* Biochemistry Department, University of Geneva, 1211-Geneva-4, Switzerland; and Center for Microscopy and Microanalysis,
Department of Physiology and Pharmacology, and Center for Molecular and Cellular Biology, University of Queensland,
Queensland 4072, Brisbane, Australia
In the present paper, we show that transport
from early to late endosomes is inhibited at the restrictive temperature in a mutant CHO cell line (ldlF) with
a ts-defect in coatomer protein (
COP), although internalization and recycling continue. Early endosomes
then appear like clusters of thin tubules devoid of the
typical multivesicular regions, which are normally destined to become vesicular intermediates during transport to late endosomes. We also find that the in vitro
formation of these vesicles from BHK donor endosomes is inhibited in cytosol prepared from ldlF cells incubated at the restrictive temperature. Although
COP is rapidly degraded in ldlF cells at the restrictive temperature, cellular amounts of the other COP-I subunits
are not affected. Despite the absence of
COP, we find
that a subcomplex of
,
, and
COP is still recruited
onto BHK endosomes in vitro, and this binding exhibits
the characteristic properties of endosomal COPs with
respect to stimulation by GTP
S and sensitivity to the
endosomal pH. Previous studies showed that
and
COP are not found on endosomes. However,
COP,
which is normally present on endosomes, is no longer recruited when
COP is missing. In contrast, all COP
subunits, except obviously
COP itself, still bind BHK
biosynthetic membranes in a pH-independent manner
in vitro. Our observations thus indicate that the biogenesis of multivesicular endosomes is coupled to early endosome organization and depends on COP-I proteins.
Our data also show that membrane association and
function of endosomal COPs can be dissected: whereas
,
, and
COP retain the capacity to bind endosomal
membranes, COP function in transport appears to depend on the presence of
and/or
COP.
AFTER internalization, cell surface proteins and lipids,
as well as solutes, first appear in peripheral early
endosomes. Depending on their fate, internalized
molecules can then either be recycled back to the cell surface for reutilization or transported to late endosomes and
then lysosomes for degradation (Gruenberg and Maxfield, 1995 In previous in vivo and in vitro studies, we have identified and characterized intermediates (endosomal carrier
vesicles [ECVs]), which mediate transport from early to
late endosomes (Gruenberg et al., 1989 Candidates responsible for COP-I binding to biosynthetic membranes include proteins normally retained in or
retrieved to the endoplasmic reticulum via a KKXX motif
(Cosson and Letourneur, 1994 In the present paper, we have studied the role of COP
proteins in ECV/MVB biogenesis, using the ldlF mutant
CHO cell line with a ts mutation in the gene encoding for
Cell Culture and Immunological Reagents
Monolayers of baby hamster kidney cell line (BHK-21) were grown and
maintained as described (Gruenberg et al., 1989 The M3A5 and maD monoclonal antibodies against Internalization and Recycling of Endocytosed Markers
in ldlF Cells
Cells were grown onto 10-cm dishes at the permissive temperature (34°C)
for 3 d and then subsequently incubated for 6 h at the same temperature
or at the restrictive temperature (40°C). To measure continuous HRP uptake, cells were washed twice with PBS and incubated in internalization
medium (MEM, 25 mM glucose, 10 mM Hepes, pH 7.4) containing 3 mg/
ml HRP and 2 mg/ml BSA for 5, 15, 30, 60, or 120 min at either temperature. Cells were extensively washed for 10 min six times with PBS containing 5 mg/ml BSA (PBS-BSA) on ice, scraped off the dish, and collected by
centrifugation at 450 g. The cell pellet was solubilized for 30 min in 400 µl homogenization buffer (HB; 250 mM sucrose, 3 mM imidazole) containing 0.2% Triton X-100, and both HRP activity and protein content were quantified. To quantify HRP recycling, cells were incubated as above with
internalization medium containing 0.5 mg/ml HRP for 5 min at either temperature and then washed on ice as above. Then, cells were reincubated in
10 ml internalization medium containing 2 mg/ml BSA for 10, 20, 30, or 40 min at the corresponding temperature. The medium was collected, cells
were processed as above, and HRP activity of cell extract and medium
was measured. In some experiments, ldlF cells were transiently transfected with the cDNA encoding for the human transferrin receptor (Zerial et al., 1987 Subcellular Fractionation of ldlF Cells
Cells were grown onto 12 × 10 cm dishes at the permissive temperature
(34°C) for 3 d. When needed, six dishes were subsequently incubated at
the restrictive temperature (40°C) for 6 h. In either case, cells were rinsed
in PBS and incubated 5 min with 4 mg/ml HRP in internalization medium
to label early endosomes. To label late endosomes, cells were washed three
times for 10 min on ice in PBS containing 5 mg/ml BSA and then reincubated for 40 min at the desired temperature (34 or 40°C) in internalization
medium containing 2 mg/ml BSA. In all cases, cells were then washed
twice with PBS, homogenized in HB containing 1 mM EDTA, and centrifuged at 1,300 g for 10 min to prepare a postnuclear supernatant (PNS). The
PNS was brought to 30% sucrose, 3 mM imidazole, 1 mM EDTA up to a
volume of 2 ml, loaded on top of a 1-ml cushion of 40.6% sucrose, 3 mM
imidazole, 1 mM EDTA in a SW60 tube (Beckman Instruments, Fullerton, CA), and overlaid with 1.5 ml of HB containing 1 mM EDTA. The
gradient was then centrifuged at 35,000 rpm for 90 min, and fractions were
collected. Early and late endosomal fractions were recovered from the
30% sucrose/HB and 40.6%/30% sucrose interfaces, respectively. To ensure efficient recoveries, the 30% cushion was brought back to 34% sucrose in the same buffer and subjected to a second round of centrifugation, as used for the PNS. Early and late endosomal fractions from both
centrifugation rounds were pooled, and HRP content of the fractions were
quantified.
Cytosol Preparation
CHO and ldlF cells were grown onto 24 × 24 cm dishes at 37 and 34°C, respectively. Then, ldlF cells, as well as control CHO cells, were further incubated at the restrictive temperature (40°C) for 12 h. Cells were washed
twice with PBS on ice and homogenized. A PNS was then prepared and
centrifuged at 55,000 rpm for 35 min in a rotor (model TLS 55; Beckman
Instruments, Fullerton, CA). The supernatant containing the cytosol was
collected, aliquoted, frozen in liquid N2, and stored at Fluorescence Microscopy
Monolayers of ldlF cells were grown on glass coverslips for 2 d at 34°C to
80% confluency and then reincubated at 34 or 40°C for 6 h. The following
protocols were used: (a) cells were fixed in 2% paraformaldehyde for 20 min at room temperature, permeabilized with 1% Triton X-100, and then
labeled with human antiserum against EEA1 diluted 1:400; (b) cells were
fixed and permeabilized using MeOH at We used acridine orange (Calbiochem, La Jolla, CA) that had been
stored at a concentration of 20 mM in DMSO at Phase Contrast Light Microscopy and Electron
Microscopy of ldlF Cells
Early and late endosomes were labeled with HRP as described above, except that 10 mg/ml high activity HRP (Serva, Heidelberg, Germany) was
used to ensure proper detection of the marker. The cells were then fixed
in LG-fix (0.5% glutaraldehyde in 100 mM cacodylate, pH 7.35) for 60 min
at room temperature and washed six times for 10 min with 100 mM cacodylate, pH 7.35. The distribution of internalized HRP was revealed using a cytochemical reaction with diaminobenzidine (Sigma) and H2O2 as
substrates. Fixed cells were treated in the dark with 1 mg/ml diaminobenzidine in 200 mM cacodylate for 5 min and then with the same solution but
also containing 0.0012% H2O2 for 30 min. Samples were then washed four times for 5 min with 100 mM cacodylate and observed by phase contrast light microscopy using a 100× objective. For electron microscopy, the cells
were postfixed with 2% osmium tetroxide for 1 h at room temperature, and
processed for Epon embedding as described (Parton et al., 1992 COP Binding Assay
Cytosol from ldlF cells was prepared as described above. Membranes
were obtained from BHK cells incubated with or without 20 µg/µl brefeldin A (Sigma) for 1 h to allow disassembly of the Golgi complex to occur
(Rojo et al., 1997 In Vitro Formation of ECV/MVBs from
Early Endosomes
ECV/MVB formation from donor early endosomal membranes was measured exactly as described in Aniento et al. (1996) Other Methods
Quantification of protein was carried out using the procedure of Bradford
(1976) It has been previously shown that ts-
Endocytosis in ldlF Cells at the Restrictive Temperature
As a first step, we investigated whether early steps of the
endocytic pathway were affected at the restrictive temperature. Cells were transiently transfected with the cDNA encoding for the human transferrin receptor (Zerial et al.,
1987
We found that internalization into endosomes and recycling back to the plasma membrane of the lipid bodipy-sphingomyelin (Koval and Pagano, 1989 Inhibition of Early to Late Endosome Transport at the
Restrictive Temperature
Our previous data showed that
After the 5-min pulse, HRP was internalized within elements with the typical peripheral location of the early endosome (Fig. 3), both at the permissive and restrictive
temperatures. After the chase at the permissive temperature, HRP redistributed, as expected, to structures that were
often clustered in the perinuclear region, corresponding to
late endosomes (see Fig. 7). In contrast, little marker was
found in the cells after chase at the restrictive temperature
(Fig. 3). These observations were confirmed by fractionation on a step sucrose gradient. Both at the permissive
and restrictive temperatures, HRP, which had been internalized for 5 min (Fig. 4 A), cofractionated with rab5 (Fig.
4 B), an early endosomal marker (Chavrier et al., 1990 In our previous studies, we had observed that neutralization of the endosomal pH also caused inhibition of early to
late endosome transport (Aniento et al., 1996
Disruption of Early Endosome Organization at the
Restrictive Temperature
We then investigated whether the organization of endosomes was affected in the absence of
To further investigate the effects of Inhibition of ECV/MVB Formation in the
Absence of We have previously described an in vitro assay that measures the formation of ECV/MVBs from donor early endosomal membranes prepared from BHK cells (Aniento et al.,
1996 As shown in Fig. 9 A, cytosol prepared from ldlF cells
incubated at the permissive temperature (34°C) or from
WT CHO cells incubated at 37°C supported ECV/MVB
formation from donor membranes, and the process was
ATP-dependent as observed previously (Aniento et al.,
1996
COP Membrane Binding
The striking similarity between the effects of endosomal
pH neutralization and We used BHK cells as a source of endosomes to ensure
that membranes were fully competent to support COP association (Aniento et al., 1996
We then compared the COP-binding capacity of brefeldin A-treated and untreated early endosomal membranes.
The binding assay was initiated by mixing membranes with
rat liver cytosol. GTP To our surprise, we observed that COP binding onto endosomal membranes still occurred when
Characterization of COP Membrane Binding
Association of COPs to endosomal membranes is inhibited after neutralization of the lumenal pH (Aniento et al.,
1996 In this paper, we have studied the role of COP proteins in
endosomal membrane transport, using the ldlF cell line
with a ts defect in Phenotype of ldlF Cells
Originally, Hobbie et al. (1994) COP-I and Endosome Organization/Dynamics
Within minutes after internalization into early endosomes,
molecules destined to be recycled or degraded are rapidly
segregated into separate elements (Trowbridge et al., 1993 The existence of a close relationship between endosome
ultrastructure and ECV/MVB biogenesis is further strengthened by our previous observations that neutralization of
the endosomal pH causes very similar effects. Then, early
endosomes form clusters of thin tubules (50-60-nm diameter) that typically lack multivesicular domains, and both
ECV/MVB formation and transport to late endosomes are
inhibited (Clague et al., 1994 Recent studies indicate that, in the biosynthetic pathway, COPs can interact with the cytoplasmic domains of
transmembrane proteins (Cosson and Letourneur, 1994 COP-I Subunits Involved in Membrane Binding
and Transport
Our data indicate that the Our data also suggest that COP association to endosomal membranes occurs in more than one functional step.
Indeed, neutralization of the pH inhibits COP binding but
does not cause the release of prebound COPs. Moreover,
pH neutralization inhibits the stimulatory effects of GTP; Mellman, 1996
). These two pathways exhibit major
differences with respect to membrane organization and
dynamics. In contrast to the recycling route, transport to
late endosomes is highly selective, accounting for the bulk
of downregulated receptors but only a minor fraction
(
10%) of total internalized protein and lipid (Koval and
Pagano, 1989
; Trowbridge et al., 1993
). Endosomes along
these two pathways also exhibit marked ultrastructural
differences. Whereas elements of the recycling pathway
consist of very thin tubules (50-60 nm in diameter and up
to several microns in length), endosomes at all stages of
the degradation pathway exhibit a typical multivesicular
appearance caused by the accumulation of internal membranes within their lumen, hence the name multivesicular
body (MVB)1 (Dunn et al., 1986
; Tooze and Hollinshead,
1991
; Parton et al., 1992
; van Deurs et al., 1993
; Futter et al.,
1996
). Finally, these two pathways differ in their acidification properties. The lumenal pH decreases from 6.2 in
early endosomes to ~5.5 in endosomes of the degradation
pathway but increases to ~6.4 in recycling endosomes
(Yamashiro et al., 1984
; Mellman et al., 1986
; Sipe and
Murphy, 1987
). It is intriguing how early endosomal membranes can give rise to elements that differ so widely in
their organization, internal milieu, and protein composition.
; Bomsel et al.,
1990
; Aniento et al., 1993
, 1996
; Clague et al., 1994
; Robinson et al., 1997
). These vesicles exhibit a typical multivesicular ultrastructure and will be referred to as ECV/MVBs
in this study. We found that the formation of ECV/MVBs from early endosomes depends on
coatomer protein
(
COP) (Aniento et al., 1996
), a subunit of the COP-I coat
previously shown to mediate anterograde and/or retrograde transport at early stages of the biosynthetic pathway
(Orci et al., 1986
; Ostermann et al., 1993
; Pepperkok et al.,
1993
; Letourneur et al., 1994
). Studies comparing endosomal and biosynthetic COPs, however, revealed that these
exhibit, at least in part, different properties. Two subunits of the biosynthetic COP-I coat,
and
, are not present on
endosomes (Whitney et al., 1995
; Aniento et al., 1996
), suggesting that the composition of the endosomal coat is simpler, or that the endosomal homologues of
and
have
not been identified. In addition, we have observed that
ECV/MVB formation from early endosomes depends on
the acidic lumenal pH and that
COP binding to early endosomes is itself pH dependent (Clague et al., 1994
; Aniento et al., 1996
). In contrast, COPs in the biosynthetic
pathway are recruited onto the membranes of nonacidic
organelles.
), as well as members of a
novel family of biosynthetic membrane proteins in the
~20-25-kD range (Fiedler et al., 1996
; Sohn et al., 1996
).
Recent studies have also provided some information about
the possible role of individual COP-I subunits during binding to biosynthetic membranes. Two COP-I subcomplexes,
consisting of the
/
and
/
/
subunits, could be dissociated in vitro, and the
/
/
COP subcomplex was shown
to interact with membranes and with cytoplasmic KKXX
motifs (Lowe and Kreis, 1995
). However, studies using
peptides derived from the cytoplasmic domains of different 20-25-kD proteins suggest that an -FF- motive typical for this family is required for binding, but also that different COP-I subunits may bind to different members of this
protein family (Fiedler et al., 1996
). The physiological significance of these different mechanisms remains to be elucidated. In fact, very little is known about the role of individual COP-I subunits, in particular in the process of
driving transport itself. In the endocytic pathway, essentially nothing is known about the function of any subunit.
COP. At the restrictive temperature,
COP is rapidly degraded (Guo et al., 1994
, 1996
; Hobbie et al., 1994
). Our
data show that ECV/MVB biogenesis, hence transport to
late endosomes, is coupled to the maintenance of early endosome organization. In the absence of functional COPs, accumulation of internal membranes and ECV/MVB formation no longer occur on early endosomal membranes.
Early endosomes, then, become clusters of thin tubules.
We also find that
COP degradation does not affect the
amounts of other subunits to any significant extent and
that
,
, and
COP, but not
COP, are still recruited
onto endosomes in a pH-sensitive and GTP
S-dependent
manner. Our data show that
,
, and
COP retain the
full capacity to mediate membrane association but are not
sufficient to drive ECV/MVB biogenesis in vivo and in
vitro, and thus that
and/or
COP may confer coat activity in transport.
Materials and Methods
). For large scale endosome preparation, 24 × 24 cm dishes were used. Wide-type CHO and mutant ldlF cell line were obtained from M. Krieger (Massachusetts Institute
of Technology, Cambridge, MA). CHO and ldlF cells were grown and
maintained as described (Guo et al., 1994
), using F-12 medium (Nutrient
mixture F-12 HAM; Sigma, Buchs, Switzerland) containing 5% FCS
(Sera-Tech, St. Salvator, Germany).
COP peptide were
a gift of T. Kreis (University of Geneva, Geneva, Switzerland). The antibodies against all other COP-I subunits were a gift of F. Wieland (Ruprecht
Karl University, Heidelberg, Germany). Human serum against EEA1 was
a gift of B.H. Toh (Monash Medical School, Victoria, Australia). The
polyclonal antibody against the mannose-6-phosphate receptor (Man6P-R)
was obtained from B. Hoflack (Institut Pasteur, Lilles, France), and the
polyclonal antibody against calnexin from Ari Helenius (Yale University,
New Haven, CT). The antibody against BHKp23 was raised after injection
of the RLEDLSESIVNFAY lumenal peptide of BHKp23 into rabbits
(LP2; Rojo et al., 1997
). The antibodies against rab5 and rab7 were raised
after injection of the corresponding COOH-terminal peptides into rabbits,
as in Chavrier et al. (1990)
. FITC-labeled secondary antibodies were from
Dianova (Hamburg, Germany).
; Harder and Gerke, 1993
). Transfected cells were then incubated at the permissive or restrictive temperature for 6 h and then
reincubated in internalization medium containing 50 µg/ml rhodamine-transferrin (Sigma) for 5 min at the corresponding temperature. Cells were extensively washed with PBS-BSA, fixed in 3% paraformaldehyde at room
temperature for 20 min, and analyzed by fluorescence microscopy with
100× objective.
80°C.
20°C and then labeled with a
rabbit antiserum against Man6P-R diluted 1:200, the maD monoclonal antibody against
COP diluted 1:3,000, or the rabbit antiserum against calnexin diluted 1:500; (c) cells were first permeabilized with 0.004% digitonin for 5 min, fixed with 2% paraformaldehyde for 20 min at room
temperature, blocked with 2% fish skin gelatin, and then labeled with a
rabbit antiserum against rab7 diluted 1:100. In all cases, the bound antibodies were revealed using FITC-conjugated secondary antibodies. Samples were processed as described (Kreis, 1986
; Mu et al., 1994
) and viewed
using an inverted fluorescence light microscope (model Axiovert 135 TV;
Carl Zeiss, Inc., Thornwood, NY) and a 100× objective.
20°C. We also used two
forms of LysoSensor (Molecular Probes, Eugene, OR), which detect pH
values in the 4.5-6.0 (DND-189) and 6.5-8.0 (DND-153) ranges, respectively, and were stored and used according to the manufacturer's instructions. Cells were washed twice with PBS++ (1 mM CaCl2, 1 mM MgCl2) at
room temperature, incubated for 10 min in PBS++ containing 5 µM of the
dye and 5 mM glucose, and then observed by fluorescence microscopy using a 100× objective.
a).
). Cells were then washed, and endosomes were prepared
using a step flotation gradient, as described (Gruenberg and Gorvel,
1992
). Briefly, cells were grown onto four 24 × 24 cm dishes and homogenized to prepare a PNS. The PNS was adjusted to 40.6% sucrose, 3 mM
imidazole, pH 7.4, and loaded at the bottom of six SW40 tubes (Beckman
Instruments). Each one was then overlaid sequentially with 4.5 ml of 35%
sucrose, 3 ml of 25% sucrose in 3 mM imidazole, pH 7.4, and 3 ml of HB (250 mM sucrose, 3 mM imidazole, pH 7.4). The gradients were centrifuged for 90 min at 35,000 rpm using an SW40 rotor (Beckman Instruments). Early endosomes and biosynthetic membranes were then collected at the 35%/25% and 40.6%/35% interfaces, respectively. In the binding
assay, 500 µg cytosol was mixed on ice with 50 µg of each membrane fraction and complemented with 12.5 mM HEPES, 1.5 mM MgOAc2, 1 mM
DTT, 65 mM KCl, and 15 µl of an ATP-regenerating system (Gruenberg
and Howell, 1986
) in a total volume of 350 µl. The mixture was then incubated at 37°C for 15 min. When early endosomes were being analyzed, the
whole reaction mixture was adjusted to 40.6% sucrose, 3 mM imidazole
and loaded on the bottom of a TLS 55 tube, overlaid with 500 µl of 35%
sucrose, 3 mM imidazole, and HB, and centrifuged at 45,000 rpm for 45 min. Early endosomal membranes were collected at the 35%/HB interface. For the analysis of biosynthetic membranes, the reaction mixture
was loaded on top of a step gradient formed by 200 µl of 50% sucrose, 3 mM imidazole, and 500 µl of 20% sucrose, 3 mM imidazole in a TLS 55 tube. After centrifugation at 45,000 rpm for 45 min, biosynthetic membranes were recovered at the 20%/50% sucrose interface. In all cases, proteins were then precipitated with CHCl3/methanol, solubilized in SDS gel
sample buffer (2% SDS, 10% glycerol, 100 mM DTT, 60 mM Tris, pH 6.8, 0.001% bromophenol blue) and separated by gel electrophoresis. After
transfer to nitrocellulose, analysis of COP binding was then carried out by
Western blotting using antibodies against each COPI subunit.
. Briefly, BHK cells were
incubated for 5 min with 5 mg/ml HRP to provide a marker of the early
endosomal content, and then cells were homogenized and a PNS was prepared. The PNS was fractionated as described above. Early endosomes
were the collected from the 35%/25% interface, well separated from ECVs
and late endosomes (25%/HB interface; Aniento et al., 1993
). In the assay, 300-500 µg of early endosomal protein in a final volume of 1.4-2.3 ml
was incubated for 30 min at 37°C in 12.5 mM Hepes, pH 7.0, 1 mM DTT,
1.5 mM MgOAc, 60 mM KCl, and supplemented with an ATP-regenerating system and 4 mg/ml cytosol. The cytosol was prepared from wild-type
(WT) CHO cells or from ldlF cells, as above. Then, the mixture containing
both donor early endosomes and vesicles formed in vitro was brought to
25% sucrose, 3 mM imidazole, pH 7.4, loaded at the bottom of an SW60
tube and overlaid with HB. After 1 h of centrifugation at 35,000 rpm, donor early endosomes and budded vesicles were recovered from the pellet
and the 25% sucrose/HB interface, respectively. Both fractions were recentrifuged for 30 min at 100,000 g to sediment membranes, and the HRP
activity was quantified in the pellets.
or with bicinchoninic acid (Pierce Chemical Co., Rockford, IL).
SDS-PAGE was performed according to Laemmli (1970)
. HRP activity
was measured as in Gruenberg and Gorvel (1992)
. Western blot analysis
was carried out using peroxidase-conjugated secondary antibodies (Bio-Rad Labs, Hercules, CA) and detected by chemiluminescence using the
SuperSignalTM reagent (Pierce Chemical Co.). Blot exposure times were
always within the linear range of detection.
Results
COP is rapidly degraded in ldlF cells at the restrictive temperature (Guo et al.,
1996
) and that already at the permissive temperature,
amounts of
COP are reduced when compared with WT
CHO cells (Guo et al., 1996
; Fig. 1). The other COP subunits
(
,
,
,
, and
COP) were not affected by incubation at
the restrictive temperature, except for an approximately
twofold reduction in
COP. Amounts of each subunit
were, in fact, comparable to those found in WT CHO cells cultured at 37 or 40°C (Fig. 1), indicating that COP-I subunits or subcomplexes were stable despite the complete
degradation of one subunit.
Fig. 1.
Distribution of COP-I subunits in CHO and ldlF cells. Cytosols
were prepared from ldlF cells incubated at the permissive (34°C) or restrictive (40°C) temperature. For comparison, cytosols were also prepared
from WT CHO cells incubated at 37 or
40°C. The COP-I composition of each
cytosol was analyzed by SDS-PAGE
followed by Western blotting using antibodies against each of the COP-I components. 20 µg protein was loaded per
lane.
[View Larger Version of this Image (32K GIF file)]
; Harder and Gerke, 1993
), a well-established marker
of clathrin-dependent endocytosis (Pearse and Robinson,
1990
; Trowbridge et al., 1993
) and then incubated at the
desired temperature for 6 h. At this time,
COP was fully
degraded (Guo et al., 1996
; data not shown), yet all other
COP-I subunits were present (see Fig. 1). Then, the cells
were incubated with human rhodamine-transferrin for only 5 min at the corresponding temperature, to allow one
wave of receptor-mediated endocytosis to occur. As shown
in Fig. 2 A, transferrin appeared to be internalized as efficiently at the restrictive or permissive temperature. Similarly CD4, which is endocytosed through clathrin-coated
pits in T cell lines and transfected HeLa cells, was internalized at similar rates in stably transfected ldlF cells incubated at permissive or restrictive temperature (Bowers,
K., and M. Marsh personal communication). These data indicate that clathrin-dependent endocytosis continued in
the absence of
COP.
Fig. 2.
Endocytosis in ldlF cells. Cells were incubated at the
permissive (34°C) or restrictive (40°C) temperature for 6 h. (A)
Transferrin internalization. Cells had been transiently transfected
with the cDNA encoding for the human transferrin receptor before incubation at 34 or 40°C. Transferrin internalization was visualized by fluorescence microscopy after 5 min incubation with
50 µg/ml rhodamine-transferrin at the corresponding temperature. (B) Continuous internalization of HRP. Cells were incubated with 3 mg/ml HRP at the corresponding temperature for 5, 15, 30, 60, or 120 min. The amounts of endocytosed HRP were
quantified and expressed as OD U/min/mg cellular protein. (C)
Recycling of internalized HRP. Cells were incubated with 0.5 mg/
ml HRP for 5 min at the corresponding temperature, washed, and
then reincubated for 10, 20, 30, or 40 min. At each time point, cells and the media were collected. At each time point, HRP remaining associated to the monolayer is expressed as a percentage
of the total (cell-associated and regurgitated). In B and C, each
panel shows the mean of two representative series of experiments. Bar, 5 µm.
[View Larger Versions of these Images (96 + 12K GIF file)]
; Pagano et al., 1991
)
was not changed by the temperature shift (Kobayashi, T.,
F. Gu, K. Bowers, M. Marsh, and J. Gruenberg, unpublished observations). We then measured whether fluid phase
endocytosis of HRP was affected at the restrictive temperature. Cells were incubated with HRP for increasing time periods, and the amounts of cell-associated HRP were quantified. At the permissive temperature, HRP accumulated intracellularly with time, as expected (Fig. 2 B). At the
restrictive temperature, HRP was clearly taken up, but the
process exhibited a classical saturation profile. Uptake was
somewhat reduced after short times (~60 -70% of the unshifted control) and reached a plateau after longer times,
indicating that intracellular accumulation did not occur. Recycling back to the cell surface was then measured by following regurgitation of HRP, which had been preinternalized into early endosomes for a short (5 min) period of
time. At the permissive temperature, recycling was rapid,
~60% of HRP being regurgitated within 10 min (Fig. 2 C),
in good agreement with previous observations (Besterman
et al., 1981
; Parton et al., 1992
b). At the restrictive temperature, however, ~80% of preinternalized HRP was regurgitated within 10 min. These observations suggest that
when intracellular HRP accumulation was impaired, the
endosomal content was recycled back into the medium.
COP is present on early
endosomes and is required for the formation of vesicles
that mediate transport from early to late endosomes in
vitro (Aniento et al., 1996
). We therefore investigated
whether early to late endosome transport still occurred in
ldlF cells incubated at the restrictive temperature for 6 h in
vivo. Early endosomes were labeled with HRP internalized for 5 min from the medium. To label late endosomes, HRP was subsequently chased for 30 min in marker-free
medium (Gruenberg and Howell, 1989
; Aniento et al., 1993
).
The subcellular distribution of HRP was analyzed by subcellular fractionation (Fig. 4), as well as by light (Fig. 3)
and electron (Figs. 7 and 8) microscopy.
Fig. 4.
Subcellular fractionation of ldlF endosomes.
(A) Cells prepared as in Fig.
3 were incubated at the permissive (34°C) or restrictive (40°C) temperature with medium containing 4 mg/ml
HRP for 5 min (pulse) or
subsequently reincubated
for 40 min in the absence of
marker (chase). Early (EE)
and late (LE) endosomes
were then separated by subcellular fractionation. The total HRP content of the fractions was quantified using a
colorimetric reaction and is
expressed as OD U/min.
HRP recovery in the fractions corresponded to ~40%
of the total, latent cell-associated activity (~50% lost to
the nuclear pellet after gentle
homogenization) and HRP
latency after homogenization
was always >70%. (B) The recovery of rab5 and rab7 in fractions
(EE or LE at each temperature) prepared as in A was analyzed
by SDS gel electrophoresis, followed by Western blotting with indicated antibodies. Each lane contained 20% of the total protein
in the corresponding fraction.
[View Larger Version of this Image (16K GIF file)]
Fig. 3.
Distribution of endocytosed HRP in ldlF cells.
Cells were maintained at the
permissive (34°C) or restrictive (40°C) temperature for
6 h. Then, they were incubated at the corresponding
temperature in the presence
of 10 mg/ml high activity
HRP for 5 min to label early
endosomes (pulse). Late endosomes were labeled after
reincubation of the cells for
30 min in the absence of
HRP, at the corresponding temperature (chase). After
cell fixation, intracellular
HRP distribution was revealed using a cytochemical
reaction and analyzed by
phase contrast light microscopy. Bar, 10 µm.
[View Larger Version of this Image (97K GIF file)]
Fig. 7.
Ultrastructure of ldlF endosomes at the permissive
temperature. Cells cultured at 34°C were incubated with HRP for
5 min to label early endosomes and then either fixed immediately
(A and B) or further incubated at 34°C for 30 min to label late endosomes (C). Cells were then processed for plastic sections, and
semithick (~150 nm; A and C) or ultrathin sections (50 nm; B)
were prepared. Early endosomal compartments (A and B) are
comprised of tubular and cisternal regions (arrows) and vesicular
domains (arrowheads), as in other cells. B shows a higher magnification view of the Golgi (g) area. After further incubation for 30 min (C), HRP was rarely observed within tubular domains. As
expected, it was distributed within larger multivesicular elements
concentrated in the Golgi area (arrowheads), which presumably
correspond to late endosomes. Bars, 0.5 µm.
[View Larger Version of this Image (96K GIF file)]
Fig. 8.
Ultrastructure of
ldlF endosomes at the restrictive temperature. Cells
cultured at 40°C for 6 h were
incubated with HRP for 5 min and then either fixed immediately (A and B) or further incubated at 40°C for 30 min (C-E). Semithick (A and
C) or ultrathin (B, D, and E)
sections of the cell pellet
were prepared as in Fig. 8.
Early endosomal compartments (A and B) were composed of small tubular and
vesicular elements that were
predominantly in discrete clusters (arrows). Few large
vesicular profiles were evident (compare with Fig. 7, A
and B). After further incubation for 30 min, little HRP remained in the cells (see Figs.
2-4). However, when detected (C-E), the bulk of
HRP was still observed
within clusters of tubular and
vesicular elements (arrows),
which appeared identical to
those labeled after the 10 min pulse. Few vesicular elements were labeled (arrowhead). Note the clear difference when compared with
cells incubated for the same
time at the permissive temperature (Fig. 7 C). As
shown at higher magnification (D and E), labeled elements comprise vesicles and
short tubules, which appear
discontinuous from the analysis of both semithick (C)
and thin (D and E) sections. Bars, 0.5 µm.
[View Larger Version of this Image (152K GIF file)]
).
Consistent with our HRP uptake experiments (Fig. 2 B), cells
incubated at the restrictive temperature contained ~60-
70% of the HRP internalized at the restrictive temperature. After the chase at the permissive temperature, the bulk of HRP (Fig. 4 A) then cofractionated with rab7 (Fig.
4 B), a late endosomal marker (Chavrier et al., 1990
), as
expected. However, after the chase at the restrictive temperature, HRP was reduced both in the total cell extracts
and in the late endosomal fractions (Fig. 4 A) containing
rab7 (Fig. 4 B). These data indicate that the physical properties of early and late endosomes were not affected by the
temperature shift. These observations also show that
HRP, which was internalized at the restrictive temperature, was not transported to late endosomes.
; Clague et al.,
1994
). We thus investigated whether ldlF cells exhibited an
acidification defect at the restrictive temperature, using the
pH-sensitive dyes acridine orange and two forms of LysoSensor, which detect pH values in the 4.5- 6.0 and 6.5-8.0
ranges, respectively. As shown in Fig. 5, no difference
could be observed between cells incubated at the permissive or restrictive temperature for 6 h. In fact, we did not
observe any difference with anyone of the three dyes when cells were incubated at permissive or restrictive temperature over a time period ranging from 1 to 12 h (not shown).
These experiments show that, after
COP degradation,
major differences in the acidification properties of endosomes and lysosomes could not be detected using these
dyes. Altogether, our biochemical and morphological data indicate that transport from early to late endosomes is inhibited in vivo, at the restrictive temperature.
Fig. 5.
Acidification in ldlF cells. Cells prepared as in Fig. 3 at
the permissive or restrictive temperature were treated with acridine orange (A.O.), LysoSensor acidic (detection range pH 4.5-6;
LS. a) and LysoSensor neutral (detection range pH 6.5-8; LS. n)
for 10 min to reveal acidic compartments. The intrinsic fluorescence of each dye after accumulation within acidic endosomes and
lysosomes was observed by fluorescence microscopy. Bar, 5 µm.
[View Larger Version of this Image (70K GIF file)]
COP. First, we used
antibodies against EEA1, a recently discovered protein that
localizes to early endosomal vesicles (Mu et al., 1994
). At
the permissive temperature, EEA1-positive elements exhibited a highly punctate distribution (Fig. 6 A), which is
characteristic for this protein. Although the overall topology was similar at the restrictive temperatures, EEA1-
labeled elements then appeared more clustered or tubular even at the light microscopy level, as illustrated in higher
magnification images (Fig. 6 A). In contrast, the distribution of a late endosomal marker, the small GTPase rab7
(Chavrier et al., 1990
), remained unchanged after incubation at the restrictive temperature for 6 h (Fig. 6 B), as was
the distribution of the cation-independent mannose-6-phosphate receptor, which localizes primarily to late endosomes
and TGN (Brown et al., 1986
; Griffiths et al., 1988
), and
calnexin, a marker of the endoplasmic reticulum (Wada
et al., 1991
). As reported by Guo et al. (1994)
, we also observed some redistribution of
COP at the restrictive temperature, using an antibody that recognizes primarily biosynthetic
COP by immunofluorescence.
Fig. 6.
Distribution of different markers in ldlF cells. (A) Cells
prepared as in Fig. 3 were processed for immunofluorescence microscopy using human antiserum against EEA1. Arrows point at
changes in the appearance of early endosomal elements at 40°C,
when compared with 34°C. low, low magnification; high, high magnification. (B) Cells were processed for immunofluorescence microscopy as in A using the maD antibody against COP or a rabbit antiserum against either the Man6P-R, rab7, or calnexin. The
maD antibody does not label endosomal
COP to any significant
extent under these conditions. Bars, 5 µm.
[View Larger Versions of these Images (120 + 85K GIF file)]
COP degradation
on early endosome ultrastructure, cells containing internalized HRP were analyzed by electron microscopy. At the
permissive temperature (Fig. 7), HRP distributed within
early endosomes after 5 min and reached structures with
the typical appearance and topology of late endosomes after the chase, as expected. At the restrictive temperature
(Fig. 8), however, the structure of early endosomes containing HRP endocytosed for 5 min was dramatically
changed into characteristic clusters of thin 50-60 nm tubules lacking multivesicular domains. These appeared essentially identical to early endosomes observed after neutralization of the endosomal pH (Clague et al., 1994
). In
addition, little, if any, HRP could be detected in late endosomes (and elsewhere in the cells) after the chase, in agreement with our biochemical and light microscopy data. However, in the rare cases where some HRP could still be detected intracellularly, the marker had remained primarily within tubular clusters (Fig. 8 C-E), identical to those
labeled after the pulse. Altogether these data show that
degradation of
COP at the restrictive temperature has little influence on bulk internalization into and recycling
from early endosomes, but inhibits early to late endosome
transport, and causes a dramatic change in the general organization of early endosomal elements.
COP
). Using this assay, we found that vesicles formed in
vitro excluded early endosomal markers and that vesicle
formation depended on
COP. Here, we used the same
assay to test whether cytosol prepared from ldlF cells incubated at the restrictive temperature supported ECV/MVB formation in vitro. Early endosomes were prepared from
BHK cells to ensure that donor membranes were fully
transport-competent (Aniento et al., 1996
). BHK cells
were incubated for 5 min at 37°C in the presence of HRP
to label the early endosomal content. Early endosomes
were then separated from the lighter ECV/MVBs and late
endosomes by flotation on a step sucrose gradient (Aniento et al., 1993
, 1996
). In the assay, early endosomes were
incubated at 37°C in the presence of ATP and cytosol, and
then vesicles that may have formed in vitro were separated
from the denser donor membranes on a similar flotation
gradient. The percentage of the total early endosomal content entrapped within vesicles formed in the assay was
quantified by measuring the HRP activity in both fractions.
). Approximately 10% of the tracer was packaged
within ECV/MVBs formed in the assay, a value that compares well with our previous in vitro and in vivo observations (Aniento et al., 1996
). In contrast to donor early
endosomal membranes, ECV/MVBs formed in vitro acquired the capacity to undergo fusion with late endosomes
(Aniento et al., 1993
, 1996
). They contained both
and
COP (Fig. 9 B) but excluded the early endosomal marker
rab5, in agreement with our previous studies (Aniento
et al., 1996
). These experiments were repeated using cytosol prepared from WT CHO cells incubated at 40°C, corresponding to the ldlF cell restrictive temperature, as a control (Fig. 9). Then, a slight but reproducible increase in
ECV/MVB formation was observed, presumably reflecting temperature effects on the activity of some cytosolic
factors. In contrast, ECV/MVB formation was inhibited
when using cytosol prepared from ldlF cells incubated at
40°C. Inhibition was not complete, perhaps because BHK
membranes retained some endogenous
COP after fractionation (Aniento et al., 1996
) or because some forming
ECV/MVBs were already committed, beyond the COP-dependent step, at the time of homogenization. Indeed,
similar levels of inhibition were observed after depletion
of cytosolic coatomer (Aniento et al., 1996
).
Fig. 9.
In vitro formation
of ECV/MVBs. (A) BHK
early endosomes containing
HRP internalized for 5 min
at 37°C were prepared by flotation on a sucrose gradient
and used as donor membranes in the assay (Aniento
et al., 1996). Donor endosomes were incubated with
(+) or without (
) an ATP-regenerating system and in
the presence of cytosol. Cytosols were prepared, as indicated, from ldlF cells incubated at the permissive
(34°C) or restrictive (40°C)
temperature, or from WT
CHO cells incubated at 37 or
40°C. Vesicles formed in
vitro were then separated
from donor membranes by
flotation in a gradient, and
the HRP content of both
fractions quantified. In the
assay, ~10% of HRP originally internalized into early endosomes was entrapped within vesicles formed in vitro, as shown
previously (Aniento et al., 1996
). Efficiency of vesicle formation
is expressed as a percentage of the control with cytosol from WT
CHO cells incubated at 37°C. (B) The assay was carried out in
the presence of cytosol from CHO cells incubated at 37°C. Then,
donor endosomes (I) and ECV/MVBs formed in vitro (II) were
pelleted and analyzed by SDS gel electrophoresis and Western
blotting with antibodies against
COP,
COP, and rab5, as indicated. Each lane contained 35% of the protein content of the corresponding fraction.
[View Larger Version of this Image (14K GIF file)]
COP degradation on both endosome ultrastructure and transport suggests that both
mechanisms are coupled functionally. We have previously
shown that COPs do not bind endosomal membranes in
vivo or in vitro after pH neutralization, presumably reflecting the activity of a transmembrane pH sensor (Aniento et al., 1996
). We therefore investigated whether
COP
degradation similarly inhibited membrane binding of the
remaining COP subunits, or whether these had retained
the capacity to bind endosomes. In these experiments, biosynthetic membranes were used as a control.
). Cells were pretreated with
brefeldin A to reduce possible contamination of endosomes with biosynthetic membranes (see Pelham, 1991
).
(All subsequent steps were without the drug.) Cells were
then homogenized, and fractions were prepared on a step
flotation gradient that separates the bulk of biosynthetic membranes from both early and late endosomes (Gorvel
et al., 1991
; Aniento et al., 1996
). As shown in Fig. 10, the
distribution of the small GTPase rab5 was unaffected by
brefeldin A. However, BHKp23, a transmembrane protein
of the 24-kD family that localizes to the intermediate compartment/cis-Golgi network (Rojo et al., 1997
), was no
longer detected in the fraction after the treatment (Fig. 10).
Fig. 10.
Separation of endosomal and biosynthetic
membranes. To achieve optimal conditions for the separation of endosomal and biosynthetic membranes, BHK
cells were pretreated with
brefeldin A (+BFA). The
drug was absent from all subsequent steps. In the control, the drug was omitted
(BFA). After homogenization, the corresponding postnuclear supernatants were fractionated using a sucrose step flotation gradient (Gorvel et al., 1991
; Aniento et al., 1996
), and
fractions enriched in early endosomes were collected. The COP
binding capacity of these membranes was measured after incubating 50 µg of each fraction with 500 µg BHK cytosol for 15 min at 37°C in the presence of 10 µM GTP
S, to stimulate COP
recruitment. Membranes were then retrieved by flotation on a
step gradient and analyzed by SDS gel electrophoresis followed
by Western blotting using antibodies against
COP, rab5, and
BHKp23.
[View Larger Version of this Image (40K GIF file)]
S was added to stimulate COP membrane association (Kreis and Pepperkok, 1994
; Whitney
et al., 1995
; Aniento et al., 1996
). Membranes were then
retrieved by flotation on a step sucrose gradient, and
COPs were analyzed by SDS gel electrophoresis and Western blotting. Endogenous
COP was partially lost during
fractionation (Fig. 10) but was revealed after longer exposures of the blot (not shown), as previously observed (Whitney et al., 1995
; Aniento et al., 1996
). However, after brefeldin A treatment, endosomal membranes retained the
capacity to recruit cytosolic COPs, although amounts of
bound COPs were reduced when compared with untreated
controls, as expected after depletion of biosynthetic membranes (Fig. 10). These membranes were therefore used to
test the membrane binding capacity of ldlF COPs.
COP was missing (Fig. 11; EE, early endosome fraction). However, a
small COP subset only, consisting of
,
, and
COP, was
then recruited onto early endosomes. Previous experiments had shown that
and
COP are not present on endosomes (Whitney et al., 1995
; Aniento et al., 1996
), but in
the absence of
COP,
COP also failed to interact with endosomes. In contrast, the same cytosol preparation was
fully competent to support binding of all subunits, including
,
, and
COP, but obviously excluding
COP, to biosynthetic membranes (Fig. 11; BM, biosynthetic membrane fraction). These experiments demonstrate that some
COP subunits, namely
,
, and
COP, are still recruited
onto endosomal membranes despite the absence of
COP.
Since this subcomplex did not support membrane transport in vivo (Figs. 3, 4, 7, and 8) or efficient ECV/MVB
formation in vitro (Fig. 9), our data suggest that COP function in endosome transport requires the presence of
and/or
COP. Our data also indicate that recruitment of
COP onto endosomal membranes, but not biosynthetic
membranes, requires the presence of
COP.
Fig. 11.
COP binding to
endosomal and biosynthetic
membranes. Cytosols were
prepared from ldlF cells incubated at the restrictive
temperature, as in Fig. 1.
The COP binding capacity
of biosynthetic (BM) or early
endosomal (EE) membranes
prepared from BHK cells
pretreated with brefeldin A
was tested as in Fig 10, except that ldlF cytosol was
used with (G and NG) or
without (C) 10 µM GTPS.
When indicated (NG), the
pH of endosomes was preneutralized with 50 µM nigericin before GTP
S addition. Membranes were
retrieved and analyzed using antibodies against each COP
subunit. Western blots were developed using the ECL reaction;
exposure times were five times longer for EE than for BM membranes to ensure that signals remained in the linear detection
range.
[View Larger Version of this Image (35K GIF file)]
). Neutralization of the pH, however, does not cause
the release of pre-bound COPs (not shown), indicating
that recruitment only is acidification dependent. That COP
association to endosomes involves more than one step is illustrated by the combined effects of pH and GTP
S. COP
binding to endosomes was partially inhibited when the endosomal pH was preneutralized with nigericin before addition of GTP
S when compared with GTP
S alone (Fig.
11). The same observations were made using BHK cytosols containing all COP subunits (not shown). The treatment had no effects on biosynthetic membranes, as expected since these compartments are not acidic. The fact that the effects of nigericin and GTP
S were dependent
on the order-of-addition suggests that a pH sensor and a
GTP-binding protein are sequentially involved in COP
binding to endosomal membranes. These experiments also
show that COP membrane binding characteristics are fully
retained by the
,
, and
COP subset, suggesting that
these subunits may mediate association of the coatomer
onto endosomes.
Discussion
COP. At the restrictive temperature,
COP is degraded, whereas the other subunits are unaffected. Our data show that receptor-mediated endocytosis
as well as internalization and recycling of the fluid phase
tracer HRP continue at the restrictive temperature but
that HRP fails to accumulate intracellularly. Our biochemical and morphological observations indicate that HRP is
then internalized into early endosomes, from where it can
recycle back to the medium, but that HRP transport from
early to late endosomes is inhibited. Concomitant with this
transport inhibition, we find that the early endosome ultrastructure is changed into clusters of thin tubules, which
lack typical multivesicular domains corresponding to forming ECV/MVBs. That COPs may be directly involved in
ECV/MVB biogenesis is supported by our observations
that cytosol prepared from ldlF cells incubated at the restrictive temperature, thus lacking
COP, does not support
efficient formation of ECV/MVBs from naive early endosomes of BHK cells. Our data also indicate that COP binding to endosomal and biosynthetic membranes is differentially regulated. In the absence of
COP, all remaining
subunits bind BHK biosynthetic membranes in a pH-independent manner. In contrast, only
,
, and
subunits are
recruited onto naive endosomal membranes of BHK cells,
and the process depends on the lumenal pH. Since
,
,
and
COP retain the characteristics of endosomal COP binding with respect to pH and GTP
S, it is tempting to speculate that these subunits are involved in COP association to
endosomes, whereas
and/or
COP may drive COP function in endosome transport.
observed that the low density lipoprotein (LDL) receptor is relatively unstable in
ldlF cells at the restrictive temperature. This processing
occurs via an unconventional mechanism since it is insensitive to pH neutralization, in contrast to degradation in lysosomes and to the phenotype of other mutant CHO cell
lines, where the LDL receptor is equally unstable. One
possible explanation for LDL receptor instability in ldlF
cells comes from our observations that the early endosomal content of the lysosomal enzyme
-N-acetyl-glucosaminidase is increased approximately twofold 6 h after temperature shift when compared with unshifted controls (not
shown). The presence and activity of hydrolases in mildly
acidic early endosomes has been previously reported
(Diment and Stahl, 1985
; Ludwig et al., 1991
). Increased
amounts of enzymes may be due to recapture from the medium (Kornfeld, 1992
) combined with defective transport to late endosomes. Then, recycling LDL receptor molecules may become progressively processed during their
cycle, while passing through early endosomes.
;
Gruenberg and Maxfield, 1995
; Mellman, 1996
). Whereas
tubular elements are involved in membrane recycling back
to the cell surface, molecules destined to be degraded are
selectively incorporated within multivesicular portions
(Geuze et al., 1983
), which correspond to forming ECV/ MVBs (Aniento et al., 1993
, 1996
). Very little is known
about the mechanisms controlling the morphogenesis of
early endosomal membranes into elements with such a
strikingly different organization. We find that the absence
of a functional COP coat inhibits ECV/MVB biogenesis
and therefore also inhibits the accumulation of internal membranes and transport from early to late endosomes.
We also find that early endosomal membranes are then
changed into typical clusters of thin tubules. Similarly, in
the biosynthetic pathway, COPs are required for transport,
but also for maintenance of Golgi organization (see Guo
et al., 1994
; Kreis and Pepperkok, 1994
).
; Aniento et al., 1996
). Our data indicate that both pH- and COP-mediated effects are,
in fact, coupled functionally. COP association to endosomal membranes is itself pH dependent, both in vivo and in
vitro (Aniento et al., 1996
), as is the recruitment of the
,
, and
COP subcomplex in the absence of
COP. Moreover, endosome dynamics, including ultrastructural organization and transport, appear to be affected in the same
way when the COP coat is absent after pH neutralization or when a nonfunctional coat is recruited after
COP degradation. We can conclude that maintenance of early endosome membrane organization and ECV/MVB biogenesis are coupled and that these processes are controlled by
the pH-dependent association of a functional COP coat.
We previously proposed that pH-dependent COP association may reflect the activity of a transmembrane pH sensor (Aniento et al., 1996
). We propose that this pH sensor
functions as a molecular checkpoint signaling, via coat recruitment, the onset of the degradation pathway.
;
Fiedler et al., 1996
; Sohn et al., 1996
), suggesting that
COPs perform sorting functions, much like the clathrin/
adaptor coats (Pearse and Robinson, 1990
; Robinson, 1992
).
A morphologically visible coat, resembling the biosynthetic COP coat (Orci et al., 1986
), has not been detected yet
on endosomes, perhaps because of the different composition of endosomal and biosynthetic COPs (Whitney et al.,
1995
; Aniento et al., 1996
). One may, however, speculate
that endosomal COPs mediate the selection of proteins
destined to be incorporated into forming ECV/MVBs. Indeed, this process is undoubtedly selective, presumably
depending on cytoplasmically exposed protein motives (see Gruenberg and Maxfield, 1995
). In line with this view,
COP could be detected at the neck of forming ECV/
MVBs in vitro (Aniento et al., 1996
). It is possible that inhibition of this sorting mechanism, via COP inactivation,
prevents ECV/MVB biogenesis and therefore also prevents accumulation of internal membranes.
,
, and
COP subunits interact with one or more endosomal membrane proteins, including perhaps the pH sensor itself; our data also indicate
that these subunits are selectively recruited from a complete cytosol when
COP is missing. This recruitment exhibits the characteristic properties of endosomal COP
binding, namely pH and GTP
S sensitivity. COP subcomplexes that may mediate membrane association to biosynthetic membranes have been identified. The
,
, and
subunits were shown to bind membranes and cytoplasmic
KKXX motifs (Lowe and Kreis, 1995
). In addition, different COP subcomplexes appear to bind to different peptides derived from the cytoplasmic domains of members of
the 20-25-kD protein family (Fiedler et al., 1996
). This apparent heterogeneity of COP subcomplexes involved in
membrane association, whether in biosynthesis or endocytosis, may reflect the existence of multiple interactions between COPs and different protein motives.
S
on COP recruitment, suggesting that a transmembrane pH
sensor (Aniento et al., 1996
) and a GTP-binding protein,
perhaps of the ARF family, are involved sequentially during COP recruitment. We also find that
COP is necessary
for
COP recruitment onto endosomal but not biosynthetic, membranes. That these two proteins may be functionally or physically linked is consistent with our observation that cellular amounts of
COP, but not other COPs, are decreased when
COP is degraded. Our data also show
that COP membrane association and function can be uncoupled. Whereas
,
, and
COP can interact with membrane components, perhaps via a receptor, the
and
COP subunits are essential for function, possibly reflecting the coat polymerization step. In conclusion, we propose that formation of the COP coat onto endosomal
membranes may occur in at least three distinct steps. Membrane recruitment may be mediated by the
,
, and
subunits through the sequential involvement of a pH sensor and a GTP-binding protein. ECV/MVB biogenesis, however, may be dependent on the presence of
and/or
COP.
Received for publication 21 May 1997 and in revised form 18 September 1997.
1. Abbreviations used in this paper: COP, coatomer protein; ECV, endosomal carrier vesicles; HB, homogenization buffer; LDL, low density lipoprotein; Man6P-R, mannose-6-phosphate receptor; MVB, multivesicular body; PNS, postnuclear supernatant; WT, wild-type.This work was supported by grant No. 31-37296.93 from the Swiss National Science Foundation (to J. Gruenberg), grant No. 961235 from the National Health and Medical Research Council of Australia (to R.G. Parton), and grant RG 355/94 from the International Human Frontier Science Program (to J. Gruenberg and R.G. Parton).
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