Supplemental Data S1

Intracellular Traffickingof Interleukin-1 Receptor IRequires Tollip

Brian Brissoni, Laetitia Agostini, Michel Kropf,Fabio Martinon, Valentin Swoboda, Saskia Lippens,

Helen Everett, Natalia Aebi, Sophie Janssens,Etienne Meylan, Michela Felberbaum-Corti,

Harald Hirling, Jean Gruenberg, Jurg Tschopp,and Kimberly Burns

Supplemental Experimental Procedures

Reagents and Cell Culture

Recombinant human IL-1b was from Alexis Biochemicals, and leu-

peptin was from Roche Diagnostic. Anti-Flag/M2 and anti-VSV

were from Sigma; anti-EGFR and anti-ubiquitin, P4D1, were from

Santa Cruz. Antiubiquitin, FK1, was from Affiniti Research Products.

Polyclonal rabbit antiserum to Tollip was raised against GST-Tollip

of mouse origin. 293T or immortalized wild-type and Tollip-deficient

MEFs [S1] were maintained in DMEM Glutamax supplemented with

10% fetal-calf serum and penicillin and streptomycin (100 mg). 293-

Flag IL-1RI (G9) cells stably expressing the Flag-tagged hIL-1RI

were cultured in 2% FCS DMEM-NUT F-12 (Gibco).

Expression Vectors

Expression vectors encoding Flag IL-1RI, Flag IL-1RAcP, IRAK-1

D340N (kinase dead), VSV Tollip, and VSV Tollip deletion constructs

have been previously described [S2]. Kinase-dead IRAK-1 was used

for interaction studies with Tollip because wild-type IRAK is auto-

phosphorylated upon overexpression and interacts poorly with

Tollip [S2]. The CUE mutant of Tollip (Tollip MF241-242AA) was

generated by double PCR and inserted into pCRIII (Invitrogen) con-

taining an N-terminal VSV tag (VSVpCRIII). N-terminal His6-Xpress-

tagged Tollip and Tollip DCUE was generated by cloning the respec-

tive DNA fragments into pcDNA-HisMaxC (Invitrogen). Tollip or

TollipMF240/241AA was inserted into pMSCV to generate vectors

for retroviral infection of Tollip 2/2 MEFs. Full-length mouse Tom1

and full-length human Tom1 and Tom1DGTA (D2122312) were am-

plified by PCR from an EST (IMAGE 5409159 and IMAGE 6470390,

respectively) and cloned into VSV pCRIII (Invitrogen). siRNAs

against Tom1 was purchased from Ambion (siRNA1 ID #187385

and siRNA2 ID #187387).

Immunoprecipitation

For binding studies, transfected 293T cells were lysed in NP-40 lysis

buffer (1% NP-40, 20 mM Tris [pH 7.9], 250 mM NaCl2, 10% glycerol,

20 mM b-glycerophosphate, 10 mM NaF, 1 mM sodium orthovana-

date, 2 mM dithiothreitol, 1 mM EDTA, and a protease inhibitor cock-

tail). To detect ubiquitination of IL-1RI, 293-Flag IL-1RI cells or trans-

fected 293T cells were lysed in RIPA buffer (20 mM Tris [pH 7.4],

150 mM NaCl2, 0.1% SDS, 0.5% NP-40, 0.5% Deoxycholate, and

a protease inhibitor cocktail). After a preclearing step with Sephar-

ose-6 Beads (Sigma), cell extracts were incubated overnight at

4�C with (1 mg) M2 antibodies preincubated with protein G Sephar-

ose (Amersham Biosciences). The beads were then washed six

times with lysis buffer, separated by SDS-PAGE, transferred to

Nitrocellulose, and analyzed by immunoblotting. Where indicated,

the Flag-IL-1RI/VSV-Tollip complex was dissociated in 50 ml of

PBS containing 1% SDS by boiling for 10 min and diluted 20-fold

with lysis buffer before a second immunoprecipitation with Flag

antibody.

Immunofluorescence Microscopy and Quantification of IL-1RI

Degradation

Immortalized MEFs were seeded on coverslips in six-well dishes

and the following day transfected with IL-1RI (2 mg) or IL-1RAcP (2

mg) by use of TransIT-LT1 Transfection Reagent (Mirus Corporation).

Cells were incubated with anti-IL-1RI antibody, 6B5 (Pharmingen),

and human IL-1b (50 ng/ml) 24 hr later in the presence or absence

of leupeptin (50 mg/ml) at 37�C for 3 hr. For other labeling, 24 hr after

transfection, cells were incubated at 4�C or 37�C with IL-1RI

antibody and human IL-1b (50 ng/ml) at 37�C for the indicated

time. Cells were then washed with PBS, fixed with 3.7% fomalde-

hyde in PBS for 12 min, and washed and permeabilized with PBS

containing 2% goat serum and 0.1% saponin for 30 min. The cells

were further incubated with Alexa-Fluor-488-conjugated antibodies

against rat IgG (Molecular Probes). For costaining of internalized IL-

1RI with LBPA, after IL-1RI labeling, cells were incubated with anti-

LBPA [S3] and then with Alexa-Fluor-546-conjugated antibodies

against mouse IgG (Molecular Probes). The coverslips were

mounted in FluorSave (Calbiochem) containing 5 mM of Draq 5

(Alexis Biochemicals) for nuclear staining.

For quantification of degradation of IL-1RI, two coverslips per cell

type were seeded and treated in an identical manner, except that

leupeptin was also added to one coverslip to block degradation dur-

ing internalization and labeling of IL-1RI. Images were acquired on

a Leica TCS SP2 AOBS (Leica Microsystems AG) inverted confocal

microscope with an oil objective 403 (HC PL_APO 403/1.25-0.75

OIL CS). 512 3 512 pixels images were acquired, with a zoom of

13. A stack of three Z frames was acquired. Gain and offset of the

microscope were set with control cells (leupeptin treated), and

care was taken to not saturate the image; gain and offset of the mi-

croscope were kept constant throughout the acquisition of both leu-

peptin-treated and untreated cells. Z stacks were transformed into

a 2D average projection with the Leica software (Leica Microsys-

tems AG). Fluorescence intensity was determined as described

[S4]. In brief, it was analyzed with the Metamorph Software (Molec-

ular Devices Corporation) by application of a threshold of 80 to all

images. Integrated fluorescence density was normalized to the

number of cells in the images (visualized by IL-1R staining and

Draq 5). The mean fluorescence intensities (MFI) per cell are re-

ported as a percentage, relative to the MFI of leupeptin-treated cells

(100%). The Student’s t test was used to evaluate the significance of

the differences in MFI.

IgG degradation was measured in wild-type or Tollip-deficient

cells as described previously [S5].

Reagents and Cell Culture

EGF and GST-ubiquitin were obtained from Alexis Biochemicals.

Anti-EGFR was from Santa Cruz. Polyclonal rabbit antiserum to

Tollip was raised against GST-Tollip of mouse origin. 293T or immor-

talized wild-type and Tollip-deficient MEFs [S1] were maintained in

DMEM Glutamax supplemented with 10% fetal-calf serum and pen-

icillin and streptomycin (100 mg).

Expression Vectors

Tom1DGAT (D2122312) were generated by PCR and cloned into

VSV pCRIII (Invitrogen). Myc-Hrs and EGFR-GFP were from Harold

Stenmark (Oslo, Norway) and Alexander Sorkin (Denver, Colorado),

respectively.

2D Gel Analysis

The His-Tollip pull-down was extracted by adding solubilization

buffer containing 9.0 M Urea, 4% (w/v) CHAPS (Sigma), 65 mM

1,4-dithio-DL-threitol, 0.8% (v/v) Resolytes 3-10 (BDH), 4 mM Tris

base (pH 10.5), and 0.001% (w/v) bromophenol blue and on a rocking

surface at room temperature for 30 min. The eluted sample was then

loaded by rehydratation overnight on 7-cm-long IPG strips (pH 3–10)

(Amersham Pharmacia Biotech). Isoelectric focusing was performed

at a maximum voltage of 3500 V, until a Volt$hour count of 35,000

was reached. Equilibration and transfer to the 2D SDS-PAGE

analysis were done as described [S6]. Silver staining was carried out

as described [S7].

Immunofluoresent Microscopy

For examination of the distribution of Tollip, wild-type or Tollip-re-

constituted MEFs (+Tollip MEFs) were washed with PBS, fixed

with 3.7% fomaldehyde in PBS for 12 min, and then washed and per-

meabilized with PBS containing 2% goat serum and 0.1% saponin

for 30 min. Tollip was labeled with Tollip antibodies (diluted

1:1500) and then with Alexa-Fluor-488-conjugated antibodies

against rabbit IgG (Molecular Probes). Other antibodies used for

costaining include: anti-mouse Lamp1 ID4B (Developmental Studies

Hybridoma Bank), anti-mouse LBPA [S3], or anti-rabbit EEA1 (Affin-

ity BioReagents). Colocalization with Hrs was carried out after trans-

fection of wild-type MEFs with Myc-Hrs (a gift of H. Stenmark, Oslo,

Norway). Transfected Hrs was revealed with Myc antibodies. After

immunostaining, the coverslips were mounted in FluorSave (Calbio-

chem) containing 5 mM of Draq 5 (Alexis Biochemicals) for nuclear

staining, and images were taken with an inverted confocal laser-

scanning microscope (LSM 510; Carl Zeiss).

Tollip was labeled by incubation of wild-type or Tollip reconsti-

tuted MEFs with Tollip antibodies (diluted 1:1500) and then with

Alexa-Fluor-488-conjugated antibodies against rabbit IgG (Molecu-

lar Probes). Other antibodies used for costaining include: anti-

mouse Lamp1 ID4B (Developmental Studies Hybridoma Bank) and

anti-mouse LBPA [S3]. After immunostaining, the coverslips were

mounted in FluorSave (Calbiochem) containing 5 mM of Draq 5

(Alexis Biochemicals) for nuclear staining, and images were taken

with an inverted confocal laser-scanning microscope (LSM 510;

Carl Zeiss).

For examination of bulk transport, wild-type or Tollip-deficient

MEFs were seeded on coverslips in six-well dishes. Cells were incu-

bated with Cy3-conjugated dextran (Molecular Probes) 24 hr later

for 10 min at 37�C and then washed and incubated in media for 50

min. The cells were then washed with PBS, fixed, permeablized,

and then incubated with mouse anti-LBPA or rat anti-Lamp1 as

described above.

EGFR Analysis

For coimmunoprecipitation studies with EGFR, 293T cells were co-

transfected with either EGFR-GFP (1 mg) and Tollip (1 mg) or Hrs

(2 mg). Transfected 293T cells were serum-starved for 24 hr, placed

at 4�C for 2 hr, and either left untreated or stimulated with 100 nM

EGF (Alexis). Cells were then were lysed in NP-40 lysis buffer (1%

NP-40, 20 mM Tris [pH 7.9], 250 mM NaCl2, 10% glycerol, 20 mM

b-glycerophosphate, 10 mM NaF, 1 mM sodium orthovanadate,

2 mM dithiothreitol, 1 mM EDTA, and a protease inhibitor cocktail).

After a preclearing step with Sepharose-6 Beads (Sigma), the cell ex-

tracts were incubated overnight at 4�C with (1 mg) GFP antibodies (a

gift of Andreas Mayer, University of Lausanne, Switzerland) preincu-

bated with protein G Sepharose (Amersham Biosciences). After in-

cubation, the beads were washed six times with lysis buffer, sepa-

rated by SDS-PAGE, transferred to Nitrocellulose, and analyzed by

immunoblotting.

Wild-type or Tollip-deficient MEFs were stimulated with 100 ng/ml

of EGF for the indicated times. Degradation of EGFR was monitored

by western blotting with EGFR antibodies.

Ubiquitin Binding Assays

For assaying monoubiquitin binding, GST-ubiquitin (3 mg) was im-

mobilized on glutathione-Sepharose beads and then incubated

with 293T cell extracts containing either VSV Tollip or VSV Tollip

MF/AA in 1% NP-40 lysis buffer for 2 hr at 4�C. The beads were

washed five times in NP-40 lysis buffer and bound proteins eluted

with SDS-PAGE sample buffer.

Supplemental References

S1. Didierlaurent, A., Brissoni, B., Velin, D., Aebi, N., Tardivel, A.,

Kaslin, E., Sirard, J.C., Angelov, G., Tschopp, J., and Burns, K.

(2006). Tollip regulates proinflammatory responses to interleu-

kin-1 and lipopolysaccharide. Mol. Cell. Biol. 26, 735–742.

S2. Burns, K., Clatworthy, J., Martin, L., Martinon, F., Plumpton, C.,

Maschera, B., Lewis, A., Ray, K., Tschopp, J., and Volpe, F.

(2000). Tollip, a new component of the IL-1RI pathway, links

IRAK to the IL-1 receptor. Nat. Cell Biol. 2, 346–351.

S3. Kobayashi, T., Stang, E., Fang, K.S., de Moerloose, P., Parton,

R.G., and Gruenberg, J. (1998). A lipid associated with the anti-

phospholipid syndrome regulates endosome structure and

function. Nature 392, 193–197.

S4. Steiner, P., Alberi, S., Kulangara, K., Yersin, A., Sarria, J.C.,

Regulier, E., Kasas, S., Dietler, G., Muller, D., Catsicas, S.,

et al. (2005). Interactions between NEEP21, GRIP1 and GluR2

regulate sorting and recycling of the glutamate receptor subunit

GluR2. EMBO J. 24, 2873–2884.

S5. Petiot, A., Faure, J., Stenmark, H., and Gruenberg, J. (2003).

PI3P signaling regulates receptor sorting but not transport in

the endosomal pathway. J. Cell Biol. 162, 971–979.

S6. Hochstrasser, D.F., Harrington, M.G., Hochstrasser, A.C., Miller,

M.J., and Merril, C.R. (1988). Methods for increasing the resolu-

tion of two-dimensional protein electrophoresis. Anal. Biochem.

173, 424–435.

S7. Rabilloud, T., Carpentier, G., and Tarroux, P. (1988). Improve-

ment and simplification of low-background silver staining of

proteins by using sodium dithionite. Electrophoresis 9, 288–291.

S2

Figure S1. Surface IL-1R1 Turnover in EL4

Cells

EL4 cells were preincubated with a rat anti-

mouse-IL1RI antibody (BD Biosciences clone

12A6) at 4�C for 1 hr so that cell surface IL-1RI

could be labeled. Unbound antibody was

then removed by washing in medium. IL-1b

was added at a concentration of 50 ng/ml at

4�C for 15 min prior to incubation at 4�C for

1 hr or 37�C for 1 or 3 hr. The positive control

sample (blue histogram) for measurement of

total surface labeling of IL-1RI was then fixed

in buffer (1% formaldehyde, 2% glucose, and

5 mM sodium azide) prior to staining with an

Alexa-488-conjugated secondary rat anti-

body (Molecular Probes) in permeabilization

buffer (PBS, 2% FCS, and 0.2% Saponin).

The negative control sample (green-filled his-

togram) was maintained at 4�C after IL-1 addition and then subjected to trypsin treatment to remove all surface proteins prior to staining for flow

cytometric analysis as above. Samples maintained at 37�C for 1 hr (green histogram) or 3 hr (pink histogram) were similarly subjected to trypsin

treatment and staining for flow cytometry. The data show progressive time-dependent loss of antibody-bound IL-1RI after internalization.

Figure S2. Antibodies against Polyubiquitin Chains Do Not Recognize Ubiquitinated IL-RI

(A) The FK1 antibody recognizes polyubiquitinated and not monoubiquitinated proteins. 293T cells were transfected with Flag IL-1Rs (IL-1RI +

IL-1RAcP) or Flag TRAF6 as a control. The Flag immunoprecipitates were probed with anti-Flag and then FK1 or P4D1, ubiquitin antibodies.

(B) IL-1RI is not recognized by ubiquitin antibody FK1. IL-1RI was immunoprecipitated from 293-Flag IL-1RI cells before and after stimulation

with IL-1b (20 ng/ml) for 30 min. Flag immunoprecipitates were probed with ubiquitin antibodies FK1 or P4D1.

S3

Figure S4. Tollip Is Ubiquitinated in a CUE-Domain-Dependent Manner

(A) 293T cells were transfected with His6-Xpress-tagged Tollip or TollipDCUE as indicated, lysed in 6 M guanidine-HCL, and incubated with

Ni2+-charged beads. His pull-downs were probed with antiubiquitin (Ub), anti-P4D1, or anti-Tollip as indicated.

(B) Proteins obtained by His Tollip pull-down were separated by 2D gel electrophoresis and analyzed by silver staining or by immunoblotting with

antibodies against Tollip or ubiquitin as indicated. Arrows indicate the Tollip and ubiquitin reactive spots. The other major Tollip reactive spots are

likely to be phosphorylated forms of Tollip because they migrate at the expected molecular weight for Tollip with increasing isoelectric point (pI).

Figure S3. Mutation of Tollip’s CUE Domain Abolishes Ubiquitin Binding

(A) Tollip but not TollipMF/AA binds endogenous ubiquitin and ubiquitinated proteins. 293T cells were cotransfected with VSV-Tollip or VSV-Toll-

ipMF/AA. VSV immunoprecipitates were probed with antiubiquitin and then VSV antibodies.

(B) Cellular extracts of 293T cells, expressing VSV-Tollip or VSV-TollipMF/AA, were mixed with GST-Ub or GST. The GST pull-downs (PD) were

analyzed by immunoblotting with antibodies against VSV or GST.

S4

Figure S5. Transfected IL-1RI Is Sorted to the

Cell Surface

(A) Twenty-four hours after transfection with

IL-1RI and IL-1RAcP, wild-type MEFs were

incubated with antibodies against IL-1RI

(6B5) for 1 hr at 4�C and then incubated at

37�C for 30 min in medium without (left) or

with (right) 50 ng/ml IL-1b. IL-1RI is shown

in green.

S5

Figure S6. A Significant Pool of Tollip Localizes to Late Endosomes

(A–C) A pool of Tollip localizes to late endosomes. Wild-type cells were consecutively immunostained with LBPA, LAMP1, and Tollip antibodies

as indicated. Tollip is shown in green; LAMP1 is shown in red; LBPA is shown in blue.

S6

(D–G) Merged images show that some Tollip associates with LAMP1- and LBPA-positive vesicles, characteristic of late endosomal association.

In the upper right panel, the boxed areas (numbered 1–4) have been enlarged to clearly show colocalization. Immunolabeling for Tollip was spe-

cific as confirmed by the absence of staining on Tollip-deficient cells.

(H–J) A small pool of Tollip colocalizes with EEA1-positive endosomes. +Tollip MEFs (Tollip-deficient cells reconstituted with VSV-Tollip) were

fixed, permeabilized, and costained with VSV and EEA1 antibodies. VSV-Tollip is shown in red; EEA1 is shown in green, and Draq 5 nuclear stain-

ing is shown in blue.

(K–M) Some Tollip colocalizes with Hrs. Wild-type cells were transfected with Myc-Hrs and costained with Tollip and Myc antibodies. Myc-Hrs is

shown in blue; Tollip is shown in green.

(N–P) Tollip does not colocalize with the transGolgi marker Golgin 97. Wild-type MEFs were costained with Golgin 97 and Tollip antibodies as

indicated. Golgin 97 is shown in red; Tollip is shown in green; Draq 5 nuclear staining is shown in blue.

(Q–S) A significant pool of Tollip localizes to LAMP1-positive vesicles in +Tollip MEFs. +Tollip MEFs were costained with Tollip and LAMP1 an-

tibodies, showing that Tollip’s has a similar distribution in +Tollip and wild-type MEFs (compare to [E]). LAMP1 is shown in red; Tollip is shown in

green; and Draq 5 nuclear staining is shown in blue. The right panel contains enlargements of boxed areas in merged images.

S7

Figure S7. Tollip Deficiency Does Not Affect Bulk Transport or Degradation of EGFR

(A) 2/2 and +/+ MEFs were pulsed with Cy3-conjugated dextran for 10 min and then chased in media for 50 min. After permeabilization, cells

were immunolabeled with LBPA or LAMP1 antibodies. LBPA or LAMP1, as indicated, is shown in green; Draq 5 nuclear staining is shown in blue;

and Dextran is shown in red. White boxes, numbered 1–4, highlight colocalization areas, which have been enlarged, in right panels.

(B) Tollip-deficient or wild-type cells were stimulated with EGF for the indicated times. Total cell extracts were prepared and EGF-induced deg-

radation of EGFR monitored by immunoblotting with EGFR antibodies. The same blot was reprobed with anti-Tollip or anti-tubulin, as indicated.

(C) Tollip does not bind EGFR. 293T cells were transfected with GFP-EGFR and VSV-Tollip or Myc-Hrs as a positive control. Transfected 293T

cells were serum starved for 24 hr prior to stimulation for 2 hr at 4�C with 100 ng/ml EGF. GFP-EGFR immunoprecipitates were revealed with

antibodies against VSV, Myc or EGFR as indicated.

S8

Figure S8. Tom1 May Link IL-1RI to the Endosomal Degradation Machinery

(A) Tom1 but not Tollip binds Hrs. 293T cells were cotransfected with either Myc Hrs and VSV-Tom1 or VSV-Tollip. The Myc immunoprecipitates

were revealed with antibodies against Myc or VSV as indicated.

(B) The Tom1 and IL-1Rs interaction is attenuated by removal of Tom1’s GAT domain (the region required for ubiquitin and Tollip interaction).

293T cells were cotransfected with either Flag IL-1Rs and VSV-Tom1 or VSV-Tom DGAT. The Flag immunoprecipitates were revealed with

antibodies against Flag or VSV as indicated.

(C) Overexpressed Tom1 partially localizes to LAMP1-positive vesicles. +Tollip MEFs were transfected with VSV-Tom1 and 24 hr later, were la-

beled with antibodies against Tollip, VSV, and LAMP1. Tollip is shown in green; LAMP1 is shown in blue; and VSV-Tom1 is shown in red. Arrows

indicate examples of Tollip, Tom1, and Lamp1 colocalization.

S9