The major histocompatibility complex (MHC) (
)class II
molecules are heterodimers (
and
chains) that associate in
the endoplasmic reticulum with the invariant chain (Ii) (Sung and
Jones, 1981; Kvist et al., 1982). Ii is a type II
transmembrane protein exposing 30 amino NH
-terminal
residues at the cytoplasmic side of the membrane, it spans the membrane
between residues 30 and 56 and has a luminal COOH-terminal domain of
160 amino acids (Claesson et al., 1983; Strubin et
al., 1984). The formation of 
Ii complexes is believed to
occur by the sequential addition of 
heterodimers to a
pre-existing core of trimeric Ii molecules (Marks et al.,
1990; Lamb and Cresswell, 1992), the final complex being a
stoichiometric nine-subunit complex (Roche et al., 1991).
Following subunit assembly, 
Ii complexes traverse the Golgi
apparatus before reaching the endosomes. It has been shown that the
endosomal sorting signal for newly synthesized 
Ii complexes
resides in the Ii cytoplasmic tail (Bakke and Dobberstein, 1990;
Lotteau et al., 1990; Roche et al., 1992; Simonsen et al., 1993) involving two autonomous sorting signals based
on the hydrophobic amino acids Leu-Ile and Met-Leu (Bremnes et
al., 1994). In 4N5Ii cells, the route followed by 
Ii
complexes may involve their transient expression at the plasma membrane
before being internalized (Roche et al., 1993). Once in
endosomes, Ii is sequentially degraded by proteases. In B
lymphoblastoid cells, degradation products of Ii were observed in the
presence of protease inhibitors (Blum and Cresswell, 1988; Nguyen and
Humphreys, 1989), whereas processing intermediates could be detected in
the absence of protease inhibitors in a human melanoma cell line
(Pieters et al., 1991). It has further been suggested that
proteolysis of Ii might be the rate-limiting step in the transport of
class II molecules back to the plasma membrane (Neefjes and Ploegh,
1992; Loss and Sant, 1993).
After transient expression in COS cells,
high levels of Ii expression induced large vesicular structures also
called macrosomes (Pieters et al., 1993; Romagnoli et
al., 1993). Morphological studies showed that, in cells containing
macrosomes, the rate of endocytic flow was delayed. In this paper,
using a biochemical approach by subcellular fractionation, we show that
supertransfection of Ii in cells already transfected with MHC class II
induces a general delayed transport between early and late endosomes,
although macrosomes are induced only in a small number of cells.
Pulse-chase experiments show that a p23 degradation form of Ii
associated with class II molecules appeared in early endosomes after
2-h chase. At 8 h of chase, further Ii degradation products were
observed in late endosomes.
EXPERIMENTAL PROCEDURES
Cell Lines
The transfected human fibroblast cell
lines were a kind gift of Drs. D. Karp and E. Long (National Institutes
of Health, Bethesda, MD). The generation of these cell lines have been
described earlier (Roche et al., 1992). In brief, human M1
cells were transfected with HLA-DRA and DRB cDNA under the SV40 early
promotor (4N5 cells). These cells were supertransfected with the major
form of the human Ii cDNA (p33Ii) or the human Ii cDNA lacking 20 amino
acids of the cytoplasmic tail (
20Ii) in a vector with
cytomegalovirus promotor. 4N5 cells transfected with Ii express small
amounts of surface Ii and the resulting 4N5Ii cells were selected using
cell sorting and magnetic beads and they are thus not cloned, but
represent a multitude of different clones (Roche et al.,
1993). Observations by phase contrast microscopy and staining by
indirect immunofluorescence with antibodies directed against Ii showed
that less than 2% of the 4N5Ii cells contained large vesicular
structures (data not shown). In contrast, cells transfected with
20Ii expressed large amounts of surface Ii (4N5
20Ii cells)
(Bakke and Dobberstein, 1990; Roche et al., 1992). Cells were
maintained in Dulbecco's modified Eagle's medium
supplemented with 2 mM glutamine and 10% fetal calf serum
(FCS).
Antibodies
The monoclonal IgG antibody VicY1
(Quaranta et al., 1984), recognizing the cytoplasmic tail of
Ii, was a gift of Dr. W. Knapp, Vienna. Rabbit anti-
and -
HLA-DR chain sera were a gift of Drs. J. Neefjes and H. Ploegh,
Amsterdam (Neefjes et al., 1990), whereas the anti-MHC class
II molecule L243 antibody was obtained from ATCC.
Separation of Early and Late Endosomal
Fractions
The separation of early (EE) and late (LE) endosomal
fractions was first described by Gorvel et al.(1991). Each
experiment was controlled by analyzing the distribution of horseradish
peroxidase (HRP) along the gradient as in Gorvel et al.(1991).
Briefly, cells were incubated for 5 min at 37 °C with 1.5 mg/ml
HRP. After several washings, HRP was chased along the endocytic pathway
by incubating the cells in internalization medium for various periods
of time at 37 °C. They were then scraped at 4 °C and pelleted.
Homogenization was performed at 4 °C in 250 mM sucrose, 3
mM imidazole, pH 7.4, 1 mM EDTA by needle breakdown.
Homogenates were centrifuged for 10 min at 3000 rpm at 4 °C and
post-nuclear supernatants (PNS) were collected. PNSs were then brought
up to 40.6% sucrose using a stock solution of 62% sucrose, 3 mM imidazole, pH 7.4, 1 mM EDTA and loaded at the bottom of
a SW60 centrifugation tube. The load was then sequentially overlaid
with 1.5 ml of 35% sucrose, 3 mM imidazole, pH 7.4, 1 mM EDTA; 1 ml of 25% sucrose, 3 mM imidazole, pH 7.4, 1
mM EDTA; and finally with 0.5 ml of homogenization buffer. The
gradient was centrifuged at 35,000 rpm for 60 min at 4 °C. Early
(EE) and late (LE) endosomal fractions were collected at the 35%/25%
sucrose interface and in the uppermost portion of the 25% sucrose
cushion, respectively. HRP quantifications were determined using a
horseradish peroxidase assay (Gorvel et al., 1991).
Electron Microscopy of Early and Late Endosomal Fractions
in 4N5Ii Cells
Endosomal membrane fractions were fixed using 2%
glutaraldehyde in 0.2 M cacodylate buffer. After
centrifugation (30 min) at 100,000
g at 4 °C in an
Airfuge (Beckman), post-fixation of membrane pellets was performed
using 2% OsO
and 1.5% potassium ferrocyanide in
H
O for 30 min followed by an overnight incubation with 0.5%
uranyl acetate in water (Parton et al., 1989). Both EE and LE
fractions were dehydrated in an ethanol series before embedding and
polymerization. Epon sections were stained using lead citrate.
Distribution and Quantification of Rab Proteins in
Endosomal Fractions
The distribution and the quantitative
analyses of Rab proteins have been studied using
[
-
P]GTP-ligand assay (Schmitt et
al., 1986; Bucci et al., 1992). Early and late endosomal
fractions were solubilized in 1% Nonidet P-40 for 30 min at 4 °C
and submitted to 100,000
g centrifugation in an
Airfuge Beckman ultracentrifuge for 30 min at 4 °C. Supernatants
were incubated overnight at 4 °C with rabbit anti-Rab antisera. 100
µl of a 50% suspension of protein A-Sepharose CL-4B beads were then
incubated with the supernatants for 1 h at 4 °C. Immunoadsorbents
were collected by centrifugation, washed three times with 1% Nonidet
P-40, 10 mM Tris-HCl, pH 7.5, 150 mM NaCl and twice
with 10 mM Tris-HCl, pH 7.5. Bound proteins were eluted by
boiling the samples at 95 °C for 5 min in SDS-PAGE sample buffer
containing 5 mM dithiothreitol. Samples were submitted to 12%
SDS-polyacrylamide gel electrophoresis. The gel was then soaked in 50
mM Tris-HCl, pH 7.5, 20% glycerol twice for 15 min and the
immunoprecipitated Rab proteins transferred onto nitrocellulose filters
using a carbonate buffer (10 mM NaHCO
, 3 mM Na
CO
, pH 9.8). Then, filters were rinsed
twice for 10 min in 50 mM NaH
PO
, pH
7.5, 10 µM MgCl
, 2 mM dithiothreitol,
4 µM ATP, and 0.3% Tween 20 (binding buffer) and incubated
with 1-2 µCi/ml [
-
P]GTP
(Amersham, 3000 Ci/mmol) in the binding buffer for 2 h at room
temperature. After several washings in the binding buffer,
filter-associated radioactivity was revealed using a PhosphorImager
(FujiX BAS 1000).
Distribution and Quantification of Plasma Membrane
Proteins along the Gradient
Plasma membrane distribution along
the gradient was analyzed by surface biotinylation (LeBivic et
al., 1989). Cells were prewashed three times for 5 min with PBS
containing 1 mM MgCl
and 0.1 mM CaCl
(PBS
) at 4 °C and then
incubated twice for 25 min with 0.5 mg/ml of Sulfo-NHS Biotin (Pierce)
at 4 °C. Free biotin was then quenched twice for 15 min at 4 °C
with 50 mM NH
Cl in PBS
. After
several washings with PBS
, cells were homogenized, PNS
was prepared and loaded on a sucrose step flotation gradient. The
different fractions of the gradient were collected and the
corresponding aliquots were loaded onto nitrocellulose filters. Filters
were prewashed in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, Triton X-100 0.2% (TBST) containing 5% milk for 1 h at room
temperature. After a TBST buffer wash containing 1% BSA (BTBST),
filters were incubated for 1 h with
I-streptavidin in
BTBST and then washed three times for 10 min with BTBST and once with 1 M NaCl, BTBST for 10 min. Filters were rinsed with PBS, dried
and the radioactivity quantified using phosphorimaging (FujiX BAS
1000).
Pulse-Chase Experiments
2
75-cm
confluent flasks of 4N5 and 4N5Ii cells were used for each
fractionation experiment. Cells were washed at 37 °C in PBS with 2%
FCS, then incubated with prewarmed cysteine/methionine-deficient RPMI
1640 medium containing 5% dialyzed FCS (pulse medium). After 30 min at
37 °C, cells were incubated with 2 ml of the pulse medium
containing 0.2 mCi of a 80% [
S]methionine and
20% [
S]cysteine mix (DuPont NEN) for 20 min at
37 °C. After radiolabeling, radioactivity was chased for various
periods of time with prewarmed RPMI 1640 medium containing 10% FCS and
5 mM of nonradioactive methionine/cysteine.
Immunoprecipitation of Ii and Class II
Molecules
After metabolic labeling, cells were removed with a
rubber policeman and rapidly centrifuged for 5 min at 1500 rpm. EE and
LE membrane fractions were prepared as described above. They were
solubilized for 1 h at 4 °C in 1% Nonidet P-40, 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1
mM phenylmethylsulfonyl fluoride (solubilization buffer).
Samples were precleared by a 2-h incubation with rabbit IgG anti-mouse
IgGs (Cappel) preadsorbed on protein A-Sepharose CL-4B beads
(Pharmacia). Immunoadsorbents were discarded after centrifugation (4000
rpm, 5 min), and precleared supernatants were incubated overnight with
anti-Ii or anti-HLA-DR antibodies. Finally, 60 µl of a 50%
suspension of protein A-Sepharose beads were incubated with the
supernatants for 1 h. Immunoadsorbents were collected by
centrifugation, washed three times with 1% Nonidet P-40, 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% SDS, 0.1%
deoxycholate, 2 mM EDTA, twice with the same buffer without
SDS and deoxycholate, twice with 0.5% Nonidet P-40, 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, and
twice with 10 mM Tris-HCl, pH 7.5. Washed beads were
resuspended in 50 µl of SDS-PAGE sample buffer containing 5 mM dithiothreitol and heated at 95 °C for 5 min. Eluted proteins
were then analyzed by SDS-PAGE in 10% gels. Gels were treated with
EnHance (DuPont NEN), dried, and submitted to fluorography.
RESULTS
In order to investigate the role of Ii in the transport of
class II molecules in endosomal compartments in 4N5, 4N5Ii, and
4N5
20Ii cells, we maximized the protocol previously used for the
separation of early (EE) and late (LE) endosomal compartments in
several cell lines (Gorvel et al., 1991; Papini et
al., 1993). The separation of these endosomal compartments was
controlled by electron microscopy and the distributions of HRP plasma
membrane and Rab GTPases were analyzed.
Characterization of Early and Late Endosomal
Compartments
4N5, 4N5Ii, and 4N5
20Ii cells were
homogenized, post-nuclear supernatants prepared and submitted to a
flotation step gradient (see ``Experimental Procedures'').
Gradient membrane fractions 2 and 4 were processed for electron
microscopy. Fig. 1shows micrographs obtained from 4N5Ii cell
line. In fraction 4, vesicular elements closely resembling the
appearance of early endosomes in intact cells were observed (Gruenberg et al., 1989; Griffiths et al., 1989). Conversely,
fraction 2 exhibited the typical morphology of late endosomes as in
intact cells (Gruenberg et al., 1989). Similar results were
obtained with 4N5 and 4N5
20Ii cells (data not shown).
Figure 1:
Electron microscopy of EE and LE in
4N5Ii cells. Cells were homogenized and fractionated onto the flotation
step gradient. Many tubular and vesicular endosomes are visualized in
the early endosomal fraction (a). The late endosomal fraction (b) contained predominantly large structures with a
multivesicular appearance. Bar represents 1
µm.
Small
GTP-binding proteins of the Rab subfamily are associated with specific
intracellular compartments of the secretory and endocytic pathways. In
the endocytic pathway, Rab5 and Rab7 have been localized in early and
late endosomes, respectively (Chavrier et al., 1990). Membrane
fraction 4 (related to EE, Fig. 1) and 2 (related to LE, Fig. 1) were solubilized and rab proteins immunoprecipitated by
using anti-Rab5- and anti-Rab7-specific antisera (Chavrier et
al., 1990). After immunoprecipitation and transfer onto
nitrocellulose filter, Rab proteins were revealed by a GTP-ligand assay (Fig. 2). Rab5 and Rab7 were specifically enriched in EE and LE,
respectively.
Figure 2:
Distribution of Rab proteins in EE and LE
of 4N5 and 4N5Ii cells. The distribution was analyzed by GTP-ligand
assay. EE and LE were prepared as described under ``Experimental
Procedures.'' Rab proteins were immunoprecipitated overnight at 4
°C with rabbit anti-Rab5 and -Rab7 antisera and submitted to 12%
SDS-polyacrylamide gel electrophoresis. GTP-ligand assay was performed
as described under ``Experimental
Procedures.''
In order to quantify plasma membrane contamination
along the gradient, cell surface biotinylation was performed (LeBivic et al., 1989). Contamination of plasma membrane both in EE and
LE fractions was less than 1% in the three cell lines. The majority of
the plasma membrane (>95%) remained within gradient fraction 6
(40.6/35% sucrose interface) and in the membrane pellet of the gradient
fraction 7 (40.6% sucrose cushion) (Fig. 3D).
Figure 3:
HRP and plasma membrane distributions
along the flotation gradient. 4N5 (A), 4N5Ii (B), or
4N5
20Ii (C) cells were incubated with HRP for 5 min at 37
°C and chased for 45 min at 37 °C. Cells were homogenized and
PNS loaded onto a flotation step gradient. In D after cell
surface biotinylation, 4N5Ii cells were fractionated as above. The
different fractions of the gradient were analyzed from the top
(fraction 1) to the bottom (fraction 7). Fraction 1 =
homogenization buffer; fraction 2 = LE; fraction 3 = 25%
sucrose cushion; fraction 4 = EE; fraction 5 = 35%
sucrose cushion; fraction 6 = 35%/40.6% interface; fraction 7
= 40.6% sucrose cushion (load).
These
results show that EE was efficiently separated from LE and as described
previously these compartments do not contain plasma membrane (Gorvel et al., 1991; Papini et al., 1993).
Kinetics of Transport between Early and Late Endosomes in
4N5 and 4N5Ii Cells
HRP has been widely used as a fluid phase
marker for the characterization of endosomal compartments in many cell
types (Griffiths et al., 1989; Gruenberg et al.,
1989; Tooze and Hollinshead, 1991; Gorvel et al., 1991; Bucci et al., 1992). 4N5, 4N5Ii, and 4N5
20Ii cells were
incubated for 5 min at 37 °C in the presence of HRP and chased for
45 min at 37 °C. They were then homogenized, and the resulting
post-nuclear supernatants were submitted to a flotation step gradient
centrifugation. HRP distribution was analyzed in the total cell
homogenates and in the membrane fractions obtained after the flotation
gradient. Table 1shows the quantification of HRP enzymatic
activities in the homogenates of the three cell lines. At 4 °C,
very little of HRP is adsorbed at the cell surface compared to the
amount of HRP detected in the cells after 5 min at 37 °C. This
shows that, as in many cell lines, HRP can be used as a fluid phase
marker in 4N5, 4N5Ii, and 4N5
20Ii cell lines. Interestingly, in
the three cell lines, HRP specific activities measured after 5-min
internalization were similar. This suggests that total HRP uptake is
not affected by the expression of Ii or
20Ii. However, different
specific activities were observed after 5-min internalization compared
with 5 min internalization followed by 45-min chase at 37 °C (Table 1). This can be explained by a loss of internal HRP which
has been recycled back to external medium. This percentage of recycled
HRP was found to be about 35% of the total HRP measured after 5-min
internalization in the three cell lines. This indicates that Ii does
not affect the recycling pathway from EE to the plasma membrane. Then,
we measured the HRP enzymatic activity along the flotation gradient in
the three cell lines (Fig. 3, A-C). In both 4N5
and 4N5
20Ii cells, after 5-min internalization, 18% of the total
HRP enzymatic activity in the PNS was present in EE. After 5-min
internalization followed by 45-min chase, total HRP in EE was shifted
to LE (Fig. 3, A and C). Strikingly, in 4N5Ii
cells, the majority of HRP remained in EE after 45 min chase (Fig. 3B). The HRP latency in EE and LE was >70%. In
contrast, the latency of fraction 7 was <5%, meaning that fraction 7
contained free HRP.
Kinetics of Transport between EE and LE Were
Examined
4N5, 4N5Ii, and 4N5
20Ii cells were incubated for 5
min at 37 °C in the presence of HRP and chased for various periods
of time at 37 °C. They were then homogenized and the resulting
post-nuclear supernatants fractionated by flotation step gradients. HRP
enzymatic activity was measured both in EE and LE. Both in 4N5 and
4N5
20Ii cells, half of HRP was transferred from EE to LE in 25 min (Fig. 4, A and C). In contrast, in 4N5Ii
cells, HRP transport was dramatically delayed taking 120 min for the
same transfer (Fig. 4B). This result shows that
overexpression of full-length Ii in 4N5Ii induces a delay in the
transfer of HRP from EE to LE.
Figure 4:
Kinetics of HRP transport between EE and
LE. Cells were incubated for 5 min at 37 °C with 1.5 mg/ml of HRP.
After extensive washing, HRP was chased at 37 °C from 15-120
min. Cells were homogenized and after flotation step gradient, EE were
recovered at the 35%/25% sucrose interface and LE in the upper region
of the 25% sucrose cushion. A, 4N5; B, 4N5Ii; C, 4N5
20Ii.
Kinetics of Ii Degradation in Early and Late Endosomes in
4N5Ii Cells
In order to investigate the degradation of Ii and
transport between EE and LE in 4N5Ii cells, pulse-chase experiments
followed by immunoprecipitation with an anti-Ii cytoplasmic tail
antibody (VicY1) were performed. A 23-kDa (p23) degradation product of
Ii was detected in EE at 2-h chase (Fig. 5). At 4-h chase,
smaller Ii degradation products (p20, p16, p14, and p13) were detected.
These degradative forms were not seen in EE after 8-h chase onwards. In
contrast, similar products were found in LE after 8-h chase, indicating
a transfer from EE to this compartment. Although Ii and class II form a
complex (see below), class II molecules were not clearly visible in
these immunoprecipitations. This may be partly due to the relative high
expression of Ii and the low content of methionine in HLA-DR as
compared with Ii. From this, we may conclude that the degradation of Ii
in these cells starts in EE and continues in LE and that the transport
of degradative products of Ii from EE to LE is a slow process. We also
immunoprecipitated class II molecules by using anti-HLA-DR antibodies
to know whether or not the Ii degradation products were associated with
HLA-DR molecules. Only p23 was found to be associated with class II
molecules at 2-h chase in EE (Fig. 5). Class II molecules
started to be detected in LE from 2-8-h chase and were devoid of
Ii degradation products.
Figure 5:
Kinetics of Ii degradation and transport
between EE and LE in 4N5Ii cells. Cells were pulse-labeled for 20 min
at 37 °C with 0.2 mCi of a
[
S]methionine/cysteine mix and incubated for 0-,
2-, 4-, 8-, and 16-h chase time. LE and EE membrane fractions were
prepared. Immunoprecipitations were performed using VicY1 and a mix of
anti-HLA-DR antibodies. Immunoprecipitates were analyzed on a 10%
SDS-polyacrylamide gel followed by
fluorography.
DISCUSSION
In order to bind fragments of endocytosed antigens, MHC class
II molecules must be sorted to endosomes, a process which may depend on
Ii and its sorting signal located in the cytoplasmic tail (Bakke and
Dobberstein, 1990; Lotteau et al., 1990; Simonsen et
al., 1993). Using subcellular fractionation, we show that, after
supertransfection of high levels of Ii, early and late endosomes have
typical properties such as being positive for the markers Rab5 (EE) and
Rab7 (LE), respectively (Chavrier et al., 1990; Gorvel et
al., 1991) and possess the same tubular and vesicular morphology
of endosomes previously observed in other cell lines (Griffiths et
al., 1989; Gorvel et al., 1991; Tooze and Hollinshead,
1991). The presence of Ii in the cells induces a dramatic delay in
transport between early and late endosomes involving both transfer of
the fluid phase marker HRP and Ii itself. These results suggest that,
in 4N5Ii cells, the role of Ii could be the retention of class II
molecules and endocytosed antigens in early endosomes. A delayed
transport to lysosomes has been reported after transient expression of
Ii in COS cells (Romagnoli et al., 1993), an effect which was
linked to the expression of large endosomal structures (macrosomes). In
4N5Ii cells only 2% of the cells contain such structures. Consequently,
this cannot explain the general transport delay induced by Ii.
On
leaving the Golgi apparatus, Ii has to be targeted to endosomes.
Indeed, it has been shown that the endosomal sorting signal for newly
synthesized 
Ii complexes resides in the Ii cytoplasmic tail
(Bakke and Dobberstein, 1990; Lotteau et al., 1990; Roche et al., 1992; Simonsen et al., 1993), and it is known
that
20Ii deletion prevents Ii to be targeted to endosomes (Bakke
and Dobberstein, 1990). By comparing results obtained from 4N5Ii and
4N5
20Ii cells, we demonstrate that Ii without an endosomal sorting
signal cannot induce an efficient endosomal retention mechanism between
EE and LE. Once in the endocytic pathway, we show that Ii plays an
important role for the retention of endocytosed material. However, it
is still unclear by which mechanism(s) endosomal retention is achieved.
Recently, it has been shown that the retention of several membrane
proteins in the Golgi apparatus might involve multimerization or
aggregation mechanisms (Chanat and Huttner, 1991; Pelham and Munro,
1993). For instance, it is known that multimerization has been
described for Ii chain forming trimers and the formation of a nonameric
structure together with three 
dimers (Roche et al.,
1991; Lamb and Cresswell, 1992). Alternatively, Ii might interact with
a molecular machinery involved in vesicular membrane traffic (Mellins et al., 1994). This Ii retention might possibly implicate
either aggregation and/or interaction with membrane components.
Recently, Zachgo et al.(1992) showed that leupeptin, a
potential Ii proteolysis inhibitor, reduces the transport of
endocytosed material from multivesicular body-like, endosomal carrier
vesicles to late endosomes. A speculative explanation would be that
high levels of Ii expression might act as a competitor with factor(s)
the proteolysis of which is required for membrane transport between EE
and LE. These results can be correlated with observations suggesting
that protease inhibitors and lysosomotropic reagents prevent deposition
of newly synthesized class II molecules to the cell surface (Neefjes
and Ploegh, 1992; Loss and Sant, 1993). The present report describes a
transport delay between EE and LE induced by a full-length Ii the
cytoplasmic tail of which is required for its sorting to endosomes.
Further investigations are required to identify the Ii retention signal
and to elucidate the underlying molecular mechanisms.