FIGURE 6.

Aggregated aberrant proteins accumulating in the absence of Rqc1 are polyubiquitinated in an Ltn1-dependent manner. Polysome extracts prepared from wild-type, rqc1Δ, ltn1Δ, and ltn1Δrqc1Δ cells were separated on a sucrose gradient (10–50%). Fractions of the gradient were TCA-precipitated, separated on a 10% acrylamide gel, and analyzed by Western blotting using monoclonal antibodies against ubiquitin (P4D1 from Covance) (1/1,000) to visualize the ubiquitinated forms of soluble (running gel) and aggregated (stacking gel) endogenous proteins. Aggregates containing polyubiquitinated proteins and accumulating in stacking gels are indicated. Ub, ubiquitin.