a, The temperature-sensitive mutant of Ubc9(Y68L) (Ubc9^ts) is folded and long-lived at 25 °C. After temperature shift to 37 °C, the Ubc9^ts protein misfolds and is degraded by the ubiquitin–proteasome pathway. b, Time-dependent changes in localization of folded and misfolded GFP–Ubc9 in wild-type (WT) and cim3-1 cells. Nuclei were visualized by co-expressing NLS–tdTomato (NLS–TFP). Ubc9 expression was shut off by addition of 2% glucose before temperature shift in all experiments. c, Quantification of Ubc9^ts localization after misfolding in wild-type and cim3-1 cells. Graphs represent three separate experiments conducted as in b. The phenotypes (see panels) of 100 cells were scored at each time point. d, Quality control of the VHL tumour suppressor. VHL folds after elongin BC binding to form the VBC complex. In the absence of elongin BC, VHL is degraded by the ubiquitin–proteasome pathway. e, VHL localization in wild-type and cim3-1 cells, and at 30 °C and 37 °C in cim3-1 cells (f). Two panels are shown for each experiment. g, Misfolded VHL, Ubc9 and actin co-localize in the same two inclusions. VHL tagged with mCherry (CHFP–VHL, red) with GFP–Ubc9^ts (green, upper panel) or with Act1–E364K–GFP (green, lower panel) in cim3-1 yeast, after 2 h at 37 °C. Images collected as a Z-series and deconvoluted are shown as a two-dimensional projection.

a, Co-localization of inclusions of the yeast prion Rnq1 (green, tagged with GFP) and misfolded Ubc9^ts (red, tagged with CHFP), and HttQ103–GFP with CHFP–Ubc9^ts(b). Ure2–GFP with CHFP–Ubc9^ts (c), in cim3-1 yeast after 2 h at 37 °C. Images were collected as a Z-series and de-convoluted. d, Co-localization of the yeast prions Ure2–GFP and Rnq1–CHFP in the peripheral inclusion. A direct fluorescence image is shown for e. e, Co-localization of HttQ103–GFP with Rnq1–CHFP in the peripheral inclusion.

a, CHFP–VHL and GFP–Ubc9^ts show diffuse localization in the absence of proteasome inhibition (upper panel), and form co-localizing perinuclear puncta and inclusions next to the endoplasmic reticulum after proteasome inhibition (+MG132, lower panel).
b, HttQ103–GFP forms one hyper-fluorescent inclusion (upper panel, note inclusion not always perinuclear). After proteasome inhibition (+MG132), CHFP–VHL forms an inclusion that is distinct from that of HttQ103–GFP (lower panel).

a, Qualitative FLIP analysis indicates that misfolded protein in the JUNQ and the IPOD exhibit different relative exchange rates with the cytosolic pool. Pre- and post-bleach images of a representative FLIP experiment with GFP–Ubc9^ts are shown. The fluorescence intensity scale is pseudocoloured as shown. A square designates the location of the photobleaching laser spot. GFP–Ubc9^ts was expressed in cim3-1 yeast. Relative fluorescence of JUNQ (orange), IPOD (blue) and cytosol (black) from ten FLIP experiments is shown over time. b, Protein in the IPOD inclusion is immobile. Pre- and post-bleach images of a representative FRAP experiment and subsequent recovery of GFP–Ubc9^ts are shown. GFP–Ubc9^ts was expressed in Δubc4/5 cells and shifted to 37 °C to form the IPOD.

a, JUNQ compartment tightly co-localizes with nuclear membrane. DNA is visualized with DAPI (blue), nucleoplasm with NLS–TFP (red). GFP–Ubc9^ts (top) at 37 °C and GFP–VHL (bottom) in cim3-1 cells. Two-dimensional projections of de-convoluted Z-series are shown. b, The JUNQ compartment, shown here for GFP–VHL, does not localize to the spindle pole body (marked by Spc42–CHFP), and is in close proximity to the endoplasmic reticulum visualized with Sec63–CHFP (c). d, The JUNQ compartment (upper panel, CHFP–VHL, red), but not the IPOD (Act1–E364K–CHFP, lower panel), concentrates 26S proteasomes (visualized with Cim5–GFP for regulatory particle and Pre6–GFP for core particle, green). e, Hsp104 localizes to both compartments. JUNQ and IPOD were formed by expressing CHFP–Ubc9^ts (upper panel) or CHFP–VHL (lower panel). Note Hsp104 also accumulates in an IPOD structure (blue arrow) independently of ectopically-expressed aggregating protein when CHFP–VHL is expressed in cim3-1 cells at 30 °C (middle panel). f, The IPOD, shown here for GFP–Ubc9^ts, co-localizes with CHFP–Atg8. Some CHFP–Atg8 can also be seen in the pre-autophagosomal structure (PAS).

a, Blocking ubiquitination of misfolded VHL prevents its localization to JUNQ, and redirects these proteins to the IPOD. b, VHL in the IPOD accumulates in a Triton-insoluble fraction. Asterisk denotes cross-reacting band unrelated to VHL. c, Deletion of Sti1, required for VHL degradation, reroutes misfolded VHL to the IPOD. d, Ubiquitination suffices to promote prion delivery to the JUNQ. Rnq1–GFP localizes exclusively to the IPOD. A ubiquitination signal (Ub–G76A) engineered in the yeast prion Rnq1–GFP causes Ub–G76A–Rnq1–GFP localization to both JUNQ and IPOD. e, Recovery of diffuse cytosolic fluorescence by thermally denatured GFP–Ubc9^ts accumulated in the JUNQ (top) but not in the IPOD (middle) after return to the permissive temperature. The recovery of GFP–Ubc9^ts requires Hsp104 disaggregase activity (bottom). f, Model for sorting of cytosolic misfolded proteins to distinct quality control compartments.