Overexpression of the essential Sis1 chaperone reduces TDP-43 effects on toxicity and proteolysis
Fig 4
TDP-43 overexpression inhibits degradation of a UPS-reporter and co-expression of Sis1 restores that degradation.
Degradation of CG*, the mutant version of the secretory protein carboxypeptidase Y lacking its signal sequence tagged with EGFP (ΔssCPY*) was used to report functionality of the ubiquitin proteasome system (UPS). Levels of CG* were determined in cell lysates [103] harvested at 0, 30, 90 and 180 minutes following inhibition of new protein synthesis with cycloheximide. [pin-] (L2910) or [PIN+] (L1749) cells expressing GAL1 regulated CG*-EGFP (p2154) and either control empty vectors (p2039, p1768) “v / v”; TDP-43 (p2055) and control “TDP-43 / v”; or TDP-43 and Sis1 (p1767) “TDP-43 / Sis1”, were grown in liquid SR-Trp-Ura-His to OD 600 ≥ 0.7. Galactose to 2% was added and cells were grown for 24 h to induce TDP-43, Sis1 and CG* expression. Protein synthesis was then inhibited with 0.75 ([pin-]) or 0.5 ([PIN+]) μg/ml cycloheximide for the times indicated. (A) A representative Western blot. Normalized cell lysates were run on SDS-PAGE followed by immunoblotting with anti-GFP (1:5,000, Roche) for CG*-EGFP level and anti-Pgk1 (1:10,000, Novex) as an internal loading control. (B) Quantification of three blots showing normalized ratio of CG*-EGFP and Pgk1. Standard error for three blots is shown.