Figure 5. Rnq1 amyloidogenic regions differentially modulate interactions with Sis1.

(A) Prediction of protein interaction sites in Rnq1 using the ANCHOR algorithm (http://anchor.enzim.hu/). Denoted at the top are the amyloidogenic regions A1–A11. (B) Co-immunoprecipitation of Sis1 and Rnq1(132-405). Sis1 was immunoprecipitated using an αSis1 antibody (gift from E. Craig) from cell lysates expressing Rnq1(132-405) in place of WT Rnq1, followed by SDS-PAGE and immunoblotting with αRnq1 and αSis1 antibodies. The input of the total cell lysate (I) represents 10% of the sample that was bound by the αSis1 antibody (B). The bound fraction from the same sample not incubated with antibody (N) was used as a control. (C) Rnq1-L94A differentially affects [RNQ+] variant propagation and is rescued by Sis1 over-expression. Cell lysates with (+) or without (−) over-expression (OE) of Sis1 and propagating the indicated [RNQ+] variant with WT Rnq1 or Rnq1-L94A were subjected to SDD-AGE and western blot using an αRnq1 antibody. (D–F) Elimination of [RNQ+] by mutation of Rnq1 amyloidogenic regions is differentially rescued by Sis1 over-expression. As in (C) for (D) cells propagating s.d. medium [RNQ+] or m.d. high [RNQ+] and expressing WT Rnq1 or the A3 mutant, or (E) cells propagating s.d. very high [RNQ+] expressing WT Rnq1 or mutants A1–A5 or A9–A11. (F) Yeast lysates propagating s.d. very high [RNQ+], over-expressing Sis1, and expressing WT Rnq1, or mutants A1–A5 or A9–A11 were incubated for five minutes at 100°C (+) or at room temperature (−), followed by SDS-PAGE and western blot using an αRnq1 antibody. Data are representative of at least three independent experiments.