Supplementary Figure 1. Both Kbss-HA-TEV-tagged US2 constructs are targeted to the ER
membrane and associate with heavy chains. a. ER-targeting and localization of HA-US2 and
HA-US2186 in U373-MG cells expressing these constructs. Analysis of the fate of tagged US2
molecules by pulse/chase in the presence of the proteasome inhibitor ZL3VS and
immunoprecipitation with anti-HA antibody followed by Endoglycosidase H (Endo H) digestion.
Continued Endo H sensitivity reveals US2 residency in the ER. The positions of the glycosylated
and deglycosylated US2 are indicated. IP, immunoprecipitation. b. HA-US2186, although
dislocation-incompetent, retains the ability to associate with HC. Inspection of US2
immunoprecipitates from HA-US2 and HA-US2186 cells analyzed by pulse/chase in the presence
of the proteasome inhibitor ZL3VS reveals the presence of associated HC molecules. IP,
immunoprecipitation. c. HC molecules that escape degradation in HA-US2186 cells enter the
secretory pathway. W6/32 immunoprecipitates obtained from HA-US2 and HA-US2186 cells
analyzed by pulse/chase in the presence of the proteasome inhibitor ZL3VS. IP,
immunoprecipitation. The W6/32 antibody recognizes assembled class I MHC trimeric
complexes composed of HC, 2microglobulin and antigenic peptide. W6/32-reactivity can be
used to assess HC progression through the secretory pathway12.
Supplementary Figure 2. Large-scale affinity purification of US2 and US2-associated proteins
from U373-MG control cells (-) or cells expressing HA-TEV-US2 or HA-TEV-US2186. Lysates
were incubated with anti-HA antibody and bound proteins were released by TEV cleavage and
revealed by silver staining. The bands indicated were excised and subject to mass spectrometry
analysis and database searching to identify the polypeptides present. Shown are the most
prominent proteins found in each band indicated. Shown in italic are proteins retrieved by both
US2 and US2186 in corresponding bands of lanes 2 and 3. We have not obtained or shown data
for all peptides present in the bands excised and have for now focused only on SPP; for most
proteins other than US2 and SPP, validation awaits further experiments.
Supplementary Figure 3. Mismatch of the SPP.1 shRNA sequence ablates its SPP knockdown
effect and confirms RNAi specificity. SPP and HC levels in cells expressing the indicated
shRNAs, assessed by immunoblot analysis with the indicated antibodies. -Actin levels were
used as loading control. Note also the dose-dependent effect of the SPP.1 shRNA: cells infected
with higher titer virus (SPP.1+) show a better SPP knockdown and a stronger impairment in HC
dislocation. Control, EGFP shRNA.
Supplementary Figure 4. Aminoacid sequences of wild type untagged US2 and all HA-tagged
US2 constructs used in these studies. The native US2 signal sequence, which has been replaced
by the Kbss-HA-TEV-spacer-tag in all tagged constructs is shown in italic. The transmembrane
domain aminoacid residues are underlined and those of the cytosolic tail, if present, are in
boldface type. Kb ss, murine H2-Kb signal sequence.
Supplementary Figure 5. The US2 TMD is dispensable for association with HC. a. Schematic
representation of Kbss-HA-TEV-tagged US2/CD4/US2 and US2TM/US2tail or CD4TM/US2tail
chimeric constructs. Kb ss, murine H2-Kb signal sequence. b. The US2/CD4/US2 chimeric
molecule associates with HC. Analysis of HA (US2) immunoprecipitates from cells expressing
HA-US2, HA-US2186, and HA-US2/CD4/US2 by pulse/chase in the presence of the proteasome
inhibitor ZL3VS. The position of HC molecules associated with US2 is indicated. IP,
immunoprecipitation.
Supplementary Figure 6. Both monomeric and dimeric forms of SPP are observed in
association with dislocation-competent US2; however the ratio of SDS-stable SPP
dimer/monomer is dependent on the treatment of samples before loading on the gel. a. SPP
immunoprecipitates obtained from SDS lysates of U373 cells radioactively labeled for 2 hr were
split into 5 aliquots, ressuspended in reducing sample buffer, and treated as indicated. IP,
immunoprecipitation; Endo H, Endoglycosidase treatment. b. Longer exposure of the western
blot shown in Fig. 2c shows that the SPP dimer is also seen in association with functional US2
and vice-versa. IP, immunoprecipitation. In this experiment as well as in those shown in
Supplementary Fig. 2 and Fig. 2b, samples were boiled for only 3 min at 1000C, which is not
sufficient to resolve the SPP monomer completely (see above).
Supplementary Figure 1. Both Kbss-HA-TEV-tagged US2 constructs are targeted to
the ER membrane and associate with heavy chains. a. ER-targeting and localization of
HA-US2 and HA-US2186 in U373-MG cells expressing these constructs. Analysis of the
fate of tagged US2 molecules by pulse/chase in the presence of the proteasome
inhibitor
ZL3VS
and
immunoprecipitation
with
anti-HA
antibody
followed
by
Endoglycosidase H (Endo H) digestion. Continued Endo H sensitivity reveals US2
residency in the ER. The positions of the glycosylated and deglycosylated US2 are
indicated. IP, immunoprecipitation. b. HA-US2186, although dislocation-incompetent,
retains the ability to associate with HC. Inspection of US2 immunoprecipitates from HAUS2 and HA-US2186 cells analyzed by pulse/chase in the presence of the proteasome
inhibitor
ZL3VS
reveals
the
presence
of
associated
HC
molecules.
IP,
immunoprecipitation. c. HC molecules that escape degradation in HA-US2186 cells enter
the secretory pathway. W6/32 immunoprecipitates obtained from HA-US2 and HAUS2186 cells analyzed by pulse/chase in the presence of the proteasome inhibitor
ZL3VS. IP, immunoprecipitation. The W6/32 antibody recognizes assembled class I
MHC trimeric complexes composed of HC, 2microglobulin and antigenic peptide.
W6/32-reactivity can be used to assess HC progression through the secretory
pathway12.
Supplementary Figure 2. Large-scale affinity purification of US2 and US2-associated
proteins from U373-MG control cells (-) or cells expressing HA-TEV-US2 or HA-TEVUS2186. Lysates were incubated with anti-HA antibody and bound proteins were
released by TEV cleavage and revealed by silver staining. The bands indicated were
excised and subject to mass spectrometry analysis and database searching to identify
the polypeptides present. Shown are the most prominent proteins found in each band
indicated. Shown in italic are proteins retrieved by both US2 and US2186 in
corresponding bands of lanes 2 and 3. We have not obtained or shown data for all
peptides present in the bands excised and have for now focused only on SPP; for most
proteins other than US2 and SPP, validation awaits further experiments.
Supplementary Figure 3. Mismatch of the SPP.1 shRNA sequence ablates its SPP
knockdown effect and confirms RNAi specificity. SPP and HC levels in cells expressing
the indicated shRNAs, assessed by immunoblot analysis with the indicated antibodies.
-Actin levels were used as loading control. Note also the dose-dependent effect of the
SPP.1 shRNA: cells infected with higher titer virus (SPP.1 +) show a better SPP
knockdown and a stronger impairment in HC dislocation. Control, EGFP shRNA.
Supplementary Figure 4. Aminoacid sequences of wild type untagged US2 and all HAtagged US2 constructs used in these studies. The native US2 signal sequence, which
has been replaced by the Kbss-HA-TEV-spacer-tag in all tagged constructs is shown in
italic. The transmembrane domain aminoacid residues are underlined and those of the
cytosolic tail, if present, are in boldface type. Kb ss, murine H2-Kb signal sequence.
Supplementary Figure 5. The US2 TMD is dispensable for association with HC. a.
Schematic representation of Kbss-HA-TEV-tagged US2/CD4/US2 and US2TM/US2tail
or CD4TM/US2tail chimeric constructs. Kb ss, murine H2-Kb signal sequence. b. The
US2/CD4/US2 chimeric molecule associates with HC. Analysis of HA (US2)
immunoprecipitates from cells expressing HA-US2, HA-US2186, and HA-US2/CD4/US2
by pulse/chase in the presence of the proteasome inhibitor ZL 3VS. The position of HC
molecules associated with US2 is indicated. IP, immunoprecipitation.
Supplementary Figure 6. Both monomeric and dimeric forms of SPP are observed in
association with dislocation-competent US2; however the ratio of SDS-stable SPP
dimer/monomer is dependent on the treatment of samples before loading on the gel. a.
SPP immunoprecipitates obtained from SDS lysates of U373 cells radioactively labeled
for 2 hr were split into 5 aliquots, ressuspended in reducing sample buffer, and treated
as indicated. IP, immunoprecipitation; Endo H, Endoglycosidase treatment. b. Longer
exposure of the western blot shown in Fig. 2c shows that the SPP dimer is also seen in
association with functional US2 and vice-versa. IP, immunoprecipitation. In this
experiment as well as in those shown in Supplementary Fig. 2 and Fig. 2b, samples
were boiled for only 3 min at 1000C, which is not sufficient to resolve the SPP monomer
completely (see above).