The NTD of Hsp42 mediates sorting of aggregated proteins to peripheral inclusions. (A) Domain organization of Hsp26, Hsp42, and their variants. Both sHSPs consist of an NTD, a conserved α-crystallin domain (alpha), and a CTE. All constructs were C-terminally fused to a FLAG tag (F). Domain boundaries are indicated by residue numbers. All constructs were placed under control of the native HSP42 promoter and integrated at the Hsp42 locus in hsp42Δ cells. (B) hsp42Δ cells expressing mCherry-VHL and the indicated sHSP constructs were grown at 30°C and shifted to 37°C (+MG132). mCherry-VHL localization (green) was determined at the indicated time points. Nuclei were visualized by coexpressing HTB1-cerulean (red). The dashed lines indicate the border of respective yeast cells. Bars, 1 µm. (C) Number (shaded bars) and localization (colored bars) of mCherry-VHL inclusions in hsp42Δ cells expressing the indicated sHSP construct after incubation at 37°C for 30 and 180 min. MG132 was added before the temperature shift. The total number of foci per cell is shown on the x axis in all diagrams. Quantifications are based on the analysis of 40–130 cells and are representatives of two experimental repeats.