Article
Propidium iodide exclusion assay and measurement of
caspase-3 activity
Cell death under hyperosmotic stress was quantified by propidium
iodide (PI) exclusion. Immediately after washing with PBS, PI (Biovi-
sion) was added to the cells at a final concentration of 50 μg ml−1 and
incubated at room temperature for 15 min, after which excess PI was
washed off with PBS. At least two random fields were imaged using
Olympus Fluoview FV3000 and processed with the MetaMorph soft-
ware (Molecular device). Cell death was quantified by calculating the
number of PI-labelled cells as a percentage of the total number of
cells observed by DIC. Early apoptotic activity was assessed using the
cleaved-caspase-3 (Asp175) antibody purchased from Cell Signaling
Technology (9661). The experiment was repeated three times.
Statistical analysis
Statistical analysis was performed using GraphPad Prism 7. All details
on statistics have been indicated in figure legends and Source Data.
Sample distribution was assessed with the D’Agostino-Pearson normal-
ity test or the Kolmogorov–Smirnov test depending on sample size.
Unpaired two-tailed Student’s t-test was used to determine statistical
significance when comparing two independent groups with normal
distribution and no significant difference in their s.d. When comparing
two independent groups in which at least one did not fit the normal-
ity criteria, unpaired two-tailed Mann–Whitney U-test was used. For
analysis of statistical significance in comparisons involving more than
two groups with normal distribution, ordinary one-way ANOVA with
Dunnett’s (when comparing the mean of each group with the mean
of a control group) multiple comparisons test was used. For multiple
comparisons in which at least one group did not comply with normal
distributed data, Kruskal–Wallis and Dunn’s post hoc tests were used.
In all cases, statistical significance was assessed with a 95% confidence
interval; therefore, P < 0.05 was considered significant.
Purification of ubiquitylated proteins and sample preparation
for mass-spectrometry analysis
HCT116 cells (1 × 10^7 ) were treated with 0.2 M sucrose for the indicated
periods of time (0, 15, 30 min) before collection. The cells were lysed in a
urea-containing lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10%
glycerol and 6 M Urea) supplemented with 10 μM bortezomib (LC Labo-
ratories), 10 mM iodoacetamide (GE Healthcare) and 1× Complete pro-
tease inhibitor cocktail (Roche, EDTA free), and sonicated on a Handy
Sonic (Tomy Seiko). The lysate was then clarified by centrifugation
(20,000g for 20 min), and the protein concentration was determined
using the BCA protein assay kit (Thermo Fisher Scientific). To capture
ubiquitylated proteins, we used a biotinylated trypsin-resistant tandem
ubiquitin-binding entity (BIOTR-TUBE)^41. BIOTR-TUBE (10 μg) and 5 mg of
cell lysate were incubated for 1 h at 4 °C and incubated for an additional
for 1 h at 4 °C with 50 μl of Dynabeads MyOne streptavidin C1 (Thermo
Fisher Scientific). After two washes with lysis buffer containing 6 M urea
and two washes with urea-free lysis buffer, the beads were suspended
in 50 μl of 50 mM ammonium bicarbonate (AMBC) and 0.1% RapiGest
SF (Waters). Proteins on beads were reduced in 5 mM TCEP for 30 min
at 50 °C and then alkylated with 10 mM methylmethanethiosulfonate
for 10 min at room temperature. The alkylated proteins were digested
overnight at 37 °C with 1 μg of trypsin Gold (Promega). After tryptic
digestion, samples were acidified to ~pH 2 with trifluoroacetic acid
(TFA) and desalted by solid-phase extraction using GL-Tip SDB and
GL-Tip GC (GL Sciences). Ubiquitinated peptides were enriched using
the PTMScan ubiquitin Remnant Motif (K-ε-GlyGly) Kit (Cell Signal-
ing Technology)^43. After three washes with 200 mM triethanolamine
(pH 8.2), antibody-coupled beads were cross-linked with 20 mM dime-
thyl pimelimidate in 200 mM triethanolamine (pH 8.2) for 1 h at room
temperature. The beads were then washed three times with 50 mM
Tris-HCl (pH 7.5), 100 mM NaCl, and stored at 4 °C until use. Trypsinized
peptides prepared as described above were suspended in 0.2 ml IAP
buffer (50 mM MOPS, pH 7.2, 10 mM sodium phosphate and 50 mM
NaCl) and incubated with 10 μl of antibody-coupled beads for 2 h at
4 °C. After two washes with 250 μl of IAP buffer and three washes with
250 μl of Milli-Q water (Millipore), di-Gly peptides were eluted with
3 × 20 μl of 0.15% TFA. The eluted peptides were desalted to 0.1% TFA
using GL-Tip SDB and GL-Tip GC (GL Sciences), and analysed by mass
spectrometry.Purification of proteasome-interacting proteins for mass-
spectrometry analysis
After addition of 0.2 M sucrose and incubation for 30 min, HCT116
PSMD6-eGFP-3×FlagKI/KI cells were cross-linked with 0.2% formaldehyde
(Thermo Scientific) for 10 min, after which the reaction was quenched
with 0.25 M glycine. After two washes with PBS, the cells were lysed with
lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10% glycerol, 4 mM
ATP, 10 mM MgCl 2 , 0.2% NP-40, 1 × Protein Complete Inhibitor-EDTA,
1 mM bortezomib, 1 mM iodoacetamide) with sonication. Cleared
lysates (2 mg) were incubated with 10 μl of anti-DDDDK antibody-cou-
pled agarose (MBL) for 1 h at 4 °C. After the beads were washed five times
with lysis buffer, proteins were eluted with 3×Flag peptide (Sigma). The
eluted proteins were mixed with 3× LDS NuPAGE sample buffer and
incubated for 30 min at 95 °C for uncrosslinking. Proteins were then
electrophoresed 1 cm by 4–12% NuPAGE. The gels were stained with Bio-
Safe Coomassie Stain (Bio-Rad). After washing extensively with Milli-Q
water, the gels were excized, cut into 1-mm^3 pieces, destained twice
for 1 h each with 1 ml 50 mM AMBC/50% acetonitrile (ACN), with agita-
tion, and then dehydrated with 100% ACN. Trypsin solution (Promega,
20 ng μl−1 in 50 mM AMBC/5% ACN) was added to the gel pieces, and the
samples were incubated at 37 °C overnight. Digests were extracted with
100 μl of 50% ACN/0.1% TFA. The extracted peptides were concentrated
using a speed-vac and resuspended in 0.1% TFA.Mass-spectrometry analysis
Shotgun mass-spectrometry analysis was performed essentially as
described^41 ,^44. An Easy nLC 1000 system (Thermo Fisher Scientific) was
connected online to a Q Exactive mass spectrometer (Thermo Fisher
Scientific) with a nanoelectrospray ion source. The mobile phases
were 0.1% formic acid (FA) in water (solvent A) and 0.1% FA in 100% ACN
(solvent B). Peptides were directly loaded onto a C18 analytical column
(ReproSil-Pur 3 μm, 75-μm inner diameter and 12-cm length, Nikkyo
Technos) and separated using a 150-min three-step gradient (0–40%
solvent B for 120 min, 40–100% for 20 min and 100% for 10 min) at a
constant flow rate of 300 nl min−1. For ionization, liquid junction volt-
age was 1.8 kV and capillary temperature was 250 °C. The Q Exactive
was operated by the Xcalibur software 2.2 (Thermo Fisher Scientific)
in data-dependent MS/MS mode, and the top 10 most intense ions
with charge state +2 to +5 were selected with an isolation window of
m/z = 2.0 and fragmented by higher-energy collisional dissociation
with a normalized collision energy of 25. Resolution and automatic
gain control target for the survey scans were set to 70,000 and 3E6,
respectively. Ions selected for MS/MS were dynamically excluded for
5 s (for diGly peptide identification) or 30 s (for binding protein iden-
tification). The data were analysed using the Mascot search program
(Matrix Science) in Proteome Discoverer 1.3 (Thermo Fisher Scientific).
Maximum missed cleavage site of trypsin was set to two. Oxidation
(Met), GlyGly (Lys), phosphorylation (Ser, Thr, Tyr) and pyroglutamate
conversion (Gln) were selected as variable modifications. For the diGly
proteome, Cys-methylthio modification was set as a static modifica-
tion for database searches. Peptide identification was filtered at a false
discovery rate <0.01.Cryo-EM
For sample preparation, 25,000 cells were seeded on EM grids (R2/1, Au
200 mesh grid, Quantifoil Micro Tools) in a 35-mm dish and cultured in