Hsp104-Dependent Remodeling of Prion Complexes Mediates Protein-Only Inheritance

Inheritance of phenotypic traits depends on two key events: replication of the determinant of that trait and partitioning of these copies between mother and daughter cells. Although these processes are well understood for nucleic acid–based genes, the mechanisms by which protein-only or prion-based genetic elements direct phenotypic inheritance are poorly understood. Here, we report a process crucial for inheritance of the Saccharomyces cerevisiae prion [PSI(+)], a self-replicating conformer of the Sup35 protein. By tightly controlling expression of a Sup35-GFP fusion, we directly observe remodeling of existing Sup35([PSI+]) complexes in vivo. This dynamic change in Sup35([PSI+]) is lost when the molecular chaperone Hsp104, a factor essential for propagation of all yeast prions, is functionally impaired. The loss of Sup35([PSI+]) remodeling by Hsp104 decreases the mobility of these complexes in the cytosol, creates a segregation bias that limits their transmission to daughter cells, and consequently diminishes the efficiency of conversion of newly made Sup35 to the prion form. Our observations resolve several seemingly conflicting reports on the mechanism of Hsp104 action and point to a single Hsp104-dependent event in prion propagation

Hsp104 Inhibition Renders Sup35-GFP^{[<i>PSI+</i>]} Complexes Immobile

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

Propagon Numbers Correlate with Single-Cell Doubling Times

<p>Wild-type [<i>PSI⁺</i>] 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.</p

The In Vivo Prion Cycle Is a Multistep Pathway

<p>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).</p

Sup35-GFP^{[<i>PSI+</i>]} Complexes, Which Persist after Hsp104 Inhibition, Remain Substrates for This Disaggregase upon Its Reactivation

<p>Wild-type zygotes (SY876 × 74D-694) constitutively expressing <i>SUP35</i> and <i>GST(UGA)DsRED</i> with existing Sup35-GFP (previously expressed from P<i>_MFA1</i>) 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 (<i>n</i> = 9).</p

Efficient Incorporation of Sup35-GFP^{[<i>psi−</i>]} into Existing Sup35^{[<i>PSI+</i>]} Complexes Is Hsp104 Independent

<div><p>(A) <i>MAT<b>a</b></i> [<i>psi⁻</i>] haploids expressing untagged <i>SUP35</i> from its endogenous locus, <i>SUP35-GFP</i> (green) from P<i>_MFA1</i> and <i>GST(UGA)DsRED</i> from P<i>_GPD</i> were mated to <i>MATα</i> [<i>psi⁻</i>] haploids constitutively expressing untagged <i>SUP35</i> (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<i>_MFA1</i> is repressed upon mating [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050024#pbio-0050024-b043" target="_blank">43</a>], green fluorescence is derived from existing Sup35-GFP. Images of a representative zygote (<i>n</i> ≥ 5) from a wild-type cross (SY874 × 74D-694 <i>MATα;</i> left), from a cross in which the <i>MAT<b>a</b></i> [<i>psi⁻</i>] mating partner contained a <i>KTKT</i> chromosomal replacement (SY876 × 74D-694 <i>MATα;</i> middle) or from a wild-type cross (SY874 × 74D-694 <i>MATα</i>) in the presence of GdnHCl (right) are shown.</p> <p>(B) <i>MAT<b>a</b></i> [<i>psi⁻</i>] haploids constitutively expressing untagged <i>SUP35</i> and <i>GST(UGA)DsRED,</i> and <i>SUP35-GFP</i> (green) from P<i>_MFA1</i> were mated to <i>MATα</i> [<i>PSI⁺</i>] haploids constitutively expressing untagged <i>SUP35</i> (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 (<i>n</i> = 50). Images of representative zygotes from a wild-type cross (SY874 × 74D-694 <i>MATα;</i> left), from a cross in which the <i>MAT<b>a</b></i> [<i>psi⁻</i>] mating partner contained a <i>KTKT</i> chromosomal replacement (SY876 × 74D-694 <i>MATα;</i> middle), or from a wild-type cross (SY874 × 74D-694 <i>MATα</i>) in the presence of GdnHCl (right) are shown.</p> <p>(C) <i>MAT<b>a</b></i> [<i>PSI⁺</i>] P<i>_MFA1 SUP35-GFP</i> haploids were mated to <i>MATα</i> [<i>psi⁻</i>] haploids constitutively expressing <i>SUP35</i> (gray) in the presence or absence of Hsp104 function, as indicated in the diagram (top). Images of a representative zygote (<i>n</i> ≥ 13) from a wild-type cross (SY597 × SY582; left), from a cross in which the <i>MATα</i> [<i>psi⁻</i>] mating partner contained a <i>KTKT</i> 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).</p></div

Hsp104 Inhibition Blocks Sup35-GFP^{[<i>PSI+</i>]} Remodeling and Creates a Segregation Bias for Sup35-GFP Complexes

<div><p><i>MAT<b>a</b></i> [<i>psi⁻</i>] haploids expressing untagged <i>SUP35</i> and <i>SUP35-GFP</i> from P<i>_MFA1</i> were mated to <i>MATα</i> [<i>PSI⁺</i>] haploids constitutively expressing untagged <i>SUP35</i> in the presence or absence of functional Hsp104. For each cross, a zygote (left) and a microcolony derived from that zygote (right) are shown.</p> <p>(A) Representative images from a wild-type cross (SY360 × SY581) are shown (<i>n</i> = 10).</p> <p>(B) Shown are representative images (<i>n</i> = 5) from a <i>KTKT</i> × wild-type cross (SY876 × SY581). The originating zygote is outlined in the DIC image and marked with an arrow in the microcolony.</p> <p>(C) Shown are representative images (<i>n</i> = 6) of a wild-type cross (SY360 × SY581) in the presence of GdnHCl.</p> <p>(D) A model for Hsp104-dependent remodeling of prion complexes. Soluble Sup35-GFP^{[<i>psi−</i>]} (green ball and stick) is remodeled to the prion form (black and green ball and loop) upon encountering Sup35^{[<i>PSI+</i>]} in an Hsp104-independent process. These complexes are rapidly fragmented by Hsp104 (white hexamer) to smaller complexes, which then efficiently incorporate soluble Sup35^{[<i>psi−</i>]} (black ball and stick), effectively redistributing existing Sup35-GFP^{[<i>PSI+</i>]}.</p></div

Loss of Sup35-GFP^{[<i>PSI+</i>]} Fluorescence in Wild-Type Cells Corresponds to Redistribution of Existing Fusion Protein to New Prion Complexes

<div><p>(A) The metabolic stability of Sup35-GFP was analyzed in [<i>PSI⁺</i>] (SY81) or [<i>psi⁻</i>] (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.</p> <p>(B) Shown are representative images (<i>n</i> = 7) of a wild-type zygote (SY360 × SY581) constitutively expressing untagged <i>SUP35</i> 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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050024#pbio-0050024-g002" target="_blank">Figure 2</a>A.</p> <p>(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).</p></div

The NatA Acetyltransferase Couples Sup35 Prion Complexes to the [PSI+] Phenotype

Protein-only (prion) epigenetic elements confer unique phenotypes by adopting alternate conformations that specify new traits. Given the conformational flexibility of prion proteins, protein-only inheritance requires efficient self-replication of the underlying conformation. To explore the cellular regulation of conformational self-replication and its phenotypic effects, we analyzed genetic interactions between [PSI+], a prion form of the S. cerevisiae Sup35 protein (Sup35[PSI+]), and the three Nα-acetyltransferases, NatA, NatB, and NatC, which collectively modify ∼50% of yeast proteins. Although prion propagation proceeds normally in the absence of NatB or NatC, the [PSI+] phenotype is reversed in strains lacking NatA. Despite this change in phenotype, [PSI+] NatA mutants continue to propagate heritable Sup35[PSI+]. This uncoupling of protein state and phenotype does not arise through a decrease in the number or activity of prion templates (propagons) or through an increase in soluble Sup35. Rather, NatA null strains are specifically impaired in establishing the translation termination defect that normally accompanies Sup35 incorporation into prion complexes. The NatA effect cannot be explained by the modification of known components of the [PSI+] prion cycle including Sup35; thus, novel acetylated cellular factors must act to establish and maintain the tight link between Sup35[PSI+] complexes and their phenotypic effects