Extended Data Figure 4: The protease activity of Pim1 is important for efficient degradation of misfolded cytosolic proteins.
From: Cytosolic proteostasis through importing of misfolded proteins into mitochondria
a, b, pim1S1015A:PIM1 grows normally under fermentable (a) and non-fermentable conditions (b). c, Representative images showing that delayed split-GFP of FlucSM disappearance in pim1S1015A:PIM1 cells was not affected by CHX during the recovery phase. Top, split GFP images; bottom, MTS–mCherry-labelled mitochondria. d, Quantification of mean split-GFP/mCherry ratio for pim1S1015A:PIM1. CHX was added after heat shock. Plotted are mean and s.e.m. from 747 cells that were imaged and measured; three biological repeats. e, f, Representative immunoblots (e) and quantification (f) from three biological repeats showing that the mitochondrial import (inhibited by CCCP) is the major source for degrading stress-damaged endogenous Lsg1–HA, but vacuole-mediated degradation (inhibited by PMSF) also plays a role, while the proteasome pathway (inhibited by MG132) has the least effect. f shows data points and mean. g, h, Representative immunoblots (g) and quantification (h) of wild-type or tim23ts cells treated with CHX after heat shock showing that mitochondrial import (inhibited by tim23ts) is important for the degradation of stress-damaged FlucSM–HA. h shows data points and mean plots from three biological replicates. i, j, Representative immunoblots (i) and quantification (j) showing that without heat shock, proteasome-mediated degradation of FlucSM–HA was inhibited by MG132. j shows data points and mean plots from three biological replicates. Scale bar, 5 μm. For gel source data, see Supplementary Fig. 1.
