Fig. 5.

Bag3 and LATS1 regulate protein aggregation in response to proteasome inhibition. (A) Depletion of Bag3 reduces and depletion of LATS1 increases the association of Syn-GFP and ubiquitinated species with the particulate fraction. MCF10A cells stably expressing Syn-GFP with the indicated depletions were treated for 2 h with 2.5 μM MG132 with or without 0.5 μM emetine. The preclarified lysates were subjected to 15-min centrifugation at 16,000 × g. The pellets were washed once with a lysis buffer and then were dissolved in Laemmli sample buffer for immunoblotting with the antibodies indicated on the left. Note that both bands developed with anti-GFP antibody are specific. (B) The effects of incubation with 1 μM MG132 on the distribution of cluster sizes in naive and Bag3-depleted cells. Survival plots delineate a fraction of clusters containing at least N (indicated on the x axis) localization counts. Each plot was generated from 10 cells and represents the statistical distribution of 9,000–10,000 identified aggregates. (C) Survival plot showing the effects of LATS1 depletion on the distribution of cluster sizes in naive cells. Each plot was generated from 10 cells and represents the statistical distribution of 4,000 (LATS1) or 7,000 (control) identified aggregates. (D) A model for DRiPs sensing by the HB complex. Blue indicates elements of the pathway regulating the aggregation response. Red indicates changes upon proteasome inhibition; under these conditions the HB complex suppresses LATS1/2, thus removing its inhibition of protein aggregation.