not destroyed during or after the evident curing phase.

Fig. 1.

Effect of arresting cell growth by α-factor on Gdn·HCl-mediated loss of the [PSI⁺] prion from two different [PSI⁺] strains: 780-1D (a and c) and YJW512 (b and d). At t = 0 h, 5 mM Gdn·HCl was added to all cultures. α-Factor to a concentration of 50 μM was added to one culture of each strain (○), and the other was left without (x). At various times samples were taken, diluted, and plated on ¼YEPD to count [PSI⁺] (white and red/white sectored) and [psi⁻] (wholly red) colonies. The percentage of viable cells that were [PSI⁺] is plotted against time in hours (a and b) and the number of CFUs (c and d). In a and b, the approximate time delay in the emergence of [psi⁻] cells caused by the presence of α-factor is indicated. In c and d, the points at which [psi⁻] cells were first seen in the respected cultures are indicated by arrows.

Fig. 2.

Inviability caused by the addition of α-factor to Gdn·HCl-treated cells of strains 780-1D and YJW512. (a) Viable cells as a percentage of total cells counted in YJW512 and 780-1D growing in YEPD plus 5 mM Gdn·HCl. ◇, YJW512 in 50 μM α-factor; ▵, 780-1D in 5 μM; □, 50 μM α-factor. (b) Staining of 780-1D after 10 h cultured in the presence of α-factor. Phyloxin B staining distinguishes live cells (white) from dead (dark). Three separate images of “schmoos” (i–iii) are shown.

Fig. 3.

Analysis of [PSI⁺] and [psi⁻] CFUs in the [PSI⁺] strain 780-1D cultured in the presence or absence of α-factor. (a) Total CFUs (○ and ●) and [PSI⁺] CFUs (□ and ■) were counted after dilution and plating on ¼YEPD; [PSI⁺] CFUs were those counted after plating on SC minus adenine. Filled symbols, growth in the absence of α-factor; open symbols, cultured in the presence of 50 μM α-factor. (b) Analysis of the total (◆), [PSI⁺] (○), and [psi⁻] (▵) CFUs from the culture growing in α-factor. Also plotted is the percentage [PSI⁺] cells in the culture (dashed line). The lines drawn are exponential trend lines for [PSI⁺] and [psi⁻] CFUs and a moving average for total CFUs. CFU values are all normalized to 100 at t₀. (c) A reconstruction of CFU numbers, estimated assuming release from α-factor arrest at 6 h and a lag of 9 h before the first appearance of [psi⁻] colonies among those plated on ¼YEPD. The values for the [PSI⁺] CFUs (○) were calculated by using a rate of inviability of 7% per h taken from the data in Fig. 2. The [psi⁻] CFU values (▵) were estimated from the growth curve of [psi⁻] CFUs in Fig. 2b (t_D = 1.7 h = 60% increase per h). Also shown are the numbers of [psi⁻] colonies (●), which would have been observed assuming that they derived instead from cured [PSI⁺] cells, expressed as a percentage of the total, requiring the assumption that the observed decline in numbers of [PSI⁺] cells is because of such curing. The earliest point on this curve was given an arbitrary value of 1. The curing curve (%[PSI⁺], x) was calculated from the estimated [PSI⁺] and [psi⁻] CFUs.

Fig. 4.

Consequences of removing or adding α-factor to a culture of the [PSI⁺] strain 780-1D growing in the presence of Gdn·HCl. (a and c) Here, 50 μM α-factor was added to a culture of 780-1D 10 h after the addition of 5 mM Gdn·HCl. (b and d) In a second experiment, both α-factor and 5 mM Gdn·HCl were added at t = 0, and then the cells were transferred to fresh medium lacking α-factor but still containing 5 mM Gdn·HCl after 10 h. In both experiments samples were taken and diluted and plated on ¼YEPD to count the [PSI⁺] (white and white/red sectored) and [psi⁻] (wholly red) colonies. Counts from cells with 50 μM α-factor (+α-F, ○) and without α-factor (−α-F, X) are shown. The proportion of [PSI⁺] CFUs is plotted in a and b, and the log of the total number of CFUs is shown in c and d. The time at which α-factor was added or removed is shown by an arrow in a and b. In a, the first appearance of [psi⁻] cells is indicated by a light arrow.

Fig. 5.

Elimination of [PSI⁺] by Gdn·HCl depends on growth and cell division. (a) The [PSI⁺] strain BSC783/4a was grown to log phase in SC plus 5 mM Gdn·HCl medium and after 4 h (○), 12 h (□), and 27.5 h (◇), an aliquot of the culture was removed, washed, and resuspended in SC lacking leucine (SC-leu), and incubation continued. In addition, an aliquot of the culture that was transferred to SC-leu at 4 h was supplemented with leucine (30 μg·ml⁻¹) after 12 h and allowed to continue to grow (■). Colony counts were taken at various time points, and these data were plotted against time in hours. (b) Percentage of the resulting colonies that were [PSI⁺].