Polyglutamine toxicity and aggregation in the yeast strains with various prion compositions.

<p>A – Polyglutamine constructs used in this work. All constructs were under the control of the galactose-inducible promoter (<i>P_GAL</i>), and contained the FLAG epitope, N-terminal 17 amino acid residues and poly-Q stretch of human Htt, and were fused to the gene coding for green fluorescent protein (GFP) at C-terminus. Numbers indicate length of poly-Q stretch. Poly-QP constructs also contained the proline-rich region of Htt (designated as P), immediately following the poly-Q stretch. B – Expanded poly-Q without a P-rich region (103Q), expressed under the <i>P_GAL</i> promoter on -Ura/Gal medium, is toxic in the presence of either [<i>PIN</i>⁺] or [<i>PSI</i>⁺] (or both), with two prions showing an additive effect. In contrast, expanded poly-Q with a P-rich region (103QP) is toxic only in the presence of [<i>PSI</i>⁺]. The 25Q construct, not exhibiting toxicity under these conditions, is shown as a control. The 25QP construct (not shown) behaved in the same way as 25Q. C – 103Q and 103QP form multiple peripheral aggregates and single aggregate (aggresome), respectively, in cells containing either or both prions ([<i>PIN⁺</i>] and/or [<i>PSI⁺</i>]), as visualized by fluorescence microscopy. Perinuclear location of aggresome (not shown) was confirmed by DAPI staining as described previously <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002634#pgen.1002634-Wang1" target="_blank">[42]</a>. D – Overexpressed Sup35NM-DsRed (red) forms large clumps in the [<i>PSI⁺</i>] cells, that overlap with the 103QP-GFP aggresome (green), as pointed by arrows. E - Expression of 103Q or 103QP promotes aggregation of Sup35 in the [<i>psi⁻</i>] strain as seen by an increase of pellet (P) versus supernatant (S) fraction, in comparison to the respective strain expressing 25Q. Centrifugation analysis was followed by Western blotting and immunostaining with the Sup35 antibody. F - Expression of the Sup35 derivative, lacking the prion and middle domains (Sup35C), decreases 103Q and 103QP toxicity in the [<i>PIN⁺ PSI⁺</i>] strain but does not influence 103Q toxicity in the [<i>PIN⁺ psi⁻</i>] strain. <i>SUP35C</i> gene was under control of the endogenous <i>SUP35</i> promoter. Serial decimal dilutions were spotted onto -Ura/Gal medium.