Figure 3.
TDP-43 toxicity, aggregation, and toxicity depend on the E3 ubiquitin ligase Rsp5. (A) Representative serial dilution growth assay of WT and rsp5-3 strains transformed with the empty vector (pRB43) or untagged TDP-43 plasmid (pRB232) grown at 25°C or 30°C as indicated. N = 3. (B) WT and rsp5-3 strains were transformed with TDP-43-mRuby2 plasmid and grown to mid-logarithmic growth phase at 25°C, and samples were imaged. Cultures were then incubated at 35°C for 16 h and imaged again. **, P < 0.01 by one way analysis of variance with Tukey’s post hoc test; N = 3. Scale bar, 5 µm. (C) WT and rsp5-3 strains were transformed with TDP-43-mRuby2 plasmid and grown to mid-logarithmic growth phase at 25°C before being treated with 0.2 mg/ml cycloheximide for 0, 6, 12, and 24 h. At time 0, cultures were either shifted to 35°C or kept at 25°C for the remainder of the time course. TDP-43-mRuby2 levels were detected by western blot using anti–TDP-43 antibody and normalized to GAPDH loading control. **, P < 0.01; ***, P < 0.001 by one-way analysis of variance with Tukey’s post hoc test; N = 3. (D) Strains endogenously expressing GFP-Rsp5, GFP-Rim8, GFP-Ssh4, and GFP-Ear1 were transformed with a TDP-43-mRuby2 plasmid (pRB194) and imaged at mid-log phase growth. Percentage values indicate colocalization frequency of TDP-43-mRuby2 foci with GFP foci. N = 3; scale bar, 5 µm. (E and F) Representative serial dilution growth assay of WT strain transformed with an empty vector control (pRB43) or untagged TDP-43 plasmid (pRB232) and with the empty vector control (pRB511) or Rsp5 overexpression plasmid (pRB476). N = 3 (F) WT, vps4Δ, and vps28Δ strains were transformed with TDP-43-YFP (pRB109) and also transformed with Rsp5 overexpression (pRB476) or empty vector control plasmid (pRB511) and grown to mid-logarithmic growth phase. TDP-43-YFP (pRB109) was detected by western blot using anti–TDP-43 antibody. TDP-43 levels were normalized to GAPDH loading control. **, P < 0.01 by Welch’s two-tailed T test; N = 3. Error bars in all graphical panels represent standard error of the mean. Source data are available for this figure: SourceData F3.

TDP-43 toxicity, aggregation, and toxicity depend on the E3 ubiquitin ligase Rsp5. (A) Representative serial dilution growth assay of WT and rsp5-3 strains transformed with the empty vector (pRB43) or untagged TDP-43 plasmid (pRB232) grown at 25°C or 30°C as indicated. N = 3. (B) WT and rsp5-3 strains were transformed with TDP-43-mRuby2 plasmid and grown to mid-logarithmic growth phase at 25°C, and samples were imaged. Cultures were then incubated at 35°C for 16 h and imaged again. **, P < 0.01 by one way analysis of variance with Tukey’s post hoc test; N = 3. Scale bar, 5 µm. (C) WT and rsp5-3 strains were transformed with TDP-43-mRuby2 plasmid and grown to mid-logarithmic growth phase at 25°C before being treated with 0.2 mg/ml cycloheximide for 0, 6, 12, and 24 h. At time 0, cultures were either shifted to 35°C or kept at 25°C for the remainder of the time course. TDP-43-mRuby2 levels were detected by western blot using anti–TDP-43 antibody and normalized to GAPDH loading control. **, P < 0.01; ***, P < 0.001 by one-way analysis of variance with Tukey’s post hoc test; N = 3. (D) Strains endogenously expressing GFP-Rsp5, GFP-Rim8, GFP-Ssh4, and GFP-Ear1 were transformed with a TDP-43-mRuby2 plasmid (pRB194) and imaged at mid-log phase growth. Percentage values indicate colocalization frequency of TDP-43-mRuby2 foci with GFP foci. N = 3; scale bar, 5 µm. (E and F) Representative serial dilution growth assay of WT strain transformed with an empty vector control (pRB43) or untagged TDP-43 plasmid (pRB232) and with the empty vector control (pRB511) or Rsp5 overexpression plasmid (pRB476). N = 3 (F) WT, vps4Δ, and vps28Δ strains were transformed with TDP-43-YFP (pRB109) and also transformed with Rsp5 overexpression (pRB476) or empty vector control plasmid (pRB511) and grown to mid-logarithmic growth phase. TDP-43-YFP (pRB109) was detected by western blot using anti–TDP-43 antibody. TDP-43 levels were normalized to GAPDH loading control. **, P < 0.01 by Welch’s two-tailed T test; N = 3. Error bars in all graphical panels represent standard error of the mean. Source data are available for this figure: SourceData F3.

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