To determine the fate of cytosolic misfolded substrates, we initially followed a destabilized Ubc9 variant that misfolds above 30 °C (refs 23, 24
; ). Ubc9ts
, fused to green fluorescent protein (GFP) to facilitate detection (GFP–Ubc9ts
), was expressed under the control of a galactose-regulated promoter. Glucose addition repressed expression, allowing us to follow the fate of GFP–Ubc9ts
from the earliest stages of protein misfolding after shift to 37 °C (). At permissive temperatures, GFP–Ubc9ts
was native and diffuse, similar to wild-type GFP–Ubc9 (, 0 min, compare with wild type panel 120 min). GFP–Ubc9ts
misfolding led to degradation by the ubiquitin–proteasome pathway, as reported for untagged Ubc9ts
(, compare 5 min and 60 min; and , left panel)23,24
. During degradation we observed transient accumulation of Ubc9ts
in distinct cytosolic puncta and inclusions that were eventually cleared (for example, , 30 min and ). Most cells contained a juxtanuclear inclusion as well as smaller puncta throughout the cytosol, whereas some cells contained only the juxtanuclear inclusion (). Impairment of proteasome-mediated degradation either in cim3-1
cells or by treatment with the proteasome inhibitor MG132 stabilized GFP–Ubc9ts
and led to its reproducible accumulation in two distinct inclusions in virtually every cell (, 60 min and 120 min and Supplementary Fig. 1a
). At early time points after misfolding in proteasome-defective cells, GFP–Ubc9ts
accumulated in structures resembling those observed during degradation in control cells (, compare 15 min and 30 min). Quantification indicated that the juxtanuclear inclusion formed first, closely followed by cytosolic puncta (). However, at later incubation times at 37 °C the juxtanuclear inclusion remained, but the puncta were no longer observed. Instead, a second large perivacuolar inclusion was now formed at the periphery of the cell (). Once formed, both inclusions persisted well beyond the time course shown in . Notably, formation of both inclusions was an active process, as it was reversibly inhibited by the microtubule-depolymerizing drug benomyl (Supplementary Fig. 2
). These two inclusions may represent distinct compartments for the sequestration of misfolded proteins.
We next examined other types of cytosolic quality control substrates. We initially followed the unassembled von Hippel-Lindau (VHL) tumour suppressor25,26
. VHL only folds after binding to its cofactor elongin BC27
(). Tumour-causing mutations impairing elongin BC binding, or expression in cells lacking elongin BC, lead to misfolded VHL ubiquitination and degradation25
(), resulting in reduced levels of diffuse fluorescence (, compare left panel, misfolded without elongin BC, with right panel, folded VHL with elongin BC). Inhibition of the proteasome in cim3-1
cells (), or with MG132 (Supplementary Fig. 1c
), led to formation of a single juxtanuclear GFP–VHL inclusion. Importantly, proteasome impairment did not produce GFP–VHL inclusions under conditions leading to productive VHL folding (, plus elongin BC, right panel).
It was puzzling that at 30 °C VHL consistently formed a single juxtanuclear inclusion whereas Ubc9ts
formed two distinct inclusions. Ubc9ts
destabilization requires thermal stress, hence formation of two inclusions might result from the increased load of denatured quality control substrates at 37 °C. Indeed, when unassembled VHL was expressed at 37 °C it also accumulated in two inclusions as observed for Ubc9ts
(). Three-dimensional fluorescence deconvolution microscopy demonstrated that the inclusions formed by VHL and Ubc9ts
overlap spatially in the same compartments ( and Supplementary Movie 1
A missense mutation of actin, actin(E364K), also degraded via the ubiquitin–proteasome pathway25
, similarly accumulated in the same inclusions as Ubc9ts
(). As clearance of misfolded Ubc9, VHL and actin requires ubiquitination, we considered whether proteasome impairment or stress cause widespread aggregation of ubiquitinated proteins (Supplementary Fig. 1b
). This is not the case, as native substrates of the ubiquitin–proteasome pathway28
, such as Arg–GFP (R–GFP), Ub–G76A–GFP (Ub–GFP) and Deg1–GFP (Supplementary Fig. 1b
and data not shown), remained soluble and diffuse after proteasome impairment, even under conditions of stress (Supplementary Figs 1b and 3c
). We conclude that different classes of misfolded cytosolic proteins are sequestered in two defined cellular inclusions, one juxtanuclear and one at the periphery of the cell. The juxtanuclear inclusion seems to form first and is more prevalent under normal cellular conditions. However, stress conditions lead to protein accumulation in the second peripheral inclusion. In principle, the differential partitioning of non-native quality control substrates between these two compartments may be determined by a change in their intrinsic properties, such as aggregation state, or by their interaction with saturatable quality control components, or both.
We explored the relationship between inclusions formed by disease-related amyloidogenic proteins and those characterized here for misfolded cytosolic proteins (). The relative spatial localization of the aggregates formed by glutamine-rich yeast prion proteins Rnq1 and Ure2, as well as polyQ expanded Huntingtin (HttQ103) relative to the Ubc9ts
inclusions was determined by deconvolution microscopy. All the amyloidogenic proteins tested formed an inclusion that consistently co-localized with the perivacuolar peripheral inclusion of Ubc9ts
(; Supplementary Movie 2
). We did not observe any cases of co-localization of either the prion proteins or Htt with the juxtanuclear inclusion.
Unlike normal quality control substrates, amyloidogenic proteins (including Huntingtin (Htt) and prions) form large insoluble inclusions even in the absence of proteasome inhibition10,13,17,29
. Thus, amyloidogenic proteins were also analysed in the absence of proteasome inhibition and under normal growth temperatures (). Rnq1, Ure2 and HttQ103 also accumulated under these normal conditions in aggregates localized exclusively in the peripheral compartment (). Additionally, Rnq1 was also found in small puncta throughout the cell (). The accumulation of amyloidogenic proteins in the peripheral inclusion in the absence of either stress or proteasome impairment () indicates that this compartment can also form under normal conditions. Notably, Rnq1 always surrounded the Ure2 and HttQ103 deposits (red fluorescence in ), suggesting that Rnq1 is targeted to this perivacuolar compartment with slower kinetics than the other amyloidogenic proteins. These observations suggest that some unique feature of amyloidogenic proteins earmarks them for exclusive delivery to the peripheral inclusion.