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Shim, J. H. et al. CHMP5 is essential for late endosome function and down-regulation of receptor signaling during mouse embryogenesis. J. Cell Biol. 172, 1045-1056

Section of Immunobiology, Ludwig Institute for Cancer Research, Yale University School of Medicine, New Haven, CT 06520, USA.
The Journal of Cell Biology (Impact Factor: 9.83). 04/2006; 172(7):1045-56. DOI: 10.1083/jcb.200509041
Source: PubMed
ABSTRACT
Charged MVB protein 5 (CHMP5) is a coiled coil protein homologous to the yeast Vps60/Mos10 gene and other ESCRT-III complex members, although its precise function in either yeast or mammalian cells is unknown. We deleted the CHMP5 gene in mice, resulting in a phenotype of early embryonic lethality, reflecting defective late endosome function and dysregulation of signal transduction. Chmp5-/- cells exhibit enlarged late endosomal compartments that contain abundant internal vesicles expressing proteins that are characteristic of late endosomes and lysosomes. This is in contrast to ESCRT-III mutants in yeast, which are defective in multivesicular body (MVB) formation. The degradative capacity of Chmp5-/- cells was reduced, and undigested proteins from multiple pathways accumulated in enlarged MVBs that failed to traffic their cargo to lysosomes. Therefore, CHMP5 regulates late endosome function downstream of MVB formation, and the loss of CHMP5 enhances signal transduction by inhibiting lysosomal degradation of activated receptors.

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Available from: Changchun Xiao, May 20, 2014
THE JOURNAL OF CELL BIOLOGY
JCB: ARTICLE
© The Rockefeller University Press $8.00
The Journal of Cell Biology, Vol. 172, No. 7, March 27, 2006 1045–1056
http://www.jcb.org/cgi/doi/10.1083/jcb.200509041
JCB 1045
Introduction
Genetic studies in yeast have identi ed a subset of vps
(vacuolar protein–sorting) mutants, which are called the class
E mutants. These mutants display an exaggerated prevacuolar/late
endosome compartment, called the class E compartment, which
is caused by defects in multivesicular body (MVB) sorting
(Raymond et al., 1992). There are 17 soluble and 1 membrane
class E vps proteins in yeast, including Vps27p, the ESCRT-I, -II,
and -III complexes, Vps4p, Bro1/Vps31, Vta1, Vps60p/MOS10,
and Did2/Fti1 (Babst, 2005). A recent model has proposed
that monoubiquitinated receptors and cargo proteins are  rst
recognized by Vps27p and Hse1p, which results in the sequential
recruitment of three distinct multiprotein complexes, i.e.,
ESCRT-I, -II, and -III, to endosomal membranes from the
cytosol (Katzmann et al., 2001; Babst et al., 2002a,b; Bilodeau
et al., 2002). Although the details are unclear, these complexes
are required for the sorting of monoubiquitinated cargo for
inclusion in MVBs, as well as the formation of the MVBs them-
selves (for reviews see Katzmann et al., 2002; Raiborg et al.,
2003). The  nal step in the membrane invagination that forms
MVBs may be speci cally associated with the ESCRT-III
complex and its ability to interact with the AAA-ATPase Vps4p
(Babst et al., 1998). Doa4p is a deubiquitinating enzyme that
recycles ubiquitin by releasing monoubiquitin moieties before
the incorporation of proteins into the internal membranes of the
MVB (Amerik et al., 2000).
Several in vitro studies have investigated the protein–
protein interactions of the class E Vps proteins in yeast and
mammalian cells using yeast two-hybrid and GST pull-down
CHMP5 is essential for late endosome function
and down-regulation of receptor signaling during
mouse embryogenesis
Jae-Hyuck Shim,
1,2
Changchun Xiao,
1,2
Matthew S. Hayden,
1,2
Ki-Young Lee,
1,2
E. Sergio Trombetta,
3
Marc Pypaert,
3
Atsuki Nara,
4
Tamotsu Yoshimori,
4
Bettina Wilm,
5
Hediye Erdjument-Bromage,
6
Paul Tempst,
6
Brigid L.M. Hogan,
5
Ira Mellman,
3
and Sankar Ghosh
1,2
1
Section of Immunobiology,
2
Department of Molecular Biophysics and Biochemistry, and
3
Department of Cell Biology, Ludwig Institute for Cancer Research, Yale University
School of Medicine, New Haven, CT 06520
4
Division of Cell Genetics, National Institute for Genetics, Mishima, 411-8540, Japan
5
Department of Cell Biology, Vanderbilt University Medical Center, Nashville, TN 37232
6
Memorial Sloan-Kettering Cancer Center, New York, NY 10021
C
harged MVB protein 5 (CHMP5) is a coiled coil
protein homologous to the yeast Vps60/Mos10
gene and other ESCRT-III complex members,
although its precise function in either yeast or mammalian
cells is unknown. We deleted the CHMP5 gene in mice,
resulting in a phenotype of early embryonic lethality,
refl ecting defective late endosome function and dysre-
gulation of signal transduction. Chmp5
/
cells exhibit
enlarged late endosomal compartments that contain
abundant internal vesicles expressing proteins that are
characteristic of late endosomes and lysosomes. This is in
contrast to ESCRT-III mutants in yeast, which are defective
in multivesicular body (MVB) formation. The degradative
capacity of Chmp5
/
cells was reduced, and undigested
proteins from multiple pathways accumulated in enlarged
MVBs that failed to traffi c their cargo to lysosomes. There-
fore, CHMP5 regulates late endosome function down-
stream of MVB formation, and the loss of CHMP5 enhances
signal transduction by inhibiting lysosomal degradation
of activated receptors.
J.-H. Shim and C. Xiao contributed equally to this paper.
Correspondence to Sankar Ghosh: sankar.ghosh@yale.edu
C. Xiao’s present address is The CBR Institute for Biomedical Research, Boston,
MA 02115.
B.L.M. Hogan’s present address is Dept. of Cell Biology, Duke University Medical
Center, Durham, NC 27710.
Abbreviations used in this paper: CHMP5, charged MVB protein 5; Cl-M6PR,
cation-independent M6PR; E, embryonic day; EGFR, EGF receptor; ES, embry-
onic stem; HEK, human embryonic kidney; LAMP1, Iysosome-associated mem-
brane protein 1; LBPA, lysobisphosphatidic acid; M6PR, mannose 6-phosphate
receptor; MEF, mouse embryonic fi broblast; MHC, major histocompatibility
class; MVB, multivesicular body; SARA, Smad anchor for receptor activation;
shRNA, short hairpin RNA; siRNA, small interfering RNA; TRll, TGF receptor ll;
UIM, ubiquitin-interacting motif.
Page 1
JCB VOLUME 172 NUMBER 7 2006 1046
assays (Martin-Serrano et al., 2003; von Schwedler et al., 2003;
Bowers et al., 2004). Through a coherent protein network, the
ESCRT-I, -II, and -III complexes and associated proteins form
a large MVB-sorting complex on endosomal membranes. The
ESCRT-III complex is likely composed of two functionally
distinct subcomplexes—a membrane-associated subcomplex
(Vps20p–Snf7p) and a cytosolic subcomplex (Vps2p–Vps24p;
Babst et al., 2002a). Recently, Did2/Fti1 and Vta1p were found
to interact with Vps60p/MOS10, Vps4p, and the ESCRT-III
complex, suggesting that together with Vps60p/MOS10 they
play a role in regulating the activity of Vps4p and ESCRT-III
(Bowers et al., 2004; Babst, 2005).
Several mammalian homologues of yeast Vps proteins
have been identi ed (Katzmann et al., 2002). Many of these
share common yeast homologues, implying a greater degree
of complexity in the mammalian MVB-sorting pathway
(von Schwedler et al., 2003). This may not be surprising, given
the greater functional diversity and specialization that exists
in animal cells. Hrs is homologous to the yeast Vps27p and
re cognizes ubiquitinated receptors through a conserved ubiquitin-
interacting motif (UIM), which is essential for MVB sorting to
degradative pathways (Polo et al., 2002). Tsg101 is homolo-
gous to yeast Vps23p, which is an ESCRT I component, and
down-regulates growth factor signaling through its interaction
with Hrs (Babst et al., 2000; Lu et al., 2003). The AAA-ATPase
SKD1 (Vps4B) is homologous to the yeast Vps4p and regulates
the association/dissociation of the MVB-sorting complex in a
manner that is dependent on its ATPase activity (Babst et al.,
1998; Yoshimori et al., 2000). Human Vps34 is a phosphoinosi-
tide 3 kinase that is required for internal vesicle formation
within MVBs (Futter et al., 2001), and human Vps28 directly
interacts with Tsg101 and is recruited to human Vps4 (E235Q)-
positive endosomal membranes (Bishop and Woodman, 2001).
10 human charged MVB protein (CHMP) family proteins,
which are structurally related cytosolic proteins containing
coiled coil domains and are homologous to six yeast ESCRT-III
components, have been identi ed. They have been suggested to
play important roles in the  nal step of MVB-sorting pathways,
namely the invagination of internal vesicles in MVBs, which is
regulated by Vps4p (Babst, 2005). CHMP proteins also function
in HIV budding, a process that is topologically similar to MVB
sorting (Martin-Serrano et al., 2003; von Schwedler et al.,
2003); however, the mechanism, function, and signi cance of
CHMP proteins in mammalian MVB-sorting pathways remain
to be de ned.
Results
CHMP5 is expressed ubiquitously
and is essential for mouse embryogenesis
We originally isolated CHMP5 as a protein that copuri ed with
cytosolic NF-B–IB complexes from rabbit lung extracts.
Microsequencing of this 32-kD copurifying protein revealed that
it was identical to CHMP5, a CHMP family member and the
mammalian homologue of yeast VPS60/MOS10 (Kranz et al.,
2001). Phylogenetic analyses indicate that CHMP5 is a unique
CHMP protein that is quite divergent from other CHMP family
proteins (unpublished data). We began our analysis of CHMP5
by cloning and sequencing the murine and human cDNAs,
which were obtained by screening a mouse liver cDNA library
and by PCR from HeLa cell cDNA, respectively. CHMP5 is
highly conserved through evolution and its homologues can be
found in Drosophila melanogaster, Caenorhabditis elegans,
Arabidopsis thaliana, and yeast (Fig. 1 a).
[ID]FIG1[/ID]
CHMP5 is also
ubiquitously expressed in embryonic and adult mouse tissues
(not depicted and Fig. 1 b, respectively).
Figure 1. Characterization of CHMP5. (a) Protein sequence alignment of CHMP5 homologues from mouse, human, D. melanogaster, C. elegans, A. thaliana,
and yeast. (b) Northern blot analysis of adult mouse tissues.
Page 2
CHMP5 IS ESSENTIAL FOR LATE ENDOCYTIC TRAFFICKING SHIM ET AL. 1047
Although the biological signi cance of the association of
CHMP5 with the NF-B–IB complex remains unclear, we
decided to explore the biological function of CHMP5 by
generating Chmp5
/
mice. Chmp5 genomic DNA (29.8 kb) was
isolated from a 129/SvJ murine genomic DNA library using
Chmp5 cDNA as a probe. The Chmp5 gene contains eight small
exons and is separated by only 414 bp from the Bag1 gene
(Takayama and Reed, 2001). These two genes are encoded by
different DNA strands, and their 5 ends are positioned head-
to-head (Fig. 2 a).
[ID]FIG2[/ID]
To generate a null mutation of Chmp5,
exons 3–7 of the Chmp5 gene were replaced by a loxP- anked
neomycin-resistant gene cassette. After homologous recombi-
nation, the neo gene was deleted by crossing Chmp5
/
mice
with Splicer mice (Fig. 2 b; Koni et al., 2001). The resulting
heterozygous mice were phenotypically normal, but the homo-
zygous mice died at approximately embryonic day 10 (E10).
Most wild-type embryos and mutant littermates did not exhibit
any gross morphological difference until E7.5, after which the
mutant embryos displayed severe developmental abnormalities
in the ventral region (Fig. 3 a).
[ID]FIG3[/ID]
At E8.75, mutant embryos were
severely disorganized, with abnormal neural tube formation,
allantois-chorion fusion, and somite segmentation, although
embryonic axes and structures are normal in mutant embryos
(Fig. 3 b). To further analyze the anatomy of Chmp5
/
embry-
onic structure, we performed histological analysis of E8.5 wild-
type embryos and mutant littermates (Fig. 3 c). Consistent with
Fig. 3 b, severe developmental abnormalities of allantois, head
fold, heart, and somite, and an apparent defect of ventral folding
morphogenesis, were detected in the mutant embryos. To char-
acterize the mutant phenotype, we performed whole-mount in
situ hybridization with Nkx2.5 as a marker of heart formation
(Fig. 3 d, top) and TUNEL assay to assess cell death in E8.5 mutant
embryos (Fig. 3 d, bottom). Remarkably, mutant embryos ex-
hibited the formation of two independent hearts (cardia bi da),
accompanied by massive cell death in the ventral region. These
phenotypes are similar to those of murine and D. melanogaster
embryos lacking Hrs (Table I; Komada and Soriano, 1999; Lloyd
et al., 2002).
[ID]TBL1[/ID]
These  ndings suggest that CHMP5, like Hrs, may
play a role in regulating the endocytosis or lysosomal transport
of receptors involved in signal transduction and, therefore, is
indispensable for early embryonic development. However, a
role for a putative ESCRT-III complex in receptor traf cking in
mammalian cells is yet to be demonstrated.
CHMP5 is required for lysosomal
biogenesis from late endosomes
To evaluate the involvement of CHMP5 in endocytosis, we
isolated and cultured primary embryonic cells derived from
E8.5 Chmp5
/
embryos. Phase-contrast microscopy indicated
that Chmp5
/
cells contained enlarged vacuole-like structures
(Fig. 4 a), a phenotype similar to Hrs
/
primary embryonic
cells (Komada and Soriano, 1999). To characterize these struc-
tures, we performed immuno uorescence analysis with markers
speci c for different subcellular compartments. Immunostain-
ing with transferrin receptor (early and recycling endosomes),
EEA1 (early endosomes), TGN38 (trans-Golgi network), and
Rab8 (recycling endosomes and trans-Golgi network) did not
reveal any gross differences between wild-type and mutant cells
(Fig. 4 a and not depicted). Further coimmunostaining with
markers speci c for late endosomes-MVBs and/or lysosomes,
cation-independent mannose 6-phosphate receptor (M6PR;
CI-M6PR), lysobisphosphatidic acid (LBPA), and lysosome-
associated membrane protein 1 (LAMP1), demonstrated that
Figure 2. Generation of Chmp5
/
embryos. (a and b) Targeting of the
Chmp5 gene and analysis of ES clones. The targeting vector is described
in Materials and methods. Note that the fi rst two exons of the Bag1 gene
are represented by two horizontally lined boxes. Genomic DNA was di-
gested with XbaI or BamHI and hybridized with probe B or A, respectively.
The hybridized DNA fragments were 19 kb (XbaI) and 6.1 kb (BamHI) for
the wild-type allele and 14 kb (XbaI) and 4.5 kb (BamHI) for the targeted
allele. (c) Wild-type (/) and mutant (/) ES cell lysates were immuno-
blotted with antibodies specifi c for CHMP5 and GAPDH4.
Table I. Comparison of the phenotypes between Chmp5 and Hrs
mutant embryos
Chmp5
/
Hrs
/
Early embryonic lethality around E10
Most mutant embryos are smaller than the wild types at E7.5
Defect in ventral fording morphogenesis
Cardia bifi da and massive cell death in ventral region around E8.5
No fusion of allantois with chorion
No somite segmentation
Enlarged M6PR and LAMP1-positive
endosomal compartments
Enlarged TfR-positive endosomal
compartments
Enlarged MVBs result from heavily
packed internal vesicles
Enlarged MVBs result from defect
of vesicular invagination
Page 3
JCB VOLUME 172 NUMBER 7 2006 1048
the vacuoles visible by phase contrast in Chmp5
/
cells were
positive for CI-M6PR, LBPA, and LAMP1 (Fig. 4, b and c).
Indeed, the degree of colocalization was far more pronounced
in the mutant cells as compared with wild-type cells in which, as
expected, distributions of CI-M6PR and LAMP1 and LBPA and
LAMP1 overlapped only partially. Structures positive for CI-
M6PR and lgp/LAMP1 or for LBPA and LAMP1 are generally
de ned as being late endosomes and as distinct from lysosomes
which do not contain appreciable levels of CI-M6PR and LBPA
(Kornfeld and Mellman, 1989; Kobayashi et al., 1998; Luzio
et al., 2000). Thus, CHMP5 de ciency was accompanied by
an accumulation of structures that appear to be late endosomes.
To examine a distinct MVB-sorting pathway in Chmp5
/
cells, we performed immunostaining for major histocom-
patibility class (MHC) II. The majority of newly synthesized
MHC II molecules are diverted from the secretory pathway
upon exit from the trans-Golgi network and are directly tar-
geted to endosomes/lysosomes (Mellman and Steinman, 2001).
Mouse embryonic  broblasts (MEFs) derived from E9.0 wild-
type embryos were stimulated with IFN- to induce MHC II
expression (Fig. 4 d; Ilangumaran et al., 2002). Wild-type and
Chmp5
/
MEFs, stimulated with IFN-, were then stained with
anti-MHC II (I-A) antibody (Fig. 4 e). Chmp5
/
cells exhibit
enlarged endosomes positive for MHC II molecules, whereas
MHC II–positive endosomes are scattered throughout the periph-
eral cytoplasm in wild-type cells. Thus, CHMP5 de ciency led to
the accumulation of MHC II–positive endosomes/lysosomes.
We then used transmission electron microscopy to examine
MVBs in wild-type and mutant embryos. Strikingly, MVBs in
the endodermal cells from the mutant embryos were abnormally
enlarged and heavily packed with internal vesicles and electron-
dense content, as compared with the analogous structures in
cells from wild-type embryos (Fig. 4 f). Stereological analysis
revealed that the fraction of total cell volume occupied by
MVBs in Chmp5
/
cells was 1.5-fold that in wild-type cells
(Fig. 4 g), con rming quantitatively that the endosomes were
enlarged in the mutant cells. Interestingly, this phenotype was
most clearly manifested in endodermal cells, where endo-
cytosis is most active during embryogenesis. Collectively, these
observations suggest that CHMP5 de ciency caused a global
disruption in protein sorting to, or the formation of, lyso-
somes (M6PR
/LBPA
/LAMP1
) from late endosomes/MVBs
(M6PR
/LBPA
/LAMP1
), rather than a speci c de cit in the
sorting of select proteins into lysosomes. Surprisingly, although
CHMP5 de ciency affected the ability of target proteins to
traf c to lysosomes, it did not prevent MVB formation.
To determine whether the block in the formation of
lysosomes positive for M6PR
/LBPA
/LAMP1
would result
Figure 3. Abnormal development of Chmp5
mutant embryos. (a) Morphological analysis
of Chmp5 mutant embryos. Lateral view of
wild-type (/) embryos and mutant (/)
littermates at E7.5, E7.75, E8.5, and E9.25.
(b) Lateral, front, and ventral view of E8.75
Chmp5 mutant embryos. The yolk sac was
removed from the embryos. Al, allantois; Am,
amnion; Hf, head fold; M, midline. (c) Histo-
logical analysis of E8.5 Chmp5 mutant
embryos. Ht, heart; S, somite. (d) Abnormal
heart formation in Chmp5 mutant embryos re-
vealed by whole-mount in situ hybridization
with the cardiac marker Nkx2.5. (top) Arrows
and arrowhead indicate heart tissue. Massive
cell death in Chmp5 mutant embryos revealed
by TUNEL staining of E8.5 embryos. (bottom)
Arrow indicates apoptotic cells. Bars: (c) 50 m;
(d) 100 m.
Page 4
CHMP5 IS ESSENTIAL FOR LATE ENDOCYTIC TRAFFICKING SHIM ET AL. 1049
in impaired degradative capacity in Chmp5
/
cells, we derived
Chmp5
/
embryonic stem (ES) cells from the inner cell mass
of blastocysts obtained by breeding Chmp5 heterozygotes.
ES cells are equivalent to E3.5 embryonic cells and the Chmp5
mutant phenotype was therefore less dramatic in ES cells than
in E8.5 embryonic cells. Both wild-type and mutant ES cells
were able to internalize FITC-conjugated dextran at similar
rates (unpublished data), indicating that CHMP5 was not
required for  uid phase endocytosis by itself. However,
Chmp5
/
ES cells exhibited a greatly reduced capacity to
degrade material after internalization. After a 1-h pulse of the
uid phase marker HRP, wild-type ES cells degraded nearly all
of the internalized protein after 9 h of chase. In contrast, very
little of the HRP was degraded after uptake by Chmp5
/
ES
cells, as determined by immunoblotting (Fig. 5 a) and  ow
cytometry (Fig. 5 b).
[ID]FIG5[/ID]
Moreover, immunogold labeling of inter-
nalized HRP revealed that approximately  vefold more inter-
nalized HRP accumulated within enlarged MVBs in Chmp5
/
ES cells than in wild-type cells (Fig. 5 c). Thus, although HRP
reached canonical late endosomes/MVBs, it was degraded less
effectively in the absence of CHMP5.
Defective degradation of lysosomal contents could re ect
either a disruption in the transport of internalized proteins to
lysosomes or a lower overall proteolytic capacity. Immuno-
blotting of ES cell lysates with antibodies speci c for cathepsin
D and L revealed that they were present in equivalent amounts
in wild-type and Chmp5
/
ES cells (Fig. 5 d). Thus, it
appeared more likely that the defective degradative phenotype
re ected inef cient delivery of endocytosed proteins to
compartments containing active lysosomal hydrolases. Such a
defect might also affect the normal down-regulation of signaling
receptors, suggesting that the embryonic lethality of Chmp5
deletion might re ect hyperactive signal transduction. There-
fore, we examined the dynamics of TGF receptor II (TRII),
which has a well characterized role in early embryogenesis
(Seto et al., 2002).
CHMP5 affects turnover
and down-regulation of TGF receptors
The early embryonic lethality of Chmp5 mutant embryos
prevented us from obtaining appropriate mutant cells that could
be used to study receptor turnover and signaling in vitro.
We therefore used small interfering RNA (siRNA) technology
to generate CHMP5 knockdown cells. Two target sequences of
the Chmp5 gene (Sh1 and Sh2; murine CHMP5) were cloned
into a pSUPER.retro vector along with an H1-RNA promoter,
Figure 4. CHMP5 is essential for biogenesis of lysosomes from mature MVBs. (a–c) Immunofl uorescence analysis of Chmp5
/
primary embryonic cells.
Transferrin receptor (TfR), CA-MPR, LBPA, and LAMP1 are markers for early endosome, late endosome/MVB, and MVB/lysosome, respectively. Arrowheads
indicate phase-lucent vacuolar structures present in the Chmp5
/
cells. (d) Induction of MHC II expression by IFN-. Primary cells derived from E9.0 embryos
were incubated with the indicated amount of IFN- for 48 h (top) or incubated with 1 g IFN- for the indicated time points (bottom). Expression of MHC II
molecules was analyzed by immunoblotting with anti-MHC II (I-A) antibody. (e) Wild-type or Chmp5
/
primary embryonic cells were stained with anti-
MHC II (I-A) antibody. (f) Transmission electron microscopy analysis of MVBs in the endodermal cells of E8.25 embryos. The inset shows an internal vesicle.
(g) The fraction of MVBs per total cell volume was measured in endodermal layer of wild-type embryos (/) and mutant (/) littermates. Approximately
120 MVBs from the sections of six wild-type or Chmp5
/
embryos were counted. Error bars represent the SD. Bars: (a–c and e) 10 m; (f) 500 nm.
Page 5
JCB VOLUME 172 NUMBER 7 2006 1050
which directs synthesis of short hairpin RNA (shRNA) that can
be converted into an siRNA capable of knocking down target
gene expression (Paddison et al., 2002). Transient transfection
of the shRNAs showed that Sh2 was capable of dramatically
reducing the level of transiently expressed mouse CHMP5,
whereas Sh1 was inactive in this assay.
To determine if CHMP5 de ciency affects the postendo-
cytic fate of TRII, we transfected NIH3T3 cells with HA-
tagged TRII (HA-TRII) in the absence or presence of Sh2
(Fig. 6 a).
[ID]FIG6[/ID]
Receptor endocytosis was triggered by the addition
of TGF, and receptor fate was monitored using an anti-HA
monoclonal antibody that was added simultaneously to the
medium, as previously described (Hayes et al., 2002; Di Guglielmo
et al., 2003). Receptor distribution was analyzed relative
to LAMP1 staining. In control cells, HA-TRII was found
largely in LAMP1-negative endosomes scattered throughout
the peripheral cytoplasm, re ecting internalization of the recep-
tor and its degradation in lysosomes. In CHMP5 knockdown
cells, however, TRII was more abundant and was often associ-
ated with enlarged, LAMP1-positive structures typical of the
enlarged late endosomes/MVBs seen in Chmp5
/
cells. At least
at the level of immuno uorescence, the receptor appeared to
reside within the endosomal lumen as well as on the limiting
membrane. Consistent with this, a recent report has also shown
that GFP-tagged CHMP5 protein, which is a putative CHMP5
dominant-negative mutant, leads to accumulation of ligand-
bound EGF receptor (EGFR) in enlarged perinuclear vesicles
and a delay in EGFR degradation (Ward et al., 2005).
To determine localization of TRII in these endosomes, we
performed a protease protection assay on subcellular fractions
Figure 5. CHMP5 plays a role in endosomal transport to lysosomes. (a and b) HRP degradation is blocked in Chmp5
/
cells. ES cells cultured on 0.2%
gelatin-coated plates were incubated in medium containing 100 g/ml HRP for 1 h and then placed in normal medium (time 0). (a) Cells were lysed at the
indicated time points and immunoblotted with anti-HRP antibody. (b) Alternatively, the amount of HRP remaining inside the cells was quantitatively measured
by fl ow cytometry, and the percentage of cellular HRP was calculated based on mean fl uorescence intensity. (c) Transmission electron microscopy analysis
of internalized HRP in MVBs of Chmp5
/
cells. After incubation with HRP for 2 h, ES cells were fi xed, sectioned, and stained with gold-conjugated anti-HRP
antibody. Arrowheads indicate HRP-gold particles. (d) Lysosomal hydrolase activity in Chmp5
/
cells. Wild-type (/; 1) and mutant (/; 2 and 3)
ES cells were lysed and immunoblotted with antibodies specifi c to cathepsin D and L. Bar, 100 nm.
Page 6
CHMP5 IS ESSENTIAL FOR LATE ENDOCYTIC TRAFFICKING SHIM ET AL. 1051
of cells expressing COOH-terminal HA-TRII along with control
vector or human CHMP5 shRNA (Sh3; Fig. 6 b). Percoll gradi-
ents allowed a separation of a light fraction containing the plasma
membrane, early/recycling endosomes, and a population of
Rab7
or LAMP1
endosomes from denser fractions containing
Rab7
late endosomes, as well as LAMP1
lysosomes (Mellman,
1996). In control cells, the majority of the HA-TRII was, as
expected, detected in the light fraction (i.e., at the plasma mem-
brane) in the absence of TGF stimulation. After TGF addition
for 5 h, the total amount of receptor was greatly decreased
because of degradation, but some of the remaining HA-TRII
was detected in the dense fractions, consistent with ligand-
induced internalization and delivery to lysosomes. The majority
of HA-TRII, particularly in the light-density plasma membrane–
containing fractions, was sensitive to proteinase K treatment.
A different picture emerged in cells lacking CHMP5. First,
consistent with our analysis of steady-state (Fig. 6 d) and cell
surface expression (Fig. 6 e) of TRII receptors, HA-TRII
levels were signi cantly increased in CHMP5 knockdown cells.
Importantly, after TGF treatment the HA-TRII that appeared
in the denser fractions was now protected from degradation
by proteinase K. Thus, at least a portion of the HA-TRII that
accumulates after ligand-induced receptor internalization
appears to be localized within the lumen of Percoll-dense
compartments, likely MVBs. Together with our morphological
experiments, these data suggest that CHMP5 de ciency leads to
the accumulation of internalized TRII, at least, in part, on the
internal membranes of late endosomal MVBs.
The increased staining of the HA-TRII suggested that
CHMP5 knockdown interfered with the normal down-regulation
Figure 6. CHMP5 plays a role in down-regulation of TGF receptors. (a) Subcellular distribution of TRII in CHMP5 knockdown cells. NIH3T3 cells were
transfected with extracellularly HA-TRII in the absence or presence of murine CHMP5 shRNA (Sh2). 48 h later, cells were incubated with HA antibody,
stimulated with 5 ng/ml TGF for 1 h, and immunostained with anti-LAMP1 antibody. (b) HEK293 cells were transfected with COOH-terminal HA-TRII in
the absence or presence of human CHMP5 shRNA (Sh3). 48 h after transfection, cells were treated with 10 ng/ml TGF for 5 h or left untreated, and frac-
tionated on Percoll gradients. Transferrin receptor (TfR), Rab7, or LAMP1 was used as a marker for the plasma membrane, early or recycling endosomes,
late endosomes/MVBs, or lysosomes, respectively. Each fraction was treated with proteinase K or left untreated, and HA-TRII was analyzed by immuno-
blotting with anti-HA antibody. (c) NIH3T3 cells were transfected with TRII-HA, together with control vector or murine CHMP5 shRNA (Sh2). 48 h later, cells
were labeled with S
35
methionine in the absence or presence of 0.4 mM chloroquine, and then chased for the indicated times in medium containing unla-
beled methionine and 5 ng/ml TGF. S
35
-labeled TRII was immunoprecipitated with anti-HA antibody, and then analyzed by phosphorimaging. Alterna-
tively, the HA-TRII expression was quantifi ed and graphed as receptor quantity (percentage of time 0) versus time. (d) HEK293 cells were transfected with
control vector (Vector), CHMP5 expression vector (CHMP5), or human CHMP5 shRNAs (Sh3) and immunoblotted with antibodies specifi c to CHMP5 and
GAPDH4 (top). HEK293 cells were transfected with TRI-HA and TRII-HA, together with control vector, CHMP5, or CHMP5-Sh3. 48 h after transfection,
cells were treated with 10 ng/ml TGF for 5 h, or left untreated in the presence of 20 M cyclohexamide before lysis, and then immunoblotted with anti-
bodies specifi c to HA and GAPDH4. (e) HEK293 cells were transfected with extracellularly HA-TRII, together with control vector or human CHMP5 shRNA
(Sh3). 48 h after transfection, HA-TRII expression was analyzed with anti-HA antibody by using fl ow cytometry. (f) NIH3T3 cells were incubated with
biotin-conjugated HRP in medium containing protease inhibitors for 3 h to label preexisting lysosomes. 48 h after transfection with murine CHMP5 shRNAs
(Sh1 and Sh2), cells were incubated with streptavidin for 10 min, washed, and chased for the indicated time. Cells were lysed with biotin-containing lysis
buffer and HRP enzymatic activity was measured using ELISA on anti–streptavidin-coated plates. Endogenous murine CHMP5 protein level was analyzed
by immunoblotting with anti-CHMP5 antibody (top). Error bars represent the SD. n 3.
Page 7
JCB VOLUME 172 NUMBER 7 2006 1052
of the receptor after ligand binding. To test this hypothesis, we
transfected NIH3T3 cells with HA-TRII, together with either
control vector or Sh2, and assessed receptor turnover using
pulse-chase analysis (Fig. 6 c). Consistent with a previous
study showing that chloroquine signi cantly increases the sta-
bilization of endogenous TGF receptors (Kavsak et al., 2000),
treatment of cells with chloroquine blocked degradation of
HA-TR receptors in cells expressing either control vector or
mouse Sh2. In cells expressing the control vector, the half-life
of HA-TR receptors was 1.5 h, whereas in cells where
CHMP5 was knocked down, the half-life was extended to 3.5 h.
To further characterize the effect of CHMP5 expression on
TGF receptor turnover, we determined receptor levels in
human embryonic kidney 293 (HEK293) cells that had been
transfected with vectors encoding either wild-type CHMP5 or
Sh3. The resulting cells slightly overexpressed or dramatically
diminished the expression of CHMP5 protein, respectively
(Fig. 6 d). Consistent with the decreased rate of degradation in
NIH3T3 cells, knockdown of CHMP5 greatly increased the
steady-state levels of both TRI and TRII (Fig. 6 d, bottom).
In contrast, overexpression of CHMP5 slightly decreased the
amount of receptor protein, relative to nontransfected controls.
Similarly, using  ow cytometry we found that CHMP5 knock-
down signi cantly increased steady-state levels of HA-TR
receptors on the cell surface, relative to the control vector
(Fig. 6 e). Together, these  ndings suggest that the loss of
CHMP5 slows the degradation of internalized TGF receptors
by preventing ligand-induced down-regulation. Like internal-
ized HRP, the receptors accumulated intracellularly in late en-
dosomes/MVBs, which are structures normally associated with
receptor degradation.
To investigate whether the defect in receptor degradation
might re ect a failure of late endosomes/MVBs to fuse with
preexisting lysosomes, we performed an endosome–lysosome
fusion assay using shRNA to transiently knockdown CHMP5
expression. To label preexisting lysosomes, NIH3T3 cells
pretreated with protease inhibitors were incubated with biotin-
conjugated HRP (biotin-HRP) for 3 h, and then transfected
with Sh2. 48 h later, the cells were incubated with streptavidin
for 10 min, and the formation of streptavidin–biotin-HRP
complexes was determined after various chase intervals. Vector
and control shRNA (Sh1)–expressing cells showed increasing
complex formation over time, whereas complex formation
was dramatically inhibited in Sh2-expressing cells (Fig. 6 f).
Figure 7. CHMP5 regulates multiple signaling pathways. (a) Hyperactivation of multiple signaling pathways in Chmp5
/
embryos. Extracts from E8.5
wild-type (/) and mutant (/) embryos were immunoblotted with antibodies specifi c to phosphotyrosine (pY), phospho-Erk1/2 (P-Erk1/2), phospho-
Smad2 (P-Smad2), Smad2, Smad4, and GAPDH4. (b) Nuclear localization of phosphorylated Smad2 in Chmp5
/
primary embryonic cells. Wild-type
(/) and Chmp5
/
(/) cells were stained with anti–phospho-Smad2 antibody. Images were obtained by a fl uorescence microscope using a 40
objective. (c) NMuMg cells were transfected with the 3TP-lux reporter together with the indicated amounts of CHMP5 expression vector. Cells were treated
with 1 ng/ml TGF for 24 h before lysis and then analyzed for luciferase activity. (d) NMuMG cells were transfected with murine CHMP5 shRNAs (Sh2
and Sh1) or control vector (Vector) and assayed as in c. Endogenous CHMP5 protein level was analyzed by immunoblotting with anti-CHMP5 antibody.
(e and f) Distributions of TRII and SARA in Chmp5
/
primary embryonic cells. Wild-type (/) and Chmp5
/
(/) cells were stained with antibodies
specifi c for TRII (e) and SARA (f). (g) HEK293 cells were transfected with pBIIX-luc reporter together with the indicated amounts of CHMP5 expression
vector. Cells were treated with 10 ng/ml TNF or10 ng/ml IL-1 for 4 h before lysis and then analyzed for luciferase activity. Alternatively, cells were
transfected with pBIIX-luc reporter, CD4-TLR4 expression vector, and the indicated amounts of CHMP5 expression vector. Luciferase activity was assayed
24 h after transfection. (c, d, and g) Error bars represent the SD. n 3. Bars, 20 m.
Page 8
CHMP5 IS ESSENTIAL FOR LATE ENDOCYTIC TRAFFICKING SHIM ET AL. 1053
Therefore, streptavidin cannot easily reach previously formed
lysosomes in the absence of CHMP5, implying that CHMP5 is
required for efficient fusion of late endosomes/MVBs with
preexisting lysosomes.
CHMP5 down-regulates multiple signaling
pathways during mouse embryogenesis
If, in fact, an alteration in receptor traf cking or degradation is
responsible for the developmental phenotype associated with
CHMP5 deletion, one would expect that knockout embryos
would exhibit defects in TGF receptor signal transduction.
There is evidence that TGF receptor signaling is regulated by
endocytosis, with internalization being required for the receptor
to reach an essential adaptor component, Smad anchor for
receptor activation (SARA), in EEA1-positive endosomes, and
to be subject to normal degradation and down-regulation after
signal transmission (Hayes et al., 2002; Seto et al., 2002;
Di Guglielmo et al., 2003). Moreover, signaling by FGF and
TGF family members plays an essential role in embryogenesis
at the stage when CHMP5 de ciency causes lethality (Rossant
et al., 1997; Corson et al., 2003).
We investigated the activation state of these signaling
pathways in Chmp5
/
embryos by examining the phosphoryla-
tion status of relevant signaling proteins. We made whole cell
lysates from E8.5 wild-type and Chmp5
/
embryos and immuno-
blotted these extracts with phosphospeci c antibodies against
different proteins that are phosphorylated upon signaling.
As shown in Fig. 7 a, tyrosine phosphorylation of many proteins,
including Erk1/2 and Smad2, which function in growth factor
and TGF family member signaling, respectively, was signi-
cantly enhanced in Chmp5
/
embryos.
[ID]FIG7[/ID]
In addition, phosp-
horylated Smad2 was primarily localized in the nucleus of
Chmp5
/
cells, unlike wild-type cells in which phosphory-
lated Smad2 is rarely observed (Fig. 7 b). These results sug-
gest that signaling by growth factors and TGF family members
is hyperactivated in Chmp5
/
embryos and it is likely that such
dysregulation leads to embryonic lethality. Consistent with this
hypothesis, TGF-mediated gene induction is signi cantly in-
hibited by CHMP5 overexpression and is increased upon
CHMP5 knockdown (Fig. 7, c and d). We hypothesized that
CHMP5 de ciency may facilitate recycling of TGF receptors
to the plasma membrane by inhibiting lysosomal degradation of
receptor, thereby leading to enhanced TGF signaling in
Chmp5
/
cells. To test this hypothesis, we examined the distri-
bution of TGF receptor and its signaling adaptor SARA in
Chmp5
/
cells. Immunostaining analysis with antibodies
speci c for TRII and SARA revealed that both TRII and
SARA accumulated on the plasma membrane of Chmp5
/
cells, although TRII and SARA are relatively enriched in the
TGN and cytosol of wild-type cells (Fig. 7, e and f). This is
consistent with  ow cytometric analyses, which indicated
CHMP5 knockdown increases cell surface expression of HA-
TRII, relative to control (Fig. 6 e). Therefore, these results
suggest that CHMP5 de ciency increases the steady-state levels
of TGF receptor expression at the cell surface by decreasing
degradation of internalized receptors. Increased cell surface
expression of TGF receptor and its association with the key
signaling adaptor SARA appears to be responsible for enhanced
TGF signaling in Chmp5
/
cells.
We further demonstrated a generalized role for CHMP5
in down-regulation of receptor signaling pathways by monitor-
ing NF-B activity. The early lethality of Chmp5
/
embryos,
and the inability to generate knockout mouse embryonic
broblasts prevented us from assaying NF-B activation in
the knockout cells themselves. Therefore, we tested the role
of CHMP5 in NF-B activation in cell lines using a reporter
assay system. As shown in Fig. 7 g, CHMP5 overexpres-
sion inhibits NF-B activation induced by the in ammatory
cytokines TNF and IL-1, as well as CD4-TLR4, a domi-
nant active chimera of the toll-like receptor for the bacterial
component lipopolysaccharide (Hayden and Ghosh, 2004).
This result suggests that, similar to growth factor and TGF
signaling, signaling to NF-B is also regulated by endocytosis
and, hence, that CHMP5 plays a role in the down-regulation of
multiple signaling pathways.
Discussion
This study demonstrates that CHMP5 is essential for the  nal
step of MVB sorting, namely the maturation of late endosomes/
MVBs into lysosomes. In contrast to the related members of
the yeast ESCRT-III complex, which are thought to be required
for MVB formation, CHMP5 appears to be dispensable in this
process; CHMP5-de cient cells contain abundant, and, indeed,
enlarged, late endosomal MVBs. Instead, CHMP5 de ciency
was associated with a signi cant reduction in degradative ca-
pacity, with a resulting accumulation of cargo proteins in en-
larged MVBs. We also found that CHMP5 de ciency induces a
generalized up-regulation of multiple signaling pathways that is
attributable to the lack of degradation of internalized receptors.
The resulting hyperactivation of growth factor–mediated sig-
naling is likely responsible for the observed early embryonic
lethality of Chmp5
/
mice.
A role for CHMP5 in late
endocytic traffi cking
The fact that CHMP5 was not required for MVB formation
probably does not imply a fundamental difference between the
ESCRT-III complexes in yeast and animal cells. Instead, it
seems more likely that CHMP5 may not be a key component of
the ESCRT-III complex in MVB formation. Indeed, previous in
vitro studies have found that yeast Vps60p/MOS10, the closest
yeast homologue to mammalian CHMP5, associates with the
class E Vps protein, Vta1p, but not with the key ESCRT-III pro-
teins (Bowers et al., 2004; Shi ett et al., 2004). Vps60p/MOS10
is, however, closely related to ESCRT-III proteins by amino acid
sequence and by function, as Vps60p/MOS10 mutants exhibit
a class E vacuolar phenotype (Kranz et al., 2001). Whether
Vps60p/MOS10 is required for MVB formation in yeast is not
clear, nor is its actual function in the vacuolar pathway.
Although not essential for cell viability, deletion of
CHMP5 in mice was found to be required for normal embry-
onic development, indicating that this gene plays an important
regulatory function. At the cellular level, however, CHMP5
Page 9
JCB VOLUME 172 NUMBER 7 2006 1054
deletion (or knockdown) did cause a marked phenotype
(i.e., the accumulation of an expanded late endosomal MVB
compartment) the failure to deliver internalized content to lyso-
somes, and the reduced degradation of internalized cargo pro-
teins and receptors. Although MVB formation appeared intact
in CHMP5 knockout cells, there was a nonspeci c defect in the
transport of multiple proteins, including  uid phase markers,
MHC II, and cell surface receptors, to lysosomes. As a result,
CHMP5 function seems necessary for complete MVB sorting,
as it is responsible for the  nal conversion of late endosomal
MVBs to lysosomes, rather than for the formation of MVBs
themselves. Thus, CHMP5 may affect any of several possible
events, including delivery of lysosomal enzymes to late endo-
somes, recycling of late endosomal components, or the fusion
of late endosomes with hydrolase-rich lysosomes. Our data
favor the latter possibility because we found that the rate of
delivery of newly internalized streptavidin to lysosomal com-
partments containing previously internalized biotin-HRP was
greatly slowed by CHMP5 depletion (Fig. 6 f).
Whatever its precise role, it is clear that CHMP5 is an im-
portant element in late endosomal function. Remarkably little
is known about this protein, however. As indicated by phyloge-
netic analysis, CHMP5 (Vps60p/MOS10) is a unique CHMP
family protein that does not belong to any subfamily of CHMP
proteins. Conceivably, it may play roles beyond those described
here for the endocytic pathway. The 5 end of the Chmp5 gene
is separated by only 414 bp from opposing strands of the Bag1
gene (Takayama and Reed, 2001), suggesting that both may be
coregulated at the transcriptional level. BAG-1 is clearly in-
volved in modulating apoptosis, cell proliferation, transcription,
and cell motility as a multifunctional regulator. This implies the
possibility that CHMP5 may participate in functions modu-
lated by BAG1. In this regard, it is also interesting to note that
Vps60p/MOS10 is required for the  lament maturation of
Sacchoromyces cerevisiae by targeting speci c cargo proteins
for degradation, whereas the yeast ESCRT-III proteins are
required for early pseudohyphal growth (Kohler, 2003). We also
found that CHMP5 is required for recruitment of CHMP1A (the
mammalian homologue of the yeast Did2/Fti1) to endosomal
membranes, where CHMP5 may complete an ESCRT-III
complex formation (unpublished data). Failure of these  nal
recruitment events may prevent the newly formed MVBs from
releasing the assembled ESCRT complexes, impeding subsequent
maturation of the MVBs themselves.
A role for CHMP5 in the regulation
of signaling pathways during
mouse embryogenesis
It is well established that degradation of cell surface receptors
through endocytosis is a common mechanism of down-regulating
growth factor and TGF receptor signaling (Katzmann et al.,
2002; Seto et al., 2002). Genetic studies in D. melanogaster
have reported that deletion of Hrs caused a defect in degrada-
tion of activated receptor tyrosine kinases, leading to enhanced
tyrosine kinase signaling (Lloyd et al., 2002). However, despite
the importance of the MVB-sorting pathway in receptor down-
regulation, relatively little is known about the functional roles
of mammalian endocytic components other than Hrs and
Tsg101. Early embryonic lethality in mutant embryos has hin-
dered genetic studies of mammalian endocytic components
(Komada and Soriano, 1999; Ruland et al., 2001). Our results
indicate that CHMP5 is required for down-regulation of TGF
signaling pathways through lysosomal degradation of internal-
ized receptors, which is consistent with in vivo results that show
signaling by growth factors and TGF family members is
hyperactivated in Chmp5
/
embryos. Indeed, Chmp5
/
cells
exhibit increased steady-state levels of TGF receptor expres-
sion and accumulation of an essential TGF signaling adaptor,
SARA, perhaps suggesting that CHMP5 deletion facilitates
recycling of TGF receptors to the plasma membrane by blocking
receptor degradation in lysosomes. CHMP5 de ciency also
leads to accumulation of internalized/undigested TGF recep-
tors within the lumen, as well as on the limiting membrane of
LAMP1-positive endosomes/MVBs, after TGF stimulation.
Consistent with our data, a recent study has also shown that a
putative CHMP5-negative mutant, or a CHMP5 depletion using
siRNA, perturbed the distribution of EGFR-containing late
endosomes and EGFR traf cking, resulting in the accumulation
of EGFR in enlarged perinuclear vesicles. In addition, loss of
CHMP5 stabilized ligand-bound EGFR and increased its half-
life by twofold (Ward et al., 2005). Thus, CHMP5 plays an
essential role in traf cking and degradation of growth factor
receptors, including TRII and EGFR. Given that CHMP5
functions downstream of Hrs and TSG101 in endocytosis, and
that much of the HA-TRII in late endosomes/MVBs was
protected from extravesicular proteinase K digestion, it seems
likely that in CHMP5-de cient cells MVB sorting of internal-
ized TGF receptors by Hrs/TSG101 and invagination of internal
vesicles is normal. This would suggest that the MVBs to which
the internalized receptors are delivered were rendered cataboli-
cally defective, secondary to a block in the fusion of lysosomes
with MVBs or in the maturation of MVBs into lysosomes. Thus,
in mammalian cells, the ESCRT-III complex member CHMP5
may not be required for MVB formation or cargo selection.
Instead, it appears to control a late step in lysosomal biogenesis
required for the transfer of internalized material to a hydrolyti-
cally active compartment, one that plays a critical role in down-
regulation of signaling pathways through receptor degradation.
Materials and methods
Cells and reagents
NIH3T3 (murine fi broblast cells) and NMuMG (mammary gland cells;
CRL-1636) cell lines were purchased from the American Type Culture
Collection. The antibodies used were anti–phospho-Erk1/2 (Cell Signaling
Technology), anti–phospho-Smad2 (Cell Signaling Technology), anti-Smad4
(Santa Cruz Biotechnology, Inc; H552), anti-Smad2 (Cell Signaling Tech-
nology), anti-M6PR (Affi nity BioReagents, Inc.), anti-EEA1 (BD Biosciences),
anti-TGN38 (BD Biosciences), anti–mouse transferrin receptor (BD Biosci-
ences; C2), anti–mouse LAMP1 (BD Biosciences; ID4B), and anti-GAPDH4
(Fitzgerald Industries International). An anti-CHMP5 polyclonal antibody
was generated by immunizing rabbits with a 17-aa synthetic peptide corre-
sponding to the COOH-terminal sequence of mouse CHMP5 and affi nity
purifi ed with immobilized antigen. Antibodies specifi c to LBPA, Rab8, and
MHC II (I-A) were gifts from T. Kobayashi (Institute of Physical and Chemical
Research, Wako, Saitama, Japan) and I. Mellman (Yale University School
of Medicine, New Haven, CT). Plasmids encoding HA-TBI and HA-TBII
were a gift from J.L. Wrana (University of Toronto, Toronto, Canada).
Page 10
CHMP5 IS ESSENTIAL FOR LATE ENDOCYTIC TRAFFICKING SHIM ET AL. 1055
Cloning of Chmp5
CHMP5 was copurifi ed with IB from rabbit lung tissue extract. After
separation on SDS-PAGE and silver staining, a 32-kD band was excised
from the gel and trypsinized, and fi ve peptides were microsequenced.
One mouse EST (available from GenBank/EMBL/DDBJ under accession
no.
<GENBANK>
W51061
</GENBANK>
) matched these peptides and was used as a probe to screen
a mouse liver cDNA library. One clone containing the Chmp5 open reading
frame was obtained and an additional 5 sequence was cloned by 5
rapid amplifi cation of cDNA ends.
Generation of Chmp5
/
mice and Chmp5
/
ES cells
Four Chmp5 genomic clones were isolated from a 129/SvJ mouse genomic
DNA library (Stratagene). The targeting vector consists of a 5.3-kb 5 homol-
ogy region and a 4.7-kb 3 homology region. A loxP-PGKneo-loxP cassette
was inserted between the two regions and a PGK-TK cassette was placed
upstream of the 5 homology region, resulting in a vector designed to delete
exons 3–7 of Chmp5. Linearized targeting vector was electroporated into
TC1 ES cells. Clones resistant to G418 and gancyclovir were selected, and
homologous recombination was confi rmed by Southern blotting. Two tar-
geted clones were injected into C57BL/6 blastocysts and both produced
germline chimeras. Chimeras were mated with C57BL/6 females, and
heterozygous male offspring were bred with female splicer mice to delete the
oxed neo cassette. Their offspring were screened for the targeted allele with-
out the neo cassette and in the absence of the cre transgene. Positive mice
were interbred and maintained on a mixed 129 C57BL/6 background.
To establish Chmp5
/
ES cell lines, Chmp5
/
mice were mated,
blastocysts were collected, and ES cell lines were isolated from the inner
cell mass and cultured as previously described (Robertson, 1987).
The genotypes of ES cell lines were determined by Southern blotting and
immunoblotting with anti-CHMP5 antibody.
Whole-mount in situ hybridization and TUNEL assay
Whole-mount TUNEL assay and in situ hybridization using digoxigenin
UTP-labeled riboprobes were performed as previously described (Sasaki
and Hogan, 1993). Embryos were placed on 60-mm Petri dish containing
1% agarose that was fi lled with PBS and oriented as indicated. The
embryos were imaged on a dissecting microscope (model Stemi 2000-C;
Carl Zeiss MicroImaging, Inc.) using 16/16, NA 2.5, objectives
(AxioCam; Carl Zeiss MicroImaging, Inc.). Image data was acquired and
stored as TIFF fi les using AxioCam software.
Isolation of cells from mouse embryos
E8.5 embryos from breeding of Chmp5
/
mice were dissected free of
maternal tissues and had their Reichert’s membrane removed, after which
they were washed with PBS and incubated with 0.1% collagenase (Sigma-
Aldrich) and Trypsin-EDTA (Invitrogen) for 30 min at 37C. The cell sus-
pension was plated in 24-well plates precoated with 0.2% gelatin
(Sigma-Aldrich) and cultured in DME supplemented with 15% FBS (Sigma-
Aldrich). Chmp5
/
cells were identifi ed by immunostaining with CHMP5
antibody or PCR from extraembryonic tissues.
Transmission electron microscopy
E8.0–8.25 embryos were dissected and washed with PBS, and then fi xed
with 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium
cacodylate buffer, pH 7.2, for 1 h at room temperature. Further procedures
were performed by standard protocols.
Immunofl uorescence
For immunofl uorescence analysis, cells were grown on 0.2% gelatin-coated
coverslips, washed with PBS, fi xed with 4% paraformaldehyde for 30 min
at room temperature, and permeabilized with permeabilization buffer
(0.05% saponin, 1% FBS, 10 mM Hepes, and 10 mM glycine in PBS, pH
7.5) for 30 min at room temperature. Cells were incubated with the
indicated primary antibodies for 30 min at room temperature and then
incubated with either goat anti–rabbit or anti–mouse antibodies conju-
gated with FITC or Texas red under identical conditions. Subsequently, cells
were washed three times with PBS, mounted in GEL/MOUNT (Biomeda),
and examined under either a Plan Apochromat 63 oil objective or a Plan
Neofl uar 40 oil objective on a fl uorescence microscope (Axioplan 2; all
Carl Zeiss MicroImaging, Inc.) equipped with a charge-coupled device
camera (Orca ER; Hamamatsu). Image data was acquired and stored as
TIFF fi les using OpenLab software (Improvision, Inc.).
Generation of CHMP5 ShRNAs
shRNAs with murine CHMP5 target sequence Sh2 (5-C C T G G C C C A A-
C A G T C C T T T -3) and murine CHMP5 control sequence Sh1 (5-A A G C-
G A A A C C C A A G G C T C C -3), or human CHMP5 target sequence Sh3
(5-A A G G A C A C C A A G A C C A C G G T T -3
) were produced by chemically
synthesized DNA oligonucleotides and cloned into pSUPER.retro vector
following the manufacturer’s instruction (OligoEngine). To test ShRNAs,
Flag-tagged Chmp5 cDNA and CHMP5 ShRNAs were cotransfected into
COS1 cells. 48 h after transfection, cells were lysed and immunoblotted
with antibodies specifi c for Flag and GAPDH4.
Endosome–lysosome transport assay
NIH3T3 cells were cultured in medium containing protease inhibitors to
inhibit protein degradation in lysosomes throughout the experiment. Cells
were incubated with biotin-conjugated HRP for 3 h to label lysosomes
before being transfected with CHMP5 shRNA. 48 h after transfection, cells
were incubated with streptavidin for 10 min, washed, chased for the indi-
cated time points, and lysed with biotin-containing lysis buffer. Lysates
were applied to antistreptavidin-coated plates, incubated for 1 h, and
washed; HRP enzymatic activity was measured by colorimetric assay and
is expressed as arbitrary units.
Subcellular fractionation and protease protection assay
HEK293 cells were transfected with COOH-terminal HA-TRII in the pres-
ence or absence of human CHMP5 shRNA (Sh3). 48 h after transfection,
cells were treated with 10 ng/ml TGF for 5 h or left untreated and fraction-
ated using Percoll gradients as previously described (Marsh et al., 1987).
In brief, cells were washed and resuspended in homogenization buffer
(10 mM triethanolamine, 10 mM acetic acid, 1 mM EDTA, and 0.25 M
sucrose, pH 7.4) and disrupted with 20 strokes in a dounce homogenizer.
Microscopic analysis assured that the cell breakage was nearly complete.
The homogenate was centrifuged at 1,000 g for 5 min at 4C to remove nu-
clei and unbroken cells. The postnuclear supernatant was mixed with Percoll
(Sigma-Aldrich) in homogenization buffer to give a fi nal concentration of
27%, and the mixture was underlaid with a 27.6% nycodenz solution (Sigma-
Aldrich). The gradients were centrifuged in a SW41Ti rotor at 17,500 rpm
for 1 h at 4C, and 14 fractions were collected from the top of gradient.
Protease protection assays have traditionally been used to determine
the sidedness of proteins relative to a sealed membrane compartments
(Blobel and Sabatini, 1970; Sabatini and Blobel, 1970). These experi-
ments typically involve the complete digestion of exposed domains of
proteins on the outside of a sealed compartment and the protection of those
domains or proteins that reside on the inside of the compartment (Wu et al.,
2003; Sik et al., 2004). To apply the protease protection assay to our
system, we modifi ed the original protocol of the protease protection assay.
For protease protection assay, 25-l aliquots of each fraction were treated
with 2–5 g proteinase K for 30 min at room temperature or left untreated.
The reaction was stopped by adding SDS loading buffer, and proteinase K
was inactivated by boiling at 90C. The samples were subjected to 10%
SDS-PAGE, followed by immunoblotting with the indicated antibodies.
We would like to thank Crystal Bussey for technical assistance; Pietro De
Camilli, Scott D. Emr, Walther Mothes, and Matthew B. Greenblatt for discus-
sions; and Toshihide Kobayashi and Jeffrey L. Wrana for sharing reagents.
The work was supported by grants from the National Institutes of Health
(R37-AI33443 and R37 AI-34098), the Ludwig Institute for Cancer Research
(I. Mellman), and the American Heart Association (E.S. Trombetta). M.S.
Hayden was supported in part by the National Institute of General Medical
Sciences Medical Scientist Training Program training grant GM-07205.
Submitted: 7 September 2005
Accepted: 17 February 2006
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  • Source
    • "Targeted disruption of Bag-1 resulted in a double knockout, ablating the expression of both Bag-1 and Chmp5 (our unpublished data). The Chmp5 knockout, like the Bag-1 knockout, is embryonic [99]. This double knockout has hampered analysis of the contribution of Bag-1 to steroid receptor action in the mouse. "
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  • Source
    • "Endocytic downregulation of cell surface receptors is thought to be a key regulatory step in establishing the appropriate extent of signaling. Indeed, disruption of late endosome/multivesicular body (MVB) function by small interfering RNA (siRNA)-mediated suppression of Chmp5, a subunit of the MVB-forming complex, i.e., endosomal sorting complex required for transport III (ESCRT-III), results in defective silencing of RTKs and TGF-b signaling (Shim et al., 2006). However, in a different experimental setup, EGF signaling is attenuated normally in cells deficient in hVps24, another ESCRT-III subunit (Bache et al., 2006 ). "
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    Full-text · Article · Jun 2012 · Developmental Cell
  • Source
    • "It plays a crucial role in sorting membrane proteins that are destined for degradation or routing to the lysosome3, 4. These proteins, such as activated growth-factor, hormone and cytokine receptors, are internalized into vesicles forming an MVB, the contents of the MVB are then transferred to lysosomes for degradation2, 5, 6. CHMP5 is responsible for the final conversion of late endosomal MVB to lysosomes and plays a critical role in the downregulation of signaling pathways through receptor degradation2, 7. "
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