Existing prion complexes (black ball and loop) replicate by stimulating the conversion of either newly synthesized or non-prion conformer of the protein (gray ball and stick) to the prion form (black and gray ball and loop, step 1). Prion complexes must be stably maintained (step 2), but continually divided to generate new prion templates for additional rounds of protein-state replication (step 3). The smaller complexes generated by this division are efficiently transmitted to daughter cells (step 4).

(A) MATa [psi⁻] haploids expressing untagged SUP35 from its endogenous locus, SUP35-GFP (green) from P_MFA1 and GST(UGA)DsRED from P_GPD were mated to MATα [psi⁻] haploids constitutively expressing untagged SUP35 (gray) in the presence or absence of functional Hsp104 as shown in the schematic diagram (top). The Sup35-GFP and DsRed fluorescence patterns were then examined in isolated zygotes. Since P_MFA1 is repressed upon mating [43], green fluorescence is derived from existing Sup35-GFP. Images of a representative zygote (n ≥ 5) from a wild-type cross (SY874 × 74D-694 MATα; left), from a cross in which the MATa [psi⁻] mating partner contained a KTKT chromosomal replacement (SY876 × 74D-694 MATα; middle) or from a wild-type cross (SY874 × 74D-694 MATα) in the presence of GdnHCl (right) are shown.
(B) MATa [psi⁻] haploids constitutively expressing untagged SUP35 and GST(UGA)DsRED, and SUP35-GFP (green) from P_MFA1 were mated to MATα [PSI⁺] haploids constitutively expressing untagged SUP35 (gray dots) in the presence or absence of functional Hsp104 as shown in the diagram (top). Sup35-GFP and DsRed fluorescence were examined in isolated zygotes (n = 50). Images of representative zygotes from a wild-type cross (SY874 × 74D-694 MATα; left), from a cross in which the MATa [psi⁻] mating partner contained a KTKT chromosomal replacement (SY876 × 74D-694 MATα; middle), or from a wild-type cross (SY874 × 74D-694 MATα) in the presence of GdnHCl (right) are shown.
(C) MATa [PSI⁺] P_MFA1 SUP35-GFP haploids were mated to MATα [psi⁻] haploids constitutively expressing SUP35 (gray) in the presence or absence of Hsp104 function, as indicated in the diagram (top). Images of a representative zygote (n ≥ 13) from a wild-type cross (SY597 × SY582; left), from a cross in which the MATα [psi⁻] mating partner contained a KTKT chromosomal replacement (SY597 × SY776 middle), or from a wild-type cross in the presence of GdnHCl (SY597 × SY582; right) are shown. The formation of zygotes was confirmed by sporulation (unpublished data).

(A) FRAP time courses for Sup35-GFP^[PSI+] in wild-type (SY360 × SY581; black circles) and in +/KTKT (SY893 × SY581; white squares) zygotes are shown (n = 6). The recovery half-time for the wild-type zygote is 0.92 s (R² = 0.85).
(B) FRAP time courses for Sup35-GFP^[psi−] in wild-type (SY360 × SY582; black circles, n = 5) and in +/KTKT (SY893 × SY582; white squares, n = 6) zygotes are shown. The recovery half-time for the wild-type zygote is 0.47 s (R² = 0.85) and for the +/KTKT zygote is 0.7 s (R² = 0.89). The black arrow marks the bleach point. Error bars represent standard error of the mean.

MATa [psi⁻] haploids expressing untagged SUP35 and SUP35-GFP from P_MFA1 were mated to MATα [PSI⁺] haploids constitutively expressing untagged SUP35 in the presence or absence of functional Hsp104. For each cross, a zygote (left) and a microcolony derived from that zygote (right) are shown.
(A) Representative images from a wild-type cross (SY360 × SY581) are shown (n = 10).
(B) Shown are representative images (n = 5) from a KTKT × wild-type cross (SY876 × SY581). The originating zygote is outlined in the DIC image and marked with an arrow in the microcolony.
(C) Shown are representative images (n = 6) of a wild-type cross (SY360 × SY581) in the presence of GdnHCl.
(D) A model for Hsp104-dependent remodeling of prion complexes. Soluble Sup35-GFP^[psi−] (green ball and stick) is remodeled to the prion form (black and green ball and loop) upon encountering Sup35^[PSI+] in an Hsp104-independent process. These complexes are rapidly fragmented by Hsp104 (white hexamer) to smaller complexes, which then efficiently incorporate soluble Sup35^[psi−] (black ball and stick), effectively redistributing existing Sup35-GFP^[PSI+].

(A) The metabolic stability of Sup35-GFP was analyzed in [PSI⁺] (SY81) or [psi⁻] (SY87) yeast lysates upon inhibition of new protein synthesis with cycloheximide. An anti-Sup35 immunoblot is shown along with an anti-Pgk1 immunoblot of the same membrane as a control for loading.
(B) Shown are representative images (n = 7) of a wild-type zygote (SY360 × SY581) constitutively expressing untagged SUP35 from its endogenous locus (left image) and of the same zygote following the formation of a second daughter and first granddaughter (right image). The Sup35-GFP in these cells is that existing at the time of mating as described in Figure 2A.
(C) FRAP recovery curves for a wild-type zygote (SY360 × SY581; black circles) and its daughter (white circles), as described in (B), are shown. The respective recovery times are 0.92 s (R² = 0.85) and 0.95 s (R² = 0.84).

(A) A MATa [psi⁻] haploid with KTKT and SUP35GFP chromosomal replacements (SY751) was mated to a MATα [PSI⁺] haploid constitutively expressing SUP35 (SY581) as shown in the diagram (top). Representative images of a zygote and microcolony derived from this zygote are shown (n = 7).
(B) A MATa [psi⁻] haploid with a SUP35GFP chromosomal replacement (SY87) was mated to a MATα [PSI⁺] haploid constitutively expressing SUP35 (SY581) in the presence of GdnHCl as shown in the diagram. Representative images of a zygote and microcolony derived from this zygote are shown (n = 7). Under these conditions, the isolated zygote (left, [A] and [B]) continues to express Sup35-GFP, but lacks functional Hsp104.
(C) FRAP recovery curves for [psi⁻] (SY360, white squares), [PSI⁺] (SY597, black circles) and [PSI⁺] cells after treatment with GdnHCl for 16–24 h (SY597, gray circles), which all constitutively express Sup35-GFP, are shown. The respective recovery times are: 0.33 s (R² = 0.93), 1.16 s (R² = 0.95), and 0.39 s (R² = 0.79).

Wild-type zygotes (SY876 × 74D-694) constitutively expressing SUP35 and GST(UGA)DsRED with existing Sup35-GFP (previously expressed from P_MFA1) were isolated in the presence of GdnHCl and allowed to form microcolonies. As indicated in the schematic diagram, a portion of this colony was retained on medium containing GdnHCl (0 h, left), and the remainder of the colony was transferred to medium lacking GdnHCl (0 h, right). The cells were imaged again 4 h after the transfer. Representative DIC, GFP, and DsRed images are shown (n = 9).

Wild-type [PSI⁺] haploids (5V-H19) were analyzed for single-cell doubling time in the presence of GdnHCl and propagon numbers. The percentage of cells containing more than 1,000 (black), between 1,000 and 500 (gray), and fewer than 500 (white) propagons is indicated.