FIGURE 10:

The E3 ubiquitin ligase activity of Tom1 is not required for its role in protein aggregate prevention. (A) Western blots performed with polysome extracts prepared from tom1Δ, rqc1Δ, rqc1Δtom1Δ, and rqc1Δtom1Δltn1Δ mutant cells and separated on 10–50% sucrose gradients. The sedimentation profiles of ubiquitylated proteins in their aggregated form (stacking gel) were assessed using anti-ubiquitin antibodies. The 60S cosedimenting fractions were probed using anti-Nog1 antibodies. (B) Western blots were performed using total protein extracts and polysome extracts separated on 10–50% sucrose gradients (for which the light-sedimenting fractions [SC] and the 60S-sedimenting fractions were respectively pooled), prepared from rqc1Δ, rqc1Δtom1Δ, and rqc1Δtom1-C3235A mutant yeast cells expressing the TAP-NonStop reporter. The sedimentation profiles of the aggregated and soluble versions of the TAP-NonStop reporter were probed with PAP antibodies (middle), and the levels of aggregated polyubiquitylated proteins were assessed with anti-ubiquitin antibodies (top). Anti-G6PDH antibodies were used as a loading control (bottom).