Figure 1. Diverse Hsp104 homologs suppress TDP-43 toxicity in yeast.

(A) Phylogenetic tree constructed using EMBL-EBI Simple Phylogeny tool from a multiple sequence alignment of the indicated Hsp104 homologs generated in Clustal Omega (see also Supplemental Information for alignments) showing evolutionary relationships between Hsp104 homologs studied in this paper. C. thermophila is in green, TDP-43-specific homologs are colored in shades of blue, αSyn-specific homologs are colored in red, and non-rescuing homologs are colored in shades of gray. (B) Δhsp104 yeast transformed with plasmids encoding galactose-inducible TDP-43 and the indicated galactose-inducible Hsp104 (either wild-type Hsp104 from Saccharomyces cerevisiae, the potentiated variant A503S, or the Hsp104 homolog from Calcarisporiella thermophila (Ct)) were serially diluted 5-fold and spotted onto glucose (expression off) or galactose (expression on). (C) Δhsp104 yeast transformed with plasmids encoding galactose-inducible TDP-43 and the indicated galactose-inducible Hsp104 (either wild-type Hsp104 from S. cerevisiae, the potentiated variant A503S, or homologs from Galdieria sulphuraria (Gs), Monosiga brevicollis (Mb), Chlamydomonas reinhardtii (Cr), Populus euphratica (Pe), and Salpingoeca rosetta (Sr)) were serially diluted 5-fold and spotted onto glucose (expression off) or galactose (expression on). (D) Western blots confirm consistent expression of FLAG-tagged Hsp104s and proteotoxic protein substrates in yeast, and that neither Hsp104 expression nor TDP-43 expression induces upregulation of the endogenous heat-shock protein Hsp26. The first lane are isogenic yeast that have not been grown in galactose to induce Hsp104 and TDP-43 expression but instead have been pretreated at 37°C for 30 min to upregulate endogenous heat-shock proteins. 3-phosphoglycerate kinase (PGK) is used as a loading control. Molecular weight markers are indicated (right). (E) Expression of the indicated Hsp104-FLAG relative to PGK was quantified for each strain. Values are means ± SEM (n = 3). One-way ANOVA with Dunnett’s multiple comparisons test was used to compare expression of ScHsp104-FLAG (Sc) to all other conditions. *p<0.05; NS, not significant. (F) TDP-43 expression relative to PGK was quantified for each strain. Values are means ± SEM (n = 3). One-way ANOVA with Dunnett’s multiple comparisons test was used to compare TDP-43 levels in the ∆hsp104 control to all other conditions. NS, not significant. (G) Representative images of yeast co-expressing TDP-43-GFPS11 (and separately GFPS1-10 to promote GFP reassembly) and the indicated Hsp104 homologs. Cells were stained with DAPI to visualize nuclei (blue). Scale bar, 2.5 µm. (H) Quantification of cells where TDP-43 displays nuclear localization. Values represent means ± SEM (n = 3 trials with >200 cells counted per trial). One-way ANOVA with Dunnett’s multiple comparisons test was used to compare Δhsp104 to all other conditions. ****p<0.0001. (I) Quantification of cells with no, single, or multiple TDP-43 foci. Values represent means ± SEM (n = 3). Two-way ANOVA with Tukey’s multiple comparisons test was used to compare the proportion of cells with either no or multiple TDP-43 foci between strains expressing different Hsp104 homologs and a control strain expressing ScHsp104. *p<0.05; **p<0.01; ***p<0.001.

Figure 1—figure supplement 1. Alignment of all Hsp104 homologs investigated in this study.

Amino acid sequences of Hsp104 homologs were aligned using Clustal Omega, and the multiple sequence alignment was visualized with JalView. Positions in the alignment are colored by Clustal X convention: conserved hydrophobic positions (A,I,L,M,F,W,V,C) are blue; conserved basic residues (K,R) are red; acidic residues (E,D) are magenta; polar residues (N,Q,S,T) are green; aromatic residues (H,Y) are cyan; conserved cysteines are pink; glycines are orange, prolines are yellow. Structural elements of Hsp104 are indicated. C. thermophila and ClpG are in green, TDP-43-specific homologs are colored in shades of blue, αSyn-specific homologs are colored in red, and non-rescuing homologs are colored in shades of gray.

Figure 1—figure supplement 2. Hsp104 homologs do not typically induce temperature-dependent toxicity.

Spotting assay showing that yeast strains expressing the indicated Hsp104 homolog do not show a temperature-dependent growth defect. None of the Hsp104 homologs were toxic at 30 or 37°C with the exception of PfHsp104, which was toxic at both temperatures. PfHsp104 was even more toxic than the potentiated Hsp104 variant, Hsp104^A503V, at 37°C. Unlike PfHsp104, Hsp104^A503V was not toxic at 30°C.

Figure 1—figure supplement 3. Suppression of TDP-43 or αSyn toxicity by select Hsp104 homologs is a substrate-specific effect.

(A) Spotting assay showing that MbHsp104, SrHsp104, CrHsp104, PeHsp104, and GsHsp104, which all suppress TDP-43 toxicity, have minimal effect on αSyn or FUS toxicity. ScHsp104^A503S is included as a positive control and empty vector and ScHsp104^WT are included as negative controls. (B) Spotting assay showing that TtHsp104 and TlHsp104 have minimal effect on TDP-43 or FUS toxicity. (C) Spotting assay showing that other Hsp104 homologs investigated in this study have minimal effect on TDP-43 (left), αSyn (middle), or FUS (right) toxicity.

Figure 1—figure supplement 4. Alignment comparing ScHsp104 to Hsp104 homologs that rescue TDP-43 toxicity.

Amino acid sequences of the indicated Hsp104 homologs were aligned using Clustal Omega, and the multiple sequence alignment was visualized with JalView. Positions in the alignment are colored by Clustal X convention: conserved hydrophobic positions (A,I,L,M,F,W,V,C) are blue; conserved basic residues (K,R) are red; acidic residues (E,D) are magenta; polar residues (N,Q,S,T) are green; aromatic residues (H,Y) are cyan; conserved cysteines are pink; glycines are orange, prolines are yellow. Structural elements of Hsp104 are indicated.