Extended Data Figure 1: Aggregate purification and role of mitochondrial import in aggregate dissolution.
From: Cytosolic proteostasis through importing of misfolded proteins into mitochondria
a, Schematics of aggregate purification. Yeast strains expressing FlucSM–GFP–3×Flag or FlucSM–GFP as control were treated with heat shock at 42 °C to induce aggregate formation (green dots). Sucrose gradient centrifugation was then used to separate FlucSM–GFP monomers from aggregates in the cell lysates. The fraction enriched for protein aggregates was applied to an anti-Flag column to separate FlucSM–GFP–3×Flag aggregates from other cellular debris (grey shapes). b, Representative images (n = 8) of anti-flag resin and associated aggregates isolated from FlucSM–GFP (left) or FlucSM–GFP–3×Flag (right) strains. c, Total non-redundant peptides identified by MudPIT of independent repeats using FlucSM–GFP–3×Flag (blue) and FlucSM–GFP (red) strains. d, Representative images of examples of proteins identified by MudPIT that form aggregates after 30 min heat shock. Not3 is a negative control. Three images for each. e, Tom70–GFP did not colocalize with aggregates after 30 min heat shock. FlucSM–RFP was used as a marker for cytosolic aggregates. Image is representative of five captured. f, Anti-HA immunoblot showing co-purification of HA-tagged Tom70 and Tom40 but not Om45 with aggregates. g, Additional negative controls for experiment in f: anti-HA immunoblotting of two mitochondrial outer membrane proteins Mdm10 and Mdm34 that face the cytosol and were not identified by proteomics to be enriched in aggregates. h, Dissolution kinetics of heat shock-induced FlucSM–GFP aggregates in cells treated with CHX (green) or CHX+CCCP (red). Shown are fluorescent traces of three biological repeats for each condition. i, Measurement of ATP level under the same experimental conditions as for h using a FRET-based sensor showing that CCCP did not deplete cellular ATP. The FRET efficiencies of the CCCP+CHX-treated group and the CHX-treated group were normalized to the mean of the CHX-treated group at each indicated time point. 36 cells were measured at time 0 (before drug addition), 43 and 74 cells for the CHX condition (at 30 and 60 min, respectively), and 39 and 74 cells for CHX/CCCP (at 30 and 60 min, respectively). Shown are means and s.e.m. j, Images, representative of about 300 cells imaged, from three biological repeats of TIM23 and tim23ts cells stained with TMRM (red) after heat shock to demonstrate that the membrane potentials of these cells were similar. Scale bar, 2.5 μm for (e), 5 μm for other panels. For gel source data, see Supplementary Fig. 1.
