Figure S2.

AA of Sis1 does not trigger proteostasis collapse. (A) Left: Cells expressing Hsp104-mKate and Sis1-AA-GFP in untreated cells and following the addition of rapamycin (rapa) for 1 h to AA Sis1-AA-GFP. The two dark regions in the rapa-treated cells are the nucleus and the vacuole. Right: Cells expressing Hsp104-mKate were imaged by spinning disc confocal microscopy under non–heat shock (NHS) condition and following 15 min of heat shock (HS) at 39°C. Scale bar is 2 µm. (B) Quantification of the number of Hsp104-mKate clusters in individual cells either left untreated, following heat shock, or following Sis1 AA with rapa. Individual cells are shown, and the total number is indicated in each condition. Lines represent means, and error bars show SEM. Statistical significance was determined using a two-tailed t test without assuming equal variance (****, P < 0.0001). (C) “Total-sup-pellet” assay of Sis1-AA cells either treated with rapa or heat shocked over a 60-min time course. At each time point, cells were harvested, lysed, and fractionated to resolve the soluble (sup.) and aggregate (pellet) protein fractions. Each fraction was blotted and probed with anti-Ssa1/2 antibodies to detect Hsp70. (D) Quantification of the sup. Hsp70 fraction in each sample compared with the untreated sample. The experiment was performed in triplicate, and the error bars represent the 95% confidence bounds on the mean.