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INTRODUCTION |
Signaling from cell surface receptors is attenuated by their
ligand-induced endocytosis and degradation in lysosomes (1, 2). The
molecular machinery that diverts endocytosed membrane proteins from the
default recycling pathway and sorts them into the degradative pathway
is currently being elucidated. Two structurally related proteins, Hrs
(hepatocyte growth factor-regulated tyrosine kinase substrate) and STAM
(signal-transducing adaptor
molecule), have attracted interest in this context (3, 4).
Both these proteins are tyrosine-phosphorylated upon growth factor and
cytokine stimulation (5-8), and there is evidence that that they serve to sort endocytosed membrane proteins to lysosomes (9).
Hrs contains multiple domains, including an N-terminal VHS
domain, a FYVE domain that binds specifically to
phosphatidylinositol 3-phosphate (10), a coiled-coil domain, and a
clathrin-binding domain (11-14). The FYVE and coiled-coil
domains target Hrs specifically to early endosomes (10, 15). Electron
microscopy studies of Hrs mutant Drosophila larvae revealed
an impairment in endosome membrane invagination and formation of
multivesicular bodies (MVBs)1
(16), suggesting that Hrs may play a crucial role in MVB formation and
membrane trafficking from endosomes to degradative compartments. Ubiquitination functions as a sorting signal for the degradative endocytic pathway (17, 18), and the binding of Hrs to ubiquitinated proteins via its ubiquitin-interacting motif (UIM) appears to be
important for their sorting (19).
The two related proteins STAM1 and STAM2 (also known as Hbp
(Hrs-binding protein)) share an
N-terminal VHS domain, like Hrs. These proteins also contain a
Src homology 3 domain, a coiled-coil domain, and an immunoreceptor
tyrosine-based activation motif (7). They both interact with Janus
kinases and appear to function downstream of Janus kinases 3 and 2 in
cytokine signaling (8, 20). A double knockout of STAM1 and STAM2 in
mice is early embryonic lethal (21). Deletion mutants of STAM1 and
STAM2 inhibit cytokine signaling in transfected lymphocytes (20), and
conditional double knockout of STAM1 and STAM2 in mouse T-lymphocytes
causes abnormalities in their development and survival (21). In
contrast, mice with single knockouts of STAM1 or STAM2 are viable and
have normal lymphocyte functions, indicating that the two proteins have
redundant functions (21, 22). Besides their roles in signal
transduction, STAM proteins may also regulate endocytic membrane
trafficking. A deletion mutant of STAM2 that is unable to bind Hrs
suppresses the degradation of platelet-derived growth factor and its
receptor but not the internalization of platelet-derived growth factor (23). This suggests that an interaction of STAM2 with Hrs is essential
for the degradative sorting of platelet-derived growth factor and its receptor.
STAM1 and STAM2 form a tight interaction with Hrs via the same
coiled-coil domain that targets Hrs to the early endosomal membrane (6,
8, 23). This has raised three possible models for the membrane
recruitment of Hrs and STAM proteins. 1) STAM proteins may recruit Hrs
to endosomes. 2) Hrs may recruit STAM proteins to endosomes. 3)
Hrs-STAM complex formation and membrane association may be
mutually exclusive. In this report we have tested these models and
tried to elucidate the function of the STAM-Hrs complex. We have also
investigated the relationship between the Hrs-STAM complex and the
ubiquitin-binding protein Eps15.
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MATERIALS AND METHODS |
Antibodies--
Rabbit antibodies against recombinant Hrs have
been described before (14). Antisera against STAM1 and STAM2 were made
by Eurogentec (Herstal, Belgium) by injecting rabbits with a synthetic peptide corresponding to residues 315-329 of STAM1 coupled to keyhole
limpet hemocyanin or with recombinant GST-STAM2. The STAM2 antiserum
was affinity-purified on Affi-Gel beads (Bio-Rad) containing immobilized MBP-STAM2, and the STAM1 antiserum was affinity-purified by
coupling the same peptide that was used for injecting the rabbits to a
SulfoLink Coupling Gel (Pierce) according to the manufacturer's instructions. Control experiments demonstrated that the antibodies against Hrs, STAM1, and STAM2 did not cross-react. Anti-Myc antibodies were from the 9E10 hybridoma (24). Mouse monoclonal antibodies against
the VSV-G epitope were from Roche Molecular Biochemicals. Mouse
monoclonal antibodies against conjugated mono- and polyubiquitin (FK2)
or conjugated polyubiquitin only (FK1) (25) were from Affiniti Research
Products (Exeter, UK). Human anti-EEA1 serum (26) was a gift from
Ban-Hock Toh (Melbourne, Australia). Rabbit anti-Eps15 (C terminus) and
goat anti-clathrin were from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). Sheep anti-EGF-receptor was from Fitzgerald (Concord, MA).
Rhodamine-, fluorescein isothiocyanate-, Cy2-, Cy3-, Cy5-, and
horseradish peroxidase-labeled secondary antibodies were from Jackson
Immunoresearch (West Grove, PA).
Plasmid Constructs and Small Interfering RNA (siRNA)--
The
Hrs constructs indicated were generated by PCR with mouse Hrs (5) as
the template. The STAM constructs used were generated by PCR with human
STAM1 (7) and mouse STAM2 (Hbp) (23) as the templates. For use in the
two-hybrid system, constructs were cloned into pLexA/pBTM116 (27) as
bait and pGAD GH (Clontech, Palo Alto, CA) as prey.
For expression in mammalian cells with the T7 RNA polymerase vaccinia
virus system, constructs were cloned behind the Myc epitope of
pGEM-Myc4 (28). Mouse Skd1E235Q/Vps4E235Q was
generated by PCR amplification with pTKS-mVps4E235Q (kindly
provided by Markus Babst and Scott Emr, University of California, San
Diego, La Jolla, CA) as a template and cloned behind the VSV-G
epitope of pGEM-VSVG (28). For bacterial expression of
His6-tagged Eps15, the Eps15 sequence from pEGFP-Eps15 (29) (kindly provided by Alexandre Benmerah) was cloned into the expression vector pHAT (30). For siRNA experiments, 21-nucleotide RNA duplexes with 2-nucleotide 3'-(2-deoxy)thymidine overhangs were synthesized by
Dharmacon Research, Inc. (Lafayette, CO). RNA sequences were as
follows: sense, 5'-CGA CAA GAA CCC ACA CGU CdTdT; antisense, 5'-GAC GUG
UGG GUU CUU GUC GdTdT. Control sequence was as follows: sense, 5'-GGG
GCG AGG CAG CGG CAC CdTdT.
Cell Culture and Transfection--
BHK-21, HEp-2, and HeLa cell
cultures were maintained as recommended by ATCC. The pGEM-VSVG-STAM and
pGEM-Myc-Hrs constructs and pGEM-Myc-mVps4E235Q were
expressed in BHK-21 cells using modified Ankara T7 RNA polymerase recombinant vaccinia virus (31) and lipofection as described (32).
Transfection of HeLa cells with siRNA was performed as described (33).
The cells were first transfected with siRNA for 3 days, and then the
cells were replated and the transfection was repeated for another 3 days.
Confocal Immunofluorescence Microscopy--
BHK cells grown on
coverslips were fixed with 3% paraformaldehyde after transfections and
stained for fluorescence microscopy as described (28). When indicated,
the cells were permeabilized with 0.05% saponin prior to fixation.
Coverslips were examined using a Leica TCS NT confocal microscope
equipped with a krypton/argon laser and a PL Fluotar ×100/1.3 oil
immersion objective or a Zeiss LSM 510 META microscope equipped with a
Neo-Fluar ×100/1.45 oil immersion objective.
Protein Expression and Purification--
GST fusion proteins of
STAM1 and STAM2 was produced in E. coli BL21 (DE3) cells
transformed with the respective pGEX constructs as described previously
(14). The recombinant protein was purified on glutathione-Sepharose 4B
(Amersham Biosciences) after lysis of the bacteria in
B-PERTM reagent (Pierce) according to the instructions from
the manufacturers. Expression of MBP-Hrs has been described previously
(14). His6-Eps15 was expressed in Escherichia
coli BL21 (DE3) cells transformed with pHAT-Eps15.
Pull-down Assays--
For measurements of ubiquitin binding,
aliquots (10 µl) of protein A-agarose (Sigma) or ubiquitin-agarose
(Sigma) were washed three times with assay buffer A (25 mM
HEPES, pH 7.2, 125 mM potassium acetate, 2.5 mM
magnesium acetate, 5 mM EGTA, and 1 mM
dithiothreitol) before incubation with 0.02 nmol of GST-STAM2 in 300 µl of assay buffer A containing 10% fetal calf serum and 0.1%
Triton X-100 for 1 h at room temperature. Finally, the beads were
washed four times in assay buffer and resuspended in SDS-PAGE sample
buffer. STAM2 associated with the beads was detected by SDS-PAGE
followed by immunoblotting with the rabbit anti-STAM2 antibody. For
measurements of ternary complex formation, bacterially expressed GST,
GST-STAM1 or GST-STAM2 (30 pmol) were mixed in 1 ml of assay buffer B
(20 mM Hepes, pH 7.2, 140 mM NaCl, 1 mM dithiothreitol) with glutathione-Sepharose beads (10 µl) and recombinant His6-Eps15 (30 pmol) in the absence or presence of recombinant MBP-Hrs (30 pmol). After 1 h at
rotation at 4 °C, the beads were washed extensively with assay
buffer B and analyzed by SDS-PAGE and Western blotting with
anti-Eps15.
Preparation of Membrane and Cytosolic Fractions of HEp-2
Cells--
HEp-2 cells were washed three times with ice-cold
phosphate-buffered saline and homogenized in homogenization buffer (10 mM HEPES, 3 mM imidazole, pH 7.2, 250 mM sucrose, mammalian protease inhibitor mixture (Sigma))
by repeated passages through a 22-gauge needle at 4 °C. Membrane
particulate and cytosolic fractions were prepared from postnuclear
supernatants by ultracentrifugation for 15 min at 65,000 rpm in a
TLA-100 rotor, using a Beckman table top ultracentrifuge.
Coimmunoprecipitation of Eps15, STAM1, and STAM2 with
Hrs--
HEp-2 cells were fractionated as described, and the membrane
particulate pellet was dissolved in lysis buffer (125 mM
KAc, 25 mM HEPES, 25 mM MgAc, 5 mM
EGTA, 0.5% Nonidet P-40, 1 mM dithiothreitol, pH 7.2, mammalian protease inhibitor mixture (Sigma)). The two fractions were
incubated with protein A-Sepharose (Amersham Biosciences) preincubated
with 20 µl of affinity-purified anti-Hrs after preclearance with
protein A-Sepharose only. Immunoprecipitates were washed three times
with washing buffer (125 mM KAc, 25 mM HEPES,
2.5 mM MgAc, 5 mM EGTA, 0.1% Nonidet P-40, 1 mM dithiothreitol, pH 7.2, proteinase inhibitor mixture),
eluted in sample buffer for SDS-PAGE, and run on 10% gels. Following
SDS-PAGE, proteins were transferred to 0.45-µm pore size
polyvinylidene difluoride membranes and incubated with primary and
secondary antibodies before detection with the SuperSignal
chemiluminescence kit (Pierce).
Coimmunoprecipitation of Hrs and Eps15 with STAM2--
HEp-2
cells were starved for 4 h and stimulated or not with 100 ng/ml
EGF for 8 min. The cells were lysed, and STAM2 was immunoprecipitated as described, using 25 µl of affinity-purified anti-STAM2. Complexes of STAM2, Hrs, and Eps15 were eluted in sample buffer for SDS-PAGE and
run on 10% gels. Proteins were transferred to 0.45-µm pore size
polyvinylidene difluoride membranes following SDS-PAGE and incubated
with primary and secondary antibodies before detection with the
SuperSignal chemiluminescence kit.
Quantitation of EGF-R in Cells Treated with siRNA against
Hrs--
HeLa cells were transfected twice at 72-h intervals with a
siRNA duplex specific for nucleotides 161-180 of the Hrs coding sequence, with a sense RNA as a negative control. Hrs levels were analyzed by Western blotting of total cell lysates. The amount of total
EGF-R was measured by immunofluorescence after a 30-min pulse of 200 ng/ml EGF and undegraded EGF-R after a 3-h chase in the presence of 10 µg/ml cycloheximide to prevent synthesis of new EGF-R. The cells were
permeabilized with 0.05% saponin prior to fixation and stained with
anti-EGF-R. The mean intensity of the Cy2 EGF-R signal from 20 siRNA-treated and 20 control cells after 3 h of chase was
quantified and was presented as the percentage of total EGF-R. The mean
intensity of total EGF-R was quantified by counting 20 control and 20 siRNA cells after a 30-min EGF pulse. No significant differences were
found in the amount of total EGF-R in control and siRNA cells at this
time point. Error bars denote S.E.
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RESULTS |
STAM Proteins and Eps15 Are Coimmunoprecipitated with Hrs from
Cytosol and Membrane Fractions--
Since the coiled-coil domain of
Hrs binds STAM1/STAM2 (6, 23) and is essential for the localization of
Hrs to early endosomes (15), this raises the question of whether Hrs
can bind to both STAM proteins and the endosomal membrane at the same
time. We investigated this by immunoprecipitating Hrs from the cytosol and membrane fractions of HEp-2 cells and studied whether STAM1 and
STAM2 coprecipitated from both. As shown in Fig.
1, although Hrs was most abundant in the
cytosolic fraction, STAM1 and STAM2 coprecipitated with Hrs from both
the membranes and the cytosol. No STAM1/STAM2 was detected in the
absence of anti-Hrs (not shown). These results indicate that the
association between Hrs and STAM is not disturbed by membrane
localization and that STAM proteins and Hrs are present in a complex
both in the cytosol and on membranes.

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Fig. 1.
Hrs is found in complex with STAM proteins
and Eps15 both in membrane and cytosol fractions. HEp-2 cells were
fractionated into a membrane and cytosolic fraction as described under
"Materials and Methods." Hrs was immunoprecipitated from both
fractions, and coimmunoprecipitation of STAM1, STAM2, and Eps15 was
analyzed by SDS-PAGE and Western blotting. No STAM1, STAM2, Hrs, or
Eps15 were detected in mock immunoprecipitates without anti-Hrs (not
shown). Bands corresponding to Ig heavy chain (HC) and light
chain (LC) are indicated with arrows.
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Besides STAM proteins, Eps15, a protein that regulates endocytosis, has
also been found to interact directly with Hrs (34). To study whether
Eps15 is associated with Hrs in cytosol or on membranes, we therefore
also probed the blot with an antibody to Eps15 (Fig. 1). The result
showed that Eps15, like STAM proteins, coimmunoprecipitates with Hrs
both from cytosol and membrane fractions. Eps15 has been shown to
relocalize to the plasma membrane and subsequently to EGF-R-positive
endosomes upon activation of the EGF-R kinase (35). The finding that
Eps15 interacts with Hrs in membrane fractions suggests that Hrs could
be important in order to determine the relocalization of Eps15 during endocytosis.
Quantitative Western blotting experiments using recombinant proteins as
reference standards indicated that approximately equimolar amounts of
Eps15 were coimmunoprecipitated with Hrs, whereas the amount of STAM
proteins co- immunoprecipitated varied between equimolar amounts
and 20% (not shown). Whereas the reason for this variation is not
known, these results are consistent with the possibility that Hrs is
present in stoichiometric complexes with Eps15 and STAM1/STAM2.
STAM Proteins, Eps15, and Hrs Are Found in a Ternary
Complex--
The binding sites in Hrs for STAM2 and Eps15 are
distinct, but it is not known if Hrs is capable of interacting with
these proteins in a ternary complex. We therefore investigated whether both Hrs and Eps15 could be coimmunoprecipitated with STAM2. We found,
by analyzing the samples on SDS-PAGE and Western blotting, that all
three proteins were present in the STAM2 immunoprecipitate (Fig.
2A). The extent of
coimmunoprecipitation was independent of EGF stimulation, which is
known to cause the phosphorylation and ubiquitination of Hrs, STAM
proteins, and Eps15 (5, 6, 8, 23, 36-39). This indicates that Hrs
interacts with Eps15 and STAM2 in a manner that is independent of
phosphorylation and ubiquitination of the individual components.

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Fig. 2.
Hrs, STAM proteins, and Eps15 form a ternary
complex. A, HEp-2 cells were starved for 4 h
before stimulating (+) or not ( ) with 100 ng/ml EGF. STAM2 was
immunoprecipitated from the lysates as described under "Materials and
Methods," and the immunoprecipitates were analyzed by SDS-PAGE
followed by Western blotting with anti-STAM2, anti-Hrs, and anti-Eps15.
B, GST (lane 1), GST + MBP-Hrs
(lane 2), GST-STAM1 (lane
3), GST-STAM1 + MBP-Hrs (lane 4),
GST-STAM2 (lane 5), or GST-STAM2 + MBP-Hrs
(lane 6) were incubated with recombinant
His6-Eps15 and glutathione-Sepharose beads, and the beads
were analyzed for their content of His6-Eps15 by Western
blotting as described under "Materials and Methods." The amount of
His6-Eps15 detected in lanes 4 and
6 corresponds to about 10% of the input amount.
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Previous work has shown that the chick homolog of STAM2, EAST,
coimmunoprecipitates with Eps15 (40). Both EAST and STAM contain an
N-terminal NPF motif that, in principle, might bind to the epsin
homology domains of Eps15. Although mutagenesis of the NPF motif
in EAST did not abolish its coimmunoprecipitation with Eps15 (40), we
could not rule out the possibility that Eps15 may interact directly
with STAM1/STAM2 and thus participate in two different complexes, one
with STAM1/STAM2 and one with Hrs. To investigate this possibility, we
incubated GST-STAM1 and GST-STAM2 with recombinant Eps15 in the absence
or presence of recombinant Hrs. We retrieved the GST fusion proteins
with glutathione-Sepharose beads and analyzed their possible content of
Eps15 by Western blotting. As shown in Fig. 2B, Eps15 did
not associate with beads containing GST-STAM1 or GST-STAM2. However, in
the presence of Hrs, Eps15 was associated with both GST-STAM1 and
GST-STAM2. This indicates that Hrs is able to bridge STAM proteins with
Eps15 and that Hrs, STAM proteins, and Eps15 are present in the same complex.
STAM2 Is Recruited to Early Endosomes by Hrs but Not Vice
Versa--
Given that Hrs interacts with STAM1 and STAM2 and that the
association to endosomal membranes does not appear to interfere with
this interaction, we addressed whether Hrs recruits STAM proteins to
membranes or if STAM proteins may recruit Hrs. Because our STAM2
antibody was superior to that of STAM1 for immunocytochemistry, we
restricted our investigation to STAM2. By overexpressing STAM2, we
found that it was mainly cytosolic (Fig.
3, A and C), and it had no effect on the endosomal distribution of endogenous Hrs (Fig. 3,
B and D). When we permeabilized the cells prior
to fixation, almost all of the overexpressed STAM2 was extracted (Fig.
3C), in agreement with its predominant localization to the
cytosol. In contrast, when STAM2 was coexpressed together with Hrs
(Fig. 3, E and F), it colocalized strongly with
Hrs on clustered vesicles positively stained for the early endosomal
marker EEA1 (26) (Fig. 3G). Overexpression of Hrs alone
(Fig. 3J) gave the same pattern of Hrs on clustered
EEA1-positive vesicles (Fig. 3K). Significantly, we observed
a very strong endosomal staining of endogenous STAM2 in cells
transfected with Hrs (Fig. 3I), whereas the staining of
endogenous STAM2 in untransfected cells gave a weaker signal (Fig.
3M). This is probably due to a strong recruitment of STAM2
from cytosol to endosomes in the presence of overexpressed Hrs. Even in
untransfected cells, STAM2 colocalized extensively with EEA1 (Fig. 3,
M-O), indicating that a portion of endogenous STAM2 is
indeed associated with early endosome membranes.

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Fig. 3.
Recruitment of STAM2 to early endosomes by
Hrs. BHK cells were transfected with VSV-G-tagged STAM2 alone
(A-D), cotransfected with VSV-G-tagged STAM2 and Myc-tagged
Hrs (E-H), or transfected with Myc-tagged Hrs alone
(I-L). The cells were either fixed directly (A
and B) or permeabilized before fixation (C-O).
They were stained with anti-VSV-G (A, C, and
E), anti-Hrs (B, D, and F),
anti-Myc (J), anti-STAM2 (I and M), or
anti-EEA1 (G, K, and N).
Merged images are seen in H,
L, and O. Transfected cells are pointed out with
arrowheads. Examples of colocalizing profiles are seen in
white (H and L) or yellow
(O). Untransfected cells are pointed out with
arrows. Bar, 5 µm.
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The above results indicated that overexpression of Hrs causes an
increased recruitment of STAM2 to endosomes. To study whether Hrs is
required for the membrane recruitment of STAM2, we attempted to deplete
HeLa cells of Hrs by using siRNA (41). Control cells (treated with a
Hrs-specific sense RNA) and siRNA-treated cells were fractionated into
cytosol and membrane fractions, and the levels of Hrs and STAM2 were
analyzed by Western blotting. We loaded relatively higher amounts of
membrane fractions, since Hrs is most abundant in the cytosol (see Fig.
1). As shown in Fig. 4A
(upper panel), siRNA treatment caused a strong
reduction in Hrs levels both in the cytosol (lanes
1 and 2) and membrane fractions (lanes
3 and 4). Importantly, the decreased level of Hrs
in the membrane fraction was accompanied by a strong reduction in
membrane-associated STAM2 (middle panel,
lanes 3 and 4). This indicates that
the recruitment of STAM2 to membranes requires Hrs.

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Fig. 4.
Endosomal localization of STAM2 depends on
Hrs. A, HeLa cells treated with control RNA ( ) or
with siRNA against Hrs (+) were fractionated into membrane and
cytosolic fractions as described under "Materials and Methods." Hrs
left in the cytosol and on membranes after siRNA treatment as described
under "Materials and Methods" was analyzed by SDS-PAGE
(upper panel), and the corresponding levels of
STAM2 were shown by sequential blotting of the same membrane with
anti-STAM2 (middle panel). Because Hrs is most
abundant in the cytosol fraction (see Fig. 1), the loaded amount of
membrane fraction was 5-fold higher than that of the cytosol fraction.
To visualize transferred proteins, the blot was stained with Ponceau S
(lower panel) prior to detection of Hrs and
STAM2. B, HeLa cells treated with control RNA
(upper panels) or with siRNA against Hrs
(lower panels) were fixed on coverslips and
stained with antibodies against STAM2 and EEA1. Merged
images are shown to the right, with
yellow indicating colocalization. Bar, 5 µm.
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To assess whether the reduced association of STAM2 with the membrane
fraction of Hrs-depleted cells reflected an impaired recruitment to
endosomes, we studied the siRNA-treated cells by confocal
immunofluorescence microscopy (Fig. 4B). In control cells, STAM2 could be readily detected on EEA1-positive endosomes. By contrast, in siRNA-treated cells, a diffuse, cytosolic STAM2 staining could be observed, with only faint labeling of endosomes.
Interestingly, enlarged endosomes could be observed in siRNA-treated
cells, reminiscent to the phenotype observed in Hrs
/
mouse embryos
(42). Taken together, these results indicate that whereas Hrs is
independent of STAM2 for the binding to endosomes, Hrs is required for
the recruitment of STAM2 to endosomes.
Hrs and STAM2 Accumulate on Aberrant Endosomes Induced by
Expression of an ATPase-deficient Form of mVps4--
The yeast
homologue of Hrs, Vps27p, was identified in a screen for vps
mutants defective in vacuolar protein sorting (43-46). vps27 belongs to the class E vps mutants, which
accumulate vacuolar, endocytic, and trans-Golgi markers in
an aberrant multilamellar structure, the class E compartment (47-49).
An AAA ATPase, Vps4p, may serve to modulate interactions between other
class E proteins, and it has been demonstrated that Vps4p influences
binding of the class E proteins Vps2p, Vps20p, Vps23p, Vps24p, and
Vps32p/Snf7p to endosomal membranes (18, 50, 51). In addition, it has been shown that expression of ATPase-defective mammalian Vps4 (mVps4),
mVps4E235Q, causes a portion of TSG101 (the mammalian
homologue of Vps23p) and human VPS28 to become associated with aberrant
endosomes (52). To study whether the membrane localization of Hrs and
STAM2 is regulated in a similar manner, we transfected cells
transiently with mVps4E235Q (Fig.
5, A and D).
Confocal microscopy showed a distinct staining of both Hrs and STAM2 on
the aberrant endosomal structures induced (53, 54). Compared with
untransfected cells, in which a speckled, cytosolic staining pattern
was seen, the transfected cells contained little cytosolic Hrs and
STAM2 and showed instead a strong, vesicular staining (Fig. 5,
B and E). This staining showed strong
colocalization with EEA1 (Fig. 5, C and F), which
is consistent with the idea that mVps4 regulates the morphology and the
transport functions of endosomes (54). Based on these findings, we
suggest that mVps4 is involved in the regulation of a membrane complex
containing Hrs, STAM, and additional proteins and that ATP hydrolysis
is an important step of this regulation.

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Fig. 5.
Recruitment of Hrs and STAM2 from cytosol to
enlarged endosomes in mVps4E235Q-overexpressing BHK
cells. BHK cells were transfected with VSV-G-tagged
mVps4E235Q and fixed directly. The cells were stained with
antibodies against the VSV-G epitope (A and D),
endogenous Hrs (B), EEA1 (C and F), or
endogenous STAM2 (E). Transfected cells are pointed out with
large arrows, untransfected cells are shown with
arrowheads, and examples of mVps4E235Q-induced
endosomal structures stained for Hrs and EEA1 (B and
C) or STAM2 and EEA1 (E and F) are
pointed out with small arrows. Bar, 5 µm.
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STAM2 Binds Ubiquitin--
Sequence analyses show that Hrs, Eps15,
STAM1, and STAM2 contain UIMs (55). It has recently been demonstrated
that Hrs binds ubiquitin (16, 56) and that the binding occurs directly
via the UIM (16, 19, 38, 56). Furthermore, Hrs binds ubiquitinated receptors and colocalizes with ubiquitinated proteins in endosomal microdomains (19). The Hrs/ubiquitin interaction appears to be involved
in the sorting of receptors from the recycling to the degradative
pathway by retaining ubiquitinated receptors in a clathrin-coated area
of the sorting endosome that is not involved in recycling (19). Due to
the low affinity of Hrs to monoubiquitin (KD = 300 µM), it is possible that other proteins in complex with
Hrs may also interact with neighboring ubiquitin molecules and thereby
increase the avidity. Since also Eps15 has been found to bind ubiquitin
(38), we investigated whether STAM2 is capable of binding ubiquitin.
For this purpose, we incubated recombinant STAM2 in the presence of
agarose beads to which ubiquitin had been covalently bound. By SDS-PAGE
and Western blotting, we found that STAM2 was pelleted with the
ubiquitin beads (Fig. 6, lane
2) but not with control beads containing protein A instead of ubiquitin (Fig. 6, lane 1). This indicates
that STAM2, like Hrs and Eps15, interacts directly with ubiquitin.

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Fig. 6.
STAM2 interacts directly with ubiquitin.
Protein A-agarose (lane 1) or ubiquitin-agarose
(lane 2) was incubated with GST-STAM2. The
samples were prepared for SDS-PAGE and Western blotting, and the STAM2
associated with the beads was detected with anti-STAM2 antibody.
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Ubiquitinated Proteins Localize to the Same Microdomains as STAM2
on Endosomes--
If STAM2 is involved in the sorting of ubiquitinated
proteins together with Hrs, we would expect to find STAM2 and
ubiquitinated proteins in the same microdomains of the endosomal
membrane. When cells are transfected with a GTPase-defective mutant of
Rab5, Rab5Q79L, enlarged early endosomes are formed due to
increased fusion (57), and the detection of endosomal microdomains by
confocal immunofluorescence microscopy is facilitated (14). We
therefore transfected BHK cells with Rab5Q79L and stained
the fixed cells with antibodies that recognize endogenous STAM2 and
conjugated ubiquitin (FK2) as well as EEA1. We found that in contrast
to EEA1, which is found in microdomains distinct from STAM2 (Fig.
7, D-F),
ubiquitinated proteins colocalized with STAM2 on enlarged endosomes
(Fig. 7, A-C). This is in agreement with a previous finding
that Hrs colocalizes with ubiquitinated proteins in endosomal
microdomains (19). Due to the finding that Hrs is monoubiquitinated
(38), we included an antibody (FK1) that specifically recognizes
polyubiquitinated proteins. Polyubiquitinated proteins also showed
colocalization with both STAM2 (Fig. 7, G-I) and Hrs (Fig.
7, J-L). This indicates that the STAM2/Hrs microdomains
contain ubiquitinated proteins other than Hrs itself. A general
colocalization between STAM2 and ubiquitin-conjugated proteins on early
endosomes was also seen in untransfected cells (not shown). These
results support the idea that STAM2 not only binds ubiquitin but also
colocalizes with ubiquitinated proteins on microdomains of early
endosomes and thereby may take part in their sorting together with
Hrs.

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Fig. 7.
Localization of STAM2, EEA1, and conjugated
ubiquitin in Rab5Q79L-overexpressing BHK cells. BHK
cells were transfected with pGEM-Rab5Q79L and permeabilized
prior to fixation. The cells were stained with anti-STAM2 (A
and G), anti-EEA1 (D), or anti-Hrs (J)
and double labeled with anti-ubiquitin FK2, which recognizes both mono-
and polyubiquitinated proteins (B and E) or
anti-ubiquitin FK1, which is specific to polyubiquitinated proteins
(H and K). Merged images
are shown in C, F, I, and
L. Insets show examples of endosomes that are
sufficiently large to enable a distinction between different membrane
domains. Bar, 5 µm.
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Clathrin and Eps15 Accumulate on Aberrant Endosomes Induced by
Expression of mVps4E235Q--
Previous work has shown that
Hrs interacts with clathrin (14) and that Hrs localizes to endosomal
microdomains characterized by a flat bilayered clathrin coat (14, 58).
Given that mVps4 regulates the membrane association of Hrs, we
therefore reasoned that mVps4 may regulate the association of clathrin
with endosomes. To investigate this, we transfected cells with
mVps4E235Q and stained the fixed cells with antibodies
against Hrs and clathrin. Interestingly, the enlarged endosomes caused
by mVps4E235Q were stained strongly for clathrin (Fig.
8, A-C), indicating that
mVps4, probably through its regulation of Hrs, is able to control the
recruitment of clathrin to endosome membranes.

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Fig. 8.
Localization of clathrin and Eps15 in
mVps4E235Q-expressing cells. BHK cells were
transfected with mVps4E235Q and permeabilized before
fixation. They were then stained with anti-clathrin (A),
anti-Hrs (B), anti-Eps15 (D), or anti-EEA1
(E). Yellow in the merged
images (C and F) indicates
colocalization. Examples of colocalizing profiles are pointed out with
arrows. Bar, 5 µm.
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Because Eps15 interacts with Hrs (34) and coimmunoprecipitates with Hrs
from membranes (Fig. 1), we also investigated whether the endosomal
localization of Eps15 is controlled by mVps4. As shown in Fig. 8,
D-F, Eps15 accumulates strongly on
EEA1-positive large endosomes induced by mVps4E235Q. These
results suggest that ATP hydrolysis by mVps4 regulates the membrane
localization of an entire complex consisting of Hrs and a number of
associated proteins, in agreement with the proposed function of Vps4p
in yeast (50).
Eps15 Localizes to the Same Microdomains as Ubiquitinated Proteins
and Clathrin--
Due to the ubiquitin interacting properties of
Eps15, we asked whether Eps15, like STAM2, is present in the same
microdomains on early endosomes as Hrs and ubiquitinated proteins. We
transfected BHK cells with the Rab5Q79L mutant as described
above and stained with antibodies against Eps15, EEA1, clathrin, and
polyubiquitinated proteins (FK1). Clathrin has been shown to localize
to the same microdomains as Hrs (14), and because our antibodies
against Hrs and Eps15 are both raised in rabbit, we used a goat
anti-clathrin antibody to study whether Eps15 localizes to the
Hrs/clathrin microdomains. We found that, like Hrs and STAM2, Eps15 did
not colocalize with EEA1-containing microdomains (Fig.
9, A-C) but was instead found
in the same areas as clathrin (Fig. 9, D-F) and
polyubiquitinated proteins (Fig. 9, G-I) on enlarged early
endosomes. This illustrates that Eps15, like Hrs and STAM2, localizes
to the same microdomains as its potential ligands, ubiquitinated
proteins.

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Fig. 9.
Eps15 colocalizes with clathrin but not with
EEA1 on Rab5Q79L-enlarged endosomes. BHK cells were
transfected with pGEM-Rab5Q79L and permeabilized prior to
fixation. The cells were stained with anti-Eps15 (A,
D, and G) and double-labeled with anti-EEA1
(B), anti-clathrin (E), or anti-ubiquitin FK1,
which recognizes polyubiquitinated proteins only (H).
Merged images are shown in C,
F, and I. Bar, 5 µm.
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Hrs Is Essential for Efficient Degradation of the Epidermal Growth
Factor Receptor--
Given that Hrs is needed for recruitment of other
ubiquitin-binding proteins to the early endosome and that this process
is important for proper sorting of ubiquitinated receptors to the degradative pathway, we would expect Hrs to be important for
degradation of the EGF-R, whose down-regulation is
ubiquitin-dependent (59). In order to investigate this, we
used siRNA against Hrs and examined degradation of EGF-R 3 h after
a 30-min pulse of EGF. We found that whereas control cells had degraded
80-90% of the internalized EGF-R, siRNA-treated cells had degraded
less than 50% (Fig. 10). Some
degradation was expected in siRNA-treated cells, since a small amount
of Hrs was present even in these cells (Fig 10, inset). This
result indicates that Hrs is required for ligand-dependent degradation of EGF-R.

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Fig. 10.
Hrs is important for normal degradation of
EGF-R. HeLa cells treated with Hrs-specific RNAi or sense RNA (see
"Materials and Methods") were stimulated with EGF for 30 min. The
cells were either fixed at this point or left for a 3-h chase in the
presence of cycloheximide before fixation. The cells were then
co-stained with anti-Hrs and anti-EGF-R and examined by confocal
immunofluorescence microscopy. Quantitations of EGF-R levels were
performed as described under "Materials and Methods."
Error bars denote S.E. The effect of siRNA on Hrs
levels is shown by Western blotting (inset).
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DISCUSSION |
In the work presented here, we show that Hrs recruits STAM2 to
membranes, that Hrs can be coimmunoprecipitated with Eps15 and STAM2
from membrane fractions, and that recombinant Hrs, STAM2, and Eps15
form a ternary complex. We show that all of these proteins colocalize
with ubiquitinated proteins on endosomal microdomains and that their
membrane association is controlled by mVps4. Finally, we show that Hrs
is required both for the recruitment of STAM2 to membranes and for the
efficient degradation of endocytosed EGF-R. Our findings are consistent
with the idea that Hrs, STAM proteins, and Eps15, which all bind
ubiquitin, may participate in a multivalent complex that sorts
ubiquitinated membrane proteins into the MVB pathway.
Given that Hrs and STAM2 are found in a complex, what might its
function be? Hrs was recently shown to bind ubiquitin via its UIM with
low affinity (19, 38). Here we found also that STAM2 binds directly to
ubiquitin and that it colocalizes with ubiquitinated proteins on
microdomains of early endosomes. This suggests that STAM2 is involved
in a sorting complex on early endosomes together with Hrs. It was
recently reported that Hse1p, a yeast protein with sequence similarity
to STAM, is associated with the yeast Hrs homolog Vps27p and requires
Vps27p for its membrane targeting (9). Hse1p is thus likely to be a
homolog of STAM. Interestingly, mutations in the UIM domain of Hse1p, when combined with UIM mutations in Vps27p, have been shown to lead to
impaired sorting of a ubiquitinated reporter construct, indicating that
Hse1p functions in the sorting of ubiquitinated proteins. It is also
worth noting that a chick homologue of STAM2 (EAST) binds the activated
EGF-R (40). Since the EGF-R has been shown to be ubiquitinated (59),
this interaction might take place via the UIM in EAST and the ubiquitin
linked to EGF-R. Further evidence for this idea comes from our finding
that siRNA-mediated depletion of Hrs, which inhibits the targeting of
STAM2 to endosomes, is associated with an impaired degradation of
endocytosed EGF-R. However, there are several additional candidate
ligands for Hrs-STAM, since multiple plasma membrane and endosomal
proteins are known to be ubiquitinated (60, 61). Identification of
cellular ubiquitinated ligands for Hrs as well as another mammalian
class E Vps protein, TSG101/mammalian Vps23p, has turned out to be
difficult due to instability in the receptor-dependent
ubiquitination events in cellular extracts (56). However, in yeast,
such a ligand has been found by detection of an interaction between the
Vps23p-containing ESCRT-1 complex and carboxypeptidase S precursors
(18).
How would such a sorting complex and its assembly and disassembly from
early endosomes be regulated? In yeast, it has been shown that Vps4p,
an AAA-type ATPase necessary for efficient protein transport from an
endosomal compartment to the yeast vacuole/lysosome, catalyzes
disassembly of an endosome-bound class E Vps protein complex and
thereby functions to regulate the dynamic structure and transport
activity of the prevacuolar endosome (50). The mammalian homologue of
Vps4p, mVps4, displays a high degree of sequence identity to Vps4p, and
these proteins have been suggested to fulfill similar functions (62).
Members of the AAA ATPase family couple their ATPase cycle to the
binding and release of substrate proteins (63, 64). Changing one amino
acid in the AAA cassette (E233Q and E235Q for Vps4p and mVps4,
respectively) leads to ATPase-deficient versions of the proteins
that become constitutively membrane-associated (50). Studies of cells
expressing ATPase deficient mVps4 revealed induction of aberrant
endosomes in which cholesterol accumulated, consistent with a
mVps4-induced defect in postendosomal sorting (53). In addition,
overexpression of mVps4E235Q results in decreased amounts
of transferrin receptor on the cell surface, along with increased
amounts of the receptor in perinuclear compartments positively stained
for mVps4E235Q and EEA1 (54). These observations suggest
that transferrin receptor accumulate in compartments belonging to the
endocytic pathway in mVps4E235Q-transfected cells and
raises the possibility that the mammalian counterpart of Vps4p is
involved in the function of endosomes (54).
Although we observed Hrs and STAM2 mostly in the cytosol in
untransfected cells, these proteins translocated to the aberrant endosomes formed in mVps4E235Q-transfected cells. This
indicates that the membrane association of the Hrs-STAM complex is
regulated by mVps4. Both the expression of Rab5Q79L and
mVps4E235Q causes the formation of enlarged endosomes,
presumably by quite distinct mechanisms. Whereas punctate
Hrs-STAM-Eps15-containing microdomains can be readily distinguished on
the Rab5Q79L-induced large endosomes, the staining of
Hrs-STAM-Eps15 on mVps4E235Q-induced structures was more
evenly distributed. The reason is probably that Rab5 does not regulate
the membrane association of Hrs (15), whereas mVps4E235Q
causes a hyperrecruitment of Hrs and its associated proteins.
Previous studies have indicated that chick EAST/STAM2
coimmunoprecipitates with Eps15 (40) and that mammalian Hrs interacts directly with Eps15 (34). However, it was not known whether STAM
proteins interact with Eps15 and whether STAM proteins, Eps15, and Hrs
can be found in a ternary complex. Our present studies with recombinant
proteins indicate that STAM proteins do not interact directly with
Eps15 but that a ternary complex between STAM proteins, Eps15, and Hrs
is formed through the ability of Hrs to interact simultaneously with
STAM proteins and Eps15. This raises the possibility that Hrs, Eps15,
and STAM proteins are subunits of a larger ubiquitin-binding complex on
endosomes. Consistent with this, we found that Eps15, Hrs, and STAM2
are found in the same microdomains as polyubiquitinylated proteins on
early endosomes and recruited to endosomal membranes by
mVps4E235Q. Size exclusion gel filtration chromatography
indicates that Hrs is present in two complexes of 180 and 500-550 kDa,
respectively (56, 65). The larger complex could potentially contain one or more subunits of Hrs, Eps15, and a STAM protein (total
Mr of about 300 kDa), and it is also quite
possible that it contains additional proteins such as sorting nexin 1 (65). Recent work has shown that UIM-containing proteins, including
Hrs, Eps15, and STAM1, become monoubiquitinated (38, 39). This
intriguing finding raises the possibility that Hrs, Eps15, and STAM
could strengthen their interactions by trans-interacting
with ubiquitin moieties of other subunits of the complex. However,
given that even the recombinant, nonubiquitinated proteins appear to
interact quite strongly (6, 34), ubiquitination might alternatively serve to down-regulate the ability of these molecules to interact with
ubiquitinated cargo because of cis-interactions between the UIMs and the ubiquitin moieties of Hrs, Eps15, and STAM (38). Ubiquitination could also function to sterically regulate interactions with other components of the endocytic sorting machinery.
In addition to its interaction with STAM proteins and Eps15, Hrs binds
directly to clathrin and is found in a flat, bilayered clathrin coat of
early endosomes (14, 19, 58). This suggests that a Hrs-containing
complex could function to retain ubiquitinated proteins in
clathrin-containing microdomains. The functional relevance of the
Hrs-clathrin interaction is underscored by our finding that also
clathrin is recruited onto mVps4E235Q-containing endosomes.
We speculate that Hrs, STAM proteins, and Eps15 constitute a
multivalent ubiquitin-interacting complex on early endosomes. Although
the individual interactions between UIMs and ubiquitin are of low
affinity (19, 38), such a complex would have high avidity for proteins
that are ubiquitinated on several lysine residues. Moreover, the
restricted localization to clathrin lattices might further enhance the
efficacy of this sorting mechanism. In agreement with this hypothesis,
an accumulation of endosomal ubiquitinated proteins is observed in
dominant negative mVps4-transfected cells (56). This suggests that both
a possible sorting complex and its putative cargo are regulated by mVps4.
Hrs is an essential mammalian protein (42), but its exact functions
still remain to be elucidated. We present evidence that Hrs is required
both for the membrane localization of STAM2 and for degradation of the
EGF-R. The EGF-R is thus a likely substrate for a Hrs-STAM2-containing
sorting complex, which is consistent with the fact that Hrs mutant
Drosophila displays increased EGF signaling (16). However,
in mutant pupae the total levels of EGF-R are decreased, although the
levels of activated receptors are increased (16). In the absence of Hrs
in vivo, it is possible that compensatory mechanisms may be
activated in order to counteract the sustained signaling caused by
endocytosed growth factor receptors that do not enter into MVBs. Our
finding that Hrs is required for EGF-R degradation is consistent with
the fact that depletion of two other mammalian Vps class E proteins,
hVps28 and TSG101, causes the endosomal accumulation of ubiquitinated
proteins and inhibits EGF degradation (56).
Based on the present and previous results, we propose that a
clathrin-associated complex containing Hrs, STAM2, and Eps15 binds
ubiquitinated cargo proteins on the endosomes and excludes these from
the default recycling pathway by preventing them from entering the
tubular parts of the endosome. The relationship between this complex
and the ubiquitin-binding ESCRT-I complex (18) is not known, but one
possibility is that the two complexes act sequentially in the
recognition and sorting of ubiquitinated proteins. Dissociation of the
complexes, controlled by mVps4, may control clathrin disassembly and
allow cargo to proceed along the MVB pathway to lysosomes. In future
studies, we hope to be able to test this model.