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>