Supplementary material
The otubain YOD1 is a deubiquitinating enzyme that associates with p97 to facilitate
protein dislocation from the ER
Figure S1. Identification of p97/NPL4/UFD1 as YOD1 interacting proteins.
HeLa cells stably expressing YOD1 equipped with a C-terminal HA tag were
homogenized and subjected to immunoprecipitation with anti-HA antibodies. Nontransduced Hela cells served as control. Immunoprecipitates were eluted by TEV protease
cleavage, resolved by SDS-PAGE and subjected to silver staining. Gel pieces were
processed for trypsinization and analyzed by LC-MS/MS. Depicted are the identified
proteins that were absent from samples derived from the control (YOD1 itself was also
identified but is not depicted). The positions of the peptides that were identified are
highlighted in yellow, and in red in the corresponding amino acid sequence below. The
tables summarize parent ion masses and sequence coverage.
Figure S2. Purified YOD1 associates with p97 and is catalytically active in vitro.
(A) GST-tagged YOD1 (25 µg) and a deletion mutant lacking the UBX-domain were
incubated with purified, hexameric p97 (7.5 µg). As control served a deletion variant of
p97 lacking the N-domain. After pulldown using glutathione-beads the GST-YOD1
variants and associated p97 were eluted with SDS-sample buffer and subjected to SDSPAGE (12%).
(B) The catalytic activities of Wild-type YOD1 and YOD1 C160S (both 2.6 µM) were
determined with UbAMC (10 µM) as a substrate.
(C) YOD1 or its mutant derivatives were incubated with a 1.5-fold molar excess of HAUbVME and subjected to SDS-PAGE. HA-UbVME adduct formation was visualized by
immunoblotting using anti-YOD1 antibodies (upper panel). Adduct formation was
confirmed by immunoblotting with anti-HA antibodies (lower panel).
(D) K63-linked poly-Ub chains (2 µg) were incubated for 16 h in a total volume of 10 µl
with different YOD1 variants (7.7 µM). Poly-Ub and free Ub were detected by
immunoblotting using anti-Ub antibodies.
Figure S3. RI332 expressed in 293T cells is only partially N-glycosylated.
(A) 293T cells were transiently transfected with wild-type RI332 or a mutant version that
cannot be N-glycosylated (N275T), pulse labeled for 10 min with
SDS.
The
lysates
were
subjected
to
anti-RI
35
S and lysed in 1%
immunoprecipitation.
The
immunoprecipitated material was eluted with 1% SDS and either left untreated or
incubated with PNGase F or Endo H according to the manufactures recommendations.
The eluates were then separated by SDS-PAGE (12%) and visualized by
autoradiography.
Note
that
a
fraction
of
RI332
is
non-glycosylated
when
immunoprecipitated from 293T cells and that PNGase F treated RI332 has a slightly lower
electrophoretic mobility as compared to Endo H treated RI332 probably due to the N275D
substitution through PNGase F.
(B) Schematic representation of various glycosylated, deglycosylated, and nonglycoylated RI332 variants produced by mutagenesis and enzymatic deglycosylation. Nacetylglycosamine is represented in blue, mannose in green, and the polypeptide chain of
RI332 in orange.
Figure S4. The degree of RI332 stabilization by YOD1 C160 is comparable to
proteasomal inhbition.
(A) 293T cells were transfected with an HA-tagged RI332 variant and either YOD1 WT or
C160S. 24 hours after transfection cells were pulse-labeld with 35S for 10 min, chased for
the indicated time points, lysed in 1% SDS and subjected to immunoprecipitation with
anti-HA antibody. A separate batch of cells transfected with YOD1 WT and HA-tagged
RI332 was treated with 50 µM ZL3VS during 30 min starvation period, the pulse-labeling
and throughout the chase time to inhibit the proteolytic activity of the proteasome.
The eluates were resolved by 12% SDS-PAGE and visualized by autoradiography (upper
panel). Unbound material was immunoprecipitated with anti-Flag antibodies to verify
equal expression of the YOD1 constructs (lower panel).
(B) Densiometric quantification of recovered RI332 after indicated chase times in (A).
Figure S5. YOD1 C160S expression increases the steady state level of RI332.
(A) 293T cells were transfected with RI332 and either YOD1 WT or C160S. After 24
hours the cells were lysed with 1% SDS and subjected to immunoblotting with anti-RI
antibodies. Equal expression of YOD1 WT and C160S was verified by an immunoblot
with anti-Flag antibodies. A cross-reaction of anti-rabbit-HRP antibodies serves as
loading control.
(B) Immunoblots with anti-HA antibodies show the distribution of HA-Ub in cell lysates
prior to immunoprecipitation with anti-p97 (as shown in Figure 6). Immunoblots with
Anti-Flag and were performed to verify equal expression of Flag-tagged YOD1 and p97
variants. Equal loading is controlled by an immunoblot with anti-PDI antibodies.
Figure S6. Polyubiquitin chains of proteins associated with p97 QQ are cleaved by
purified YOD1.
(A) 293T cells were co-transfected with p97 QQ and HA-tagged ubiquitin. After 24 hours
p97 QQ and associated proteins were retrieved by immunoprecipitation with anti-Flag
antibodies. To visualize poly-ubiquitin chains, the eluates were either directly subjected
to immunoblotting with anti-HA antibodies or after a 5 h incubation with purified YOD1
variants (5 µg) at 37ºC in buffer A.
Figure S7. YOD1 deubiquitinates dislocation substrates en route to the cytosol. The
model depicts the two possible fates of dislocation substrates. In the presence of YOD1
WT, substrates are deubiquitinated to facilitate their threading through the p97 pore,
leading to a progressive movement of the substrate to the cytosol (I-III). In the presence
of catalytically inactive YOD1 C160S, the Ub chain on the substrates remains, thus
imposing an impediment on the processive threading of substrates (II b), leading to an
accumulation of misfolded species in the ER lumen (III b).
Supplementary Experimental procedures
Constructs.
For heterologous expression of GST-tagged of YOD1, its variants were cloned into the
pGEX-GP-2 expression vector (GE Healthcare) via BamHI and XhoI. The full-length
YOD1 expression construct comprised aa1-348, the YOD1 Znf deletion mutant aa1319, and the UBX YOD1 mutant aa129-348. For the expression of His-tagged p97,
cDNA of p97 was amplified via PCR and subsequently cloned into the pET28a(+) vector
system (Novagen) via NdeI and HindIII restriction sites. The N p97 construct, lacking
the N-domain, comprised aa186-806. For the expression in mammalian cells, Flag-tagged
YOD1 and HA- or non-tagged RI332 variants in 293T cells the pcDNA3.1 vector system
was employed. Constructs were equipped with a Kozak sequence (GCCACC) introduced
5’ to the start-codon. The YOD1 variants were cloned into the pcDNA3.1 vector via
HindIII and XbaI and coded for a N-terminal FLAG-tag. The TCR and the 1 AT
expression constructs were described previously. Site-directed mutagenesis of p97 was
performed with the QuikChange II mutagenesis kit (Stratagene).
Large scale YOD1 pulldown and identification of interaction partners
YOD1 bearing a C-terminal tobacco etch virus (TEV) protease cleavage site followed by
an HA-tag was stably introduced into HeLa cells analogous to described previously
procedures (Mueller et al., 2008). Immunoprecipitation, SDS PAGE, sample preparation,
and analysis via LC-MS/MS was performed according to (Mueller et al., 2008).
Protein expression and purification
Recombinant GST-tagged proteins were expressed in E. coli BL21 (DE3) Rosetta cells
and purified with glutathione beads according to standard procedures. YOD1 was eluted
from the glutathione beads by overnight incubation at 4°C with 200 µg GST-tagged
prescission protease (Roche). After affinity purification, His-tagged p97 and tag-less
YOD1 were gel filtrated on a 16/60 Superdex 200 column (GE Healthcare) equilibrated
with buffer A (25 mM Tris pH 8, 150 mM KCl, 2.5 mM MgCl2 and 5% (w/v) glycerol).
Deubiquitination assays
Ubiquitin C-terminal 7-amido-4-methycoumarin (UbAMC), diubiquitin (K48-, K63 and
linear) and polyubiquitin chains (Ub2-7, K48 and K63-linked) were purchased from
Boston Biochem.
UbAMC hydrolysis was performed in buffer A (25 mM Tris pH 8, 150 mM KCl, 2.5 mM
MgCl2 and 5% (w/v) glycerol), supplemented with 100 µg/ml bovine serum albumin
(NEB) at 25°C. Enzyme concentrations were determined with the Pierce BCA Protein
Assay Kit (Thermo). Measurements were performed in a total volume of 100 µl with a
enzyme concentration of 2.6 µM and a UbAMC concentration of 10 µM. The amount of
released AMC was quantified via its fluorescence with a 368 nm/467 nm filter pair and a
455 nm cutoff using a Spectramax M2 plate reader (Molecular Devices) and 7-amido-4methylcoumarin as a standard (Anaspec Inc.).
Hydrolysis of diUb and PolyUb was analyzed by SDS-PAGE (15%) and subsequent
immunobloting against Ub after overnight incubation (if not otherwise indicated) at 25°C
in total volume of 10 µl with 2 µg diUb and PolyUb chains, with 5.2 µM and 7.7 µM
YOD1 variants, respectively, in buffer A.
HA-UbVME labeling of purified YOD1 variants was performed essentially as described
(Borodovsky et al. 2002, Schlieker et al. 2007) with a 1.5-fold molar excess of HaUbVME.
Supplemental References
Borodovsky, A., Ovaa, H., Kolli, N., Gan-Erdene, T., Wilkinson, K. D., Ploegh, H. L.,
and Kessler, B. M. (2002). Chemistry-based functional proteomics reveals novel
members of the deubiquitinating enzyme family. Chem Biol 9, 1149-1159.
Mueller, B., Klemm, E. J., Spooner, E., Claessen, J. H., and Ploegh, H. L. (2008). SEL1L
nucleates a protein complex required for dislocation of misfolded glycoproteins. Proc
Natl Acad Sci U S A 105, 12325-12330.
Schlieker, C., Weihofen, W. A., Frijns, E., Kattenhorn, L. M., Gaudet, R., and Ploegh, H.
L. (2007). Structure of a herpesvirus-encoded cysteine protease reveals a unique class of
deubiquitinating enzymes. Mol Cell 25, 677-687.