Fig. 2: Nuclear targeting of Hsp104 depends on a motif in its flexible C-terminal tail.

a Micrographs of strains (BY4741, S288c, W303, and BY4741 from the GFP Clone Collection) endogenously expressing Hsp104^GFP at 48 h. b Quantification of nuclear accumulation of Hsp104^GFP in cells described in (a). Dot plots with mean (square) and median (line). Each dot represents one biological replicate (Ctrl.: 124 cells; S288c: 166 cells; W303: 94 cells; GFP Clone Collection: 101 cells). c Hsp104 sequences and generated mutants (Hsp104* and Hsp104^∆C). N-terminal domain (NTD); Nucleotide-binding domain 1 or 2 (NBD1/2); Middle domain (MD); C-terminal domain (CTD). d, e Micrographs (d) and corresponding quantification of nuclear accumulation (e) of cells harboring endogenously GFP-tagged Hsp104, Hsp104* or Hsp104^∆C at 48 h. Dot plots with mean (square) and median (line). Each dot represents one biological replicate (Hsp104: 105 cells; Hsp104*: 118 cells; Hsp104^∆C: 122 cells). f Immunoblot and quantification of Hsp104^GFP levels of strains harboring GFP-chimeras of wild type and mutant Hsp104. Protein levels were normalized to 6 h wild type Hsp104. Line graph with mean ± s.e.m., n = 3 biological replicates. g, h Micrographs of cells harboring endogenously GFP-tagged Hsp104 or Hsp104* and Nup133^mCherry. GFP signal intensity at 8 h was adapted to ensure visibility. h Quantification of the GFP intensity ratio between nucleus and cytoplasm of cells described in (g). Violin plots with mean (square) and median (line), each dot represents one cell (8 h Hsp104: 166 cells; 8 h Hsp104*: 204 cells; 24 h Hsp104: 166 cells; 24 h Hsp104* 124 cells; 48 h Hsp104: 105 cells; 48 h Hsp104* 118 cells). i Micrographs of cells expressing GFP fused to Hsp104_aa901-908 controlled by the TET promoter. Signal intensity at 8 h was increased to ensure visibility. j Quantification of the GFP intensity ratio between nucleus and cytoplasm of cells described in (i). Violin plots with mean (square) and median (line), each dot represents one cell (Hsp104_aa901-908: 157 cells; Hsp104*_aa901-908: 150 cells). Scale bars: 2 µm. ***p < 0.001. Source data are provided as a Source Data file. See Supplementary Table 3 for details on statistical analyses.