Whi3 mnemon association with endoplasmic reticulum membranes confines the memory of deceptive courtship to the yeast mother cell

Abstract

Prion-like proteins are involved in many aspects of cellular physiology, including cellular memory. In response to deceptive courtship, budding yeast escapes pheromone-induced cell-cycle arrest through the coalescence of the G1/S inhibitor Whi3 into a dominant, inactive super-assembly. Whi3 is a mnemon (Whi3^mnem), a protein that conformational change maintains as a trait in the mother cell but is not inherited by the daughter cells. How the maintenance and asymmetric inheritance of Whi3^mnem are achieved is unknown. Here, we report that Whi3^mnem is closely associated with endoplasmic reticulum (ER) membranes and is retained in the mother cell by the lateral diffusion barriers present at the bud neck. Strikingly, barrier defects made Whi3^mnem propagate in a mitotically stable, prion-like manner. The amyloid-forming glutamine-rich domain of Whi3 was required for both mnemon and prion-like behaviors. Thus, we propose that Whi3^mnem is in a self-templating state, lending temporal maintenance of memory, whereas its association with the compartmentalized membranes of the ER prevents infectious propagation to the daughter cells. These results suggest that confined self-templating super-assembly is a powerful mechanism for the long-term encoding of information in a spatially defined manner. Yeast courtship may provide insights on how individual synapses become potentiated in neuronal memory.

Figure 1

Sup35 prion foci are not closely linked to ER membranes, whereas Whi3 super-assemblies are (A) Single focal plane images of [PSI⁺] cells expressing Sup35-GFP and Sec61-mCherry. (B) Percentage of Sup35 foci close to ER membranes (1,180 foci analyzed from 90 cells of three independent clones, mean ± SD is presented). (C) Western blot analysis of Whi3-TAP, an ER marker (Dpm1), and a soluble marker (Pgk1) in the microsome fraction (P, pellet) or the soluble fraction (S, supernatant). (D and E) (D) Single focal plane images of cells expressing Whi3-mNG and Sec61-mCherry exposed to pheromone for 3 h, (E) 4 h. (F and G) (F) Single focal plane images of cells expressing Whi3-mNG and Sec61-mCherry exposed to pheromone for 5 h, released in a pheromone-free medium, and then imaged at 6 h and (G) 10.5 h after initial exposure to pheromone. (H) Percentage of Whi3-mNG super-assemblies close to ER membranes (3 independent clones, >300 super-assemblies from >153 cells were analyzed, mean ± SD are presented). (I) Percentage of cells with at least one Whi3-mNG super-assembly away from ER membranes (3 independent clones, >300 super-assemblies from >153 cells were analyzed, mean ± SD are presented). Scale bars: 5 μm in (A) and (C–F).

Figure 2

Whi3-3GFP super-assemblies form in the buds of mutants with a weak diffusion barrier (A) Single focal plane images of Whi3-3GFP expressing cells. Scale bars, 5 μm. (B) Quantification of buds with a detectable Whi3-3GFP super-assembly (N = 122 cells, 148 cells, 180 cells, and 165 cells for WT, bud6Δ, sur2Δ, and bud1Δ cells, respectively). Results from bud6Δ, sur2Δ, and bud1Δ are significantly different from WT (p < 0.0001, one-way ANOVA).

Figure 3

The ER diffusion barrier prevents the daughter cells from inheriting the pheromone refractory state (A) Escape of a WT, a bud6Δ, a sur2Δ, and a bud1Δ cell exposed to 7 nM pheromone. Scale bars, 5 μm. (B) Percentage of initial cells still shmooing after the indicated time (N > 154 cells). (C and D) Percentage of the daughter cells budding immediately after separation from the mother cell (N > 128 cells, N > 121 cells, N > 111 cells, N > 88 cells, and N > 59 cells for the 1^st, 2^nd, 3^rd, 4^th, and 5^th daughter cells, respectively). Mean ± SD are presented. (E) Box plot of the timing of escape of the daughter cells of indicated genotypes that are shmooing (N > 81 cells). (F) Percentage of the 1^st daughter cells budding after a second pulse of pheromone (N > 27 cells). Mean ± SD are presented. (G) Localization of Whi3-GFP and Spc42(-RFP)-GBP. Scale bars, 5 μm. (H) Percentage of the daughter cells budding immediately after separation from the mother cell (N > 81 cells, N > 50 cells, N > 40 cells, N > 28 cells, and N > 19 cells for the 1^st, 2^nd, 3^rd, 4^th, and 5^th daughter, respectively). Mean ± SD are presented. See also Figure S1.

Figure 4

ER compartmentalization by a lateral membrane diffusion barrier is not required for prion induction and curing (A) Fluorescence intensity measured by flow cytometer of the [PSI⁺] WT and [PSI⁺] sur2Δ cells treated or not treated with 0.1 and 1 mM GuHCl. (B and C) (B) Frequency of de novo [PSI⁺] appearance and (C) percentage of cells cured of [PSI⁺] over time. Graphs (A) and (B) display mean ± SD. (D) Growth curves of WT and sur2Δ strains assessed by OD_600nm.

Figure 5

A novel epigenetic phenotype, constitutive escapers (A) Serial 1/10 dilutions of exponentially growing cultures of indicated strains spotted on a YPD solid medium (left) or YPD containing pheromone (10 nM middle and 600 nM right). The bottom panel shows another bud6Δ independent clone with a high frequency of CE. (B) Frequency of appearance of CE in the indicated genotypes (median with 95% confidence interval, N > 37 clones for each, Dunn’s multiple comparisons test was used to determine significance). (C) Distribution of the logit transformed ratios (R) for each genotype. (D) Cell-size distributions of individual clones or CE isolates of indicated strains (N > 39 clones for each). (E) Maximal projection images of the sur2Δ cells expressing Whi3-GFP from 20 CE isolates and the parental strain. Isolates are in the order of the mean cell size from the smallest (orange-framed are smaller than the blue-framed parental strain) to the largest (purple are larger than the parental strain). Scale bars, 5 μm. (F) Number of Whi3-GFP bright foci per cell in the 20 CE isolates and the parental strain as a function of cell size. (G) Single focal plane images of bud6Δ cells (top) and CE^bud6Δ cells (bottom) expressing Whi3-mNG and Sec61-mCherry. Scale bars, 5 μm. (H) Percentage of Whi3-mNG granules (bud6Δ) and foci (CE^bud6Δ) localizing close to ER membranes (4 clones or isolates, 527 granules, and 359 foci in 200 cells were analyzed, mean ± SD are presented). The arrow points at a focus away from the ER and the arrowhead points at focus close to the ER. (I) Frequency of appearance of CE in the indicated genotypes (median with 95% confidence interval, N > 33 clones for each, Mann-Whitney test was used to determine significance). See also Figures S2, S3, and S7 and Methods S1.

Figure 6

The polyQ domain of Whi3 has a self-templating activity (A) Sequence of the Whi3^polyQ peptide and fibers observed after 7 days of growth. (B) Sequence of the B2M^K3 peptide and fibers observed after 7 days of growth. Scale bars: 200 nm for 25 kx magnification and 50 nm for 100 kx magnification. (C) The fibrillar growth, monitored by bisANS, of B2M^K3 (green) and Whi3^polyQ (orange) fibers is shown. (D) BisANS fluorescence monitoring of fibrillar growth for Whi3^polyQ without (orange), or with B2M^K3 seeds (green) and Whi3^polyQ seeds at two different concentrations (light and dark blue for 10 and 100 μM, respectively). (E) Number of colonies growing on YPD + pheromone (0.6 μM) after transformation with either the native Whi3^polyQ peptide or sonicated fibers of Whi3^polyQ. Mean ± SD are presented. ∗∗ denotes a p value < 0.01 using an unpaired t test. (F) Number of colonies growing on YPD + pheromone (0.6 μM) after transformation with a crude lysate from sur2Δ cells ± pheromone treatment for 5 h or CE^sur2Δ cells.Mean ± SD are presented. p values: ∗ < 0.05, ∗∗∗∗ < 0.0001 using an ANOVA. See also Figures S4, S5, and S6 and Tables S1, S2, S3, S4, S5, and S6.

Figure 7

Schematic of Whi3 behavior in different conditions (±pheromone) and genotypes (±diffusion barrier)