(Received for publication, February 26, 1997, and in revised form, April 24, 1997)
From The R. W. Johnson Pharmaceutical Research Institute, San Diego, California 92121
Major histocompatibility complex class II molecules are heterodimeric cell surface molecules which acquire antigenic peptides in the endosomal/lysosomal system. Invariant chain (Ii), a third chain which is associated with class II molecules intracellularly mediates the endosomal targeting, but it is debated whether class II molecules reach the endosomal system mainly from the trans-Golgi network or via the cell surface.
Dynamin is a cytosolic GTPase which is necessary for the formation of clathrin-coated vesicles from the plasma membrane, but which is not required for vesicle formation from the trans-Golgi network. Here we have used HeLa cells expressing a dominant negative form of dynamin to show that inhibition of clathrin-mediated uptake from the plasma membrane leads to accumulation of transfected Ii-class II complexes at the cell surface, while delivery of such complexes to endosomes/lysosomes is decreased. Our data therefore suggest that in this experimental system the majority of Ii-class II complexes traverse the cell surface before they reach the endosomal system.
Major histocompatibility complex (MHC)1 class II molecules are heterodimeric cell surface proteins which present antigenic peptides to CD4+ T cells. After synthesis in the endoplasmic reticulum, class II molecules associate with a third chain, the invariant chain (Ii) (1). Ii facilitates exit of class II molecules from the endoplasmic reticulum and mediates targeting of the Ii-class II complexes to the endosomal system where Ii is degraded and class II molecules acquire peptides (2). Complete removal of Ii and efficient peptide loading of class II molecules require the function of HLA-DM (3, 4), a class II-like molecule which is mainly, but not exclusively, located in lysosome-like MIIC compartments (MHC class II compartments) (5-8). This structure has been proposed to be the location where class II molecules acquire peptides, but the actual evidence that peptide loading occurs preferentially in the MIIC is circumstantial and other endosomal compartments containing early and late endosomal markers have also been suggested to be compartments where class II-peptide association may occur (9-11).
Membrane proteins can be sorted for delivery to the endosomal/lysosomal system in two cellular locations; from the trans-Golgi network (TGN) and from the plasma membrane (12, 13). In both cases clathrin-coated vesicles are part of the sorting machineries, but the associated adaptor complexes are distinct in the two locations (14, 15). Thus AP1 is necessary for sorting and clathrin-coated pit assembly in the TGN, while AP2 has the same function at the plasma membrane. Although adaptor proteins from both complexes have been reported to bind lysosomal targeting signals, the sorting specificities of the two adaptor complexes are likely to be different (16, 17).
The N-terminal cytoplasmic tail of Ii contains two extensively characterized dileucine-based endosomal targeting motifs (18, 19). These motifs can clearly mediate internalization from the plasma membrane, but whether Ii sorting is occurring mainly in the TGN or at the cell surface under normal conditions is unclear. Several studies have suggested that the majority of Ii-class II complexes are transported directly from the TGN to endocytic compartments and in particular to the MIIC (5, 20, 21), while other reports have come to the conclusion that a large part of Ii-class II complexes reach the endosomal system via the cell surface (11, 22). The small number of Ii-class II complexes present at the cell surface at steady state has made it difficult to determine the relative importance of these pathways.
In this study we have used HeLa cells stably transfected with a dominant negative form of dynamin, K44A, under an inducible promoter (23) to study the intracellular transport pathways of Ii and Ii-class II complexes. Dynamin is a cytosolic GTPase which is required for the formation of constricted clathrin-coated pits at the plasma membrane (24). Cells which express the K44A form of dynamin accumulate elongated clathrin-coated pits, but are unable to form clathrin-coated vesicles and are thus incapable of clathrin-mediated endocytosis. Since dynamin is not required either for transport in the secretory pathway or for the clathrin-mediated vesicular transport from the TGN to the endosomal system (23), the use of dynamin-mutant cells provides an opportunity to address the question where endosomal sorting of Ii occurs. Although HeLa cells are not antigen presenting cells and do not normally express MHC class II molecules or Ii, transfected class II molecules are sorted to endosomal/lysosomal compartments in cells expressing class II and Ii (7, 25).
In the presence of the mutant form of dynamin we found that Ii, as well as Ii-class II complexes, accumulated at the cell surface, while only a minor amount of Ii appeared to reach the endosomal system. Our data suggest that in HeLa cells the sorting of Ii-class II complexes for delivery to the endosomal system occurs mainly at the cell surface, rather than in the TGN.
The stable tTA-HeLa cell line with
tetracycline-regulated expression of dynamin mutant K44A was provided
by Sandra Schmid (23). The expression plasmids for HLA DRA, DRB, human
Iip31, Iip41, and Iip31(AA-AA) have been described (19, 26).
Iip41(AA-AA) was generated by replacing a
BsrGI-MunI fragment in the Iip31(AA-AA) plasmid
with the corresponding fragment from a Iip41 plasmid. Human CD8 and
mouse CD40 were cloned in the expression vector pCMU. Monoclonal
antibody (mAb) Bü45 was purchased from Research Diagnostics
(Flanders, NJ). The hybridoma lines DA6.147 (27) and OKT8 (28) have
been described. Rabbit antiserum K456 was raised against purified mouse
CD40Fc fusion protein. Fluorescein isothiocyanate- or Texas Red-labeled
secondary antibodies were purchased from Molecular Probes (Eugene,
OR).
K44A-transfected tTA-HeLa cells were maintained in DMEM with 10% fetal bovine serum, 400 µg/ml G418, 200 µg/ml puromycin, and 2 µg/ml tetracycline (tet). Expression of the dynamin mutant, K44A, was induced by removing tetracycline from the medium. Cells were split 6 h prior to transfection, then changed to either tetracycline-containing DMEM or DMEM without tetracycline 2 h prior to transfection. 25 µg of plasmids in various combinations were transfected using calcium phosphate precipitation (29). Cells were analyzed 72 h after transfection.
Flow CytometryTransfected cells were detached from the dishes with 2 mM EDTA in PBS. Cells were stained with mAbs in PBS containing 1% bovine serum albumin at 4 °C. Fluorescein-conjugated goat anti-mouse IgG was used as secondary reagent. Cells were analyzed on a Becton Dickinson LYSIS II instrument.
Immunofluorescence MicroscopyCells on glass coverslips were fixed with 4% formaldehyde in PBS. Antibody incubations were done in PBS with 0.2% saponin and 0.6% fish skin gelatin for permeabilized staining or with 0.6% gelatin only for non-permeabilized staining. Fluorescence microscopy was performed using a Zeiss axiophot microscope.
Invariant Chain InternalizationCells on coverslips were incubated with mAb Bü45 diluted in medium for 30 min on ice, then washed before being returned to DMEM with or without tetracycline. Cells were incubated at 37 °C for 1 h, then fixed and stained as described above with fluorescein-conjugated goat anti-mouse IgG. Cells were also co-stained with K456 followed by Texas Red-labeled goat anti-rabbit IgG.
Metabolic Labeling and ImmunoprecipitationCells were incubated in methionine-free DMEM for 15 min prior to labeling, then labeled for 30 min with 0.2 mCi of [35S]methionine (DuPont NEN) in methionine-free DMEM. Cells were lysed immediately or incubated in methionine-containing medium with or without tetracycline for various times at 37 °C. When leupeptin (Calbiochem, San Diego, CA) was used, 250 µg/ml was included in the chase medium. Cells were lysed in 1% Nonidet P-40 in PBS with protease inhibitors (Complete Protease Inhibitor Mixture Tablets, Boehringer Mannheim). Lysates were precleared with protein A-Sepharose (Pharmacia) then incubated with mAb DA6.147 at 4 °C for 2 h before addition of protein A-Sepharose. The beads were washed 5 times with 0.1% Nonidet P-40. The precipitates were boiled in SDS sample buffer before being applied to 10-15% SDS-polyacrylamide gels. Gels were fixed and dried before autoradiography or PhosphorImager Scanning (Molecular Dynamics, Inc., Sunnyvale, CA). Autoradiographs were scanned using an Agfa ArcusII scanner and composites were printed on a Kodak XLS 8600 PS printer.
Surface Biotinylation and ImmunoprecipitationCells were washed and incubated with sulfo-NHS-biotin (0.5 mg/ml) (Pierce, Rockford, IL) in PBS at 4 °C for 30 min. Cells were washed with serum-free DMEM and ice-cold PBS then lysed in 1% Nonidet P-40 with protease inhibitors. Ii-class II complexes were immunoprecipitated as above. After SDS-PAGE, the proteins were transferred to BA-S 85 filters (Schleicher and Schuell) by electroblotting. Blocking was done with 5% dry milk in TBST (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 0.05% Tween 20) followed by incubation with alkaline phosphatase-conjugated streptavidin. After washing, the filter was developed with 5-bromo-4-chloro-3-indolyl phosphate (0.33 mg/ml) and nitro blue tetrazolium (0.16 mg/ml) (Promega) in 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl2.
To determine whether invariant chain localization was
dependent on dynamin function we transiently transfected wild-type or targeting-deficient forms of invariant chain (Iip31 or Iip31AA-AA) into
HeLa cells stably transfected with the K44A dynamin mutant under a
tetracycline-regulated promoter. Overexpression of proteins with
endosomal or lysosomal targeting motifs has been reported to result in
mis-sorting and accumulation of transfected and endogenous lysosomal
proteins at the plasma membrane (30, 31). To avoid this problem a
moderately strong -globin promoter was used to control the
expression of Ii. The cells were grown in the presence of tetracycline
(to suppress K44A expression) or in the absence of tetracycline (to
induce K44A expression). 72 h after transfection the cell surface
expression of invariant chain was analyzed by flow cytometry after
staining with mAb Bü45. Fig. 1A shows
that in the presence of tetracycline (+tet) only a small
amount of Ii could be detected at the cell surface, suggesting that the capacity for Ii sorting was not exceeded. In contrast, the cell surface
expression in the absence of tetracycline (
tet) was
similar to the level of cells transfected with the targeting-deficient form of Ii (Fig. 1B). In the latter case the presence or
absence of tetracycline did not influence the cell surface levels of
Ii. Staining of a cotransfected control molecule, human CD8
, was similar in the different transfections and was not affected by the
expression of the mutant dynamin (not shown). This experiment shows
that if dynamin function is blocked, Ii accumulates at the cell
surface.
We next examined the uptake of Ii from the cell surface in the presence
or absence of mutant dynamin. Cells transfected with Ii and CD40 (as a
cell surface control) were plated on coverslips and these were
incubated on ice with mAb Bü45. After washing, the cells were
allowed to internalize the bound antibodies for 1 h in complete
medium at 37 °C. Cells were then fixed and stained with fluorescein
isothiocyanate-labeled anti-mouse IgG before or after permeabilization.
The cells were also co-stained with a rabbit antiserum directed against
CD40, followed by Texas Red-labeled anti-rabbit IgG. The stained cells
were analyzed by immunofluorescence microscopy. Fig.
2a shows the distinct vesicular staining of
non-induced cells after permeabilization. When the cells were not
permeabilized, Ii staining could not be detected (Fig. 2c),
indicating that Bü45 which had bound to the cell surface had been
internalized. CD40 staining was present at the cell surface whether the
cells had been permeabilized or not (Fig. 2, b and
d). When cells expressing the dynamin mutant were analyzed
for Ii expression, considerable cell surface expression of Ii was
detected. The Ii staining pattern after permeabilization (Fig.
2e) was not significantly different from the pattern seen in
the non-permeabilized cells (Fig. 2g) and in both cases the
staining was largely coincident with the CD40 control staining (Fig. 2,
f and h). The shape of K44A-expressing cells is
different from non-induced cells, probably due to the accumulation of
coated pits which are unable to pinch off to form vesicles (23).
Molecules which are normally internalized are concentrated in these
pits, thus giving a dotty cell surface staining pattern. The data from
these experiments show that Ii internalization from the cell surface
depends on the formation of clathrin-coated vesicles.
Functional Dynamin Is Required for Efficient Endosomal Localization of Invariant Chain
The subcellular distribution of Ii under
steady state conditions was analyzed by indirect immunofluorescence of
cells transfected with Ii and CD40 cDNAs. Staining of permeabilized
non-induced cells with mAb Bü45 showed Ii to be localized
intracellularly in reticular and vesicular structures (Fig.
3A, a). Staining of non-permeabilized cells showed that little if any Ii was located at the
cell surface (Fig. 3A, c). CD40 staining was
detectable at the cell surface in both cases (Fig. 3A,
b and d). When K44A expressing cells were stained
with mAb Bü45, Ii could only be detected at the cell surface
(coincident with the CD40-staining), whether the cells were
permeabilized or not (Fig. 3A, e and
g). No vesicular staining was seen in the permeabilized
cells, suggesting that a large part of Ii was located at the plasma
membrane. Immunofluorescence staining of cells transfected with Iip41
showed that this form of Ii, like Iip31, accumulated at the cell
surface in K44A expressing cells (compare Fig. 3, B, panel
a, with A, panel b).
K44A Expression Blocks DR-Ii Dissociation
Since dynamin only
affects internalization from the cell surface, the accumulation of Ii
in this location suggested that a fraction of the transported invariant
chain was delivered to the cell surface before it was internalized into
endosomes. To determine if the intracellular transport and maturation
of Ii-class II complexes were similarly affected by the expression of
mutant dynamin, we made pulse-chase experiments using non-induced or
induced K44A HeLa cells transiently transfected with Iip41, HLA-DRA,
and HLA-DRB. Delivery to the endosomal system is required for
dissociation of Ii from class II molecules since this process is
dependent on both acidic pH and proteolysis (32-34). Thus, the degree
of Ii dissociation from class II molecules can be used as an indirect measure of the delivery of Ii-class II complexes to the endosomal system. Iip41 was used in these transfections instead of Iip31, since
it is easier to distinguish from the class II and
chains in
SDS-PAGE gels due to its larger size. The two forms of Ii appear to be
equally efficient in promoting class II folding and targeting, although
they may influence Ii and antigen degradation in different ways (35,
36).
Non-induced or induced transfected cells were labeled for 30 min then
washed and either lysed immediately or chased in non-radioactive medium
as indicated. Radiolabeled class II molecules were immunoprecipitated from the lysates using mAb DA6.147, reactive with DR, before analysis by SDS-PAGE. Fig. 4A shows that in
non-induced cells (i.e. +tet) class II molecules
were associated with Ii after the pulse labeling and after the early
chase time points. Both the DR chains and Ii (p41+) acquired
carbohydrate modifications during the chase (as indicated by their
slower migration), indicating that the chains were transported as
expected. The amount of co-precipitated Ii decreased after 2 h of
chase and after 6 h of chase essentially no Ii was detectable in
the precipitate.
When class II molecules were immunoprecipitated from the cells
expressing K44A dynamin we found that the association of DR with Ii, as
well as the acquisition of carbohydrate modifications, were very
similar in the induced and the non-induced cells (Fig. 4A,
tet). In contrast, the dissociation of Ii from the class II
molecules was markedly delayed and after 8 h of chase the amount of co-precipitated Ii which had acquired carbohydrate modifications (Iip41+) was only slightly decreased (Fig. 4, A and
B). The slow dissociation of the DR-Ii complexes was similar
to the slow dissociation of DR molecules from mutated forms of Ii
lacking endosomal targeting information, where the DR-Ii complexes
accumulate at the cell surface (Ref. 34, and data not shown).
Although the majority of Ii was not delivered to endosomes in the presence of the mutant dynamin, it was not clear to what extent Ii-class II complexes did reach the endosomal system, since the protease sensitivity of Ii results in rapid degradation after arrival in these compartments. To estimate the endosomal delivery of Ii-class II complexes in the absence or presence of the mutant dynamin, we took advantage of the fact that in the presence of the protease inhibitor leupeptin invariant chain degradation is partly inhibited (32). The resulting Ii-derived fragments, LIP (leupeptin-induced proteins) migrating at 21-23 kDa and SLIP (small leupeptin-induced proteins) migrating at 12 kDa, remain associated with class II molecules in the endosomal system (37, 38). The relative abundance of the two fragments appear to vary depending on experimental conditions.
To quantify the amount of Ii-class II complexes that did reach the
endosomal system we made pulse-chase experiment as above, but included
leupeptin in the chase medium. Fig. 5 shows that in the
non-induced cells (i.e. +tet), a distinct LIP
fragment was present in the immunoprecipitates from leupeptin-treated
cells after 8 h of chase. No distinct SLIP fragment could be
detected. The immunoprecipitates from the induced cells
(tet) showed, as expected, that most of the Ii was still
intact. However, a small amount of LIP could be detected after 8 h
of chase also in this case. Quantification by PhosphorImager
analysis showed that the LIP fragment constituted 80% of the total
transported invariant chain remaining after 8 h in the non-induced
cells (after compensating for the lower number of methionines in the
LIP fragment; 9 versus 13) while it constituted 18% in the
induced cells. Comparison with the amount of Ii precipitated after
1 h of chase showed that only a minor amount of Ii was unaccounted
for in the leupeptin-treated cells. This experiment shows that some
Ii-class II complexes reached the endosomal system in the presence of
mutant dynamin, although the majority of complexes did not.
Accumulation of DR-Ii Complexes at the Cell Surface
Internalization of Ii was blocked by K44A expression and
it was therefore likely that the slow dissociation of Ii from DR in the
K44A expressing cells was a reflection of the blocked internalization from the plasma membrane, leading to accumulation of DR-Ii complexes at
the cell surface. To verify that this was the case we analyzed the cell
surface expression of cells transfected with DRA, DRB, and either
wild-type Iip41 or Iip41(AA-AA), which lacks endosomal targeting
information. 72 h after transfection, the cell surface proteins of
induced or non-induced cells were labeled with sulfo-NHS-biotin. After
lysis, DR molecules were immunoprecipitated with DA6.147, as above. The
samples were separated on SDS-PAGE gels, then blotted onto
nitrocellulose filters. Immunoprecipitated proteins labeled with biotin
residues (i.e. molecules derived from the cell surface) were
detected using alkaline-phosphatase-conjugated streptavidin in
combination with a colorimetric substrate. Fig. 6 shows
that little Ii was coprecipitated with the cell surface DR molecules from the non-induced cells transfected with wild-type Ii and DR (Fig.
6, lane 1). In contrast, most of the cell surface DR
molecules derived from the K44A-expressing cells were associated with
Ii (Fig. 6, lane 2). Cell surface DR molecules were also
associated with Ii in the cells transfected with Iip41(AA-AA), whether
K44A was expressed (not shown) or not (Fig. 6, lane 3).
Thus, deficient internalization due to expression of the mutant dynamin
results in accumulation of Ii-class II complexes at the plasma
membrane.
It is a well accepted fact that MHC class II molecules acquire peptides in endosomal or lysosomal compartments, although the exact location where peptide loading occurs is debated. Several studies have tried to establish whether Ii-class II complexes reach these loading compartments directly from the TGN or whether they are delivered to the endosomal system via the cell surface. Studies based on immunoelectron microscopy and subcellular fractionation as well as the use of inhibitors of proteolysis or endosomal acidification have concluded that the majority of Ii-class II complexes are delivered directly to the endosomal/lysosomal system MIIC (5, 20, 21, 37). Other reports using internalization experiments and biochemical methods have come to the opposite conclusion, that a large part of Ii-class II complexes reach the endosomal system via the cell surface (11, 22, 39). Cell type-dependent differences both in peptide-loading requirements and in intracellular transport of class II molecules may explain some of the differing results, but it appears likely that the methods used to address this question influence the conclusions reached. The subcellular distribution of proteins at steady state is a reflection of how long they dwell in different compartments as well as the sizes of the compartments. Rapid transit of molecules via the cell surface or through a certain compartment may not allow detection by morphological methods, especially if the compartment is large. Pulse-chase labelings and subcellular fractionation are unlikely to distinguish transiently occupied compartments in the later part of the secretory pathway or in the endosomal system, both due to the poor resolution of subcellular fractionation methods, and due to the fact that even a small cohort of labeled molecules created during a short pulse labeling will have become spread out by the time the molecules reach the later stages of the secretory pathway.
In this study we have addressed the question of how Ii-class II
complexes travel to endosomes by taking advantage of the fact that
formation of clathrin-coated vesicles during endocytosis from the cell
surface require the function of the cytosolic GTPase dynamin, whereas
formation of clathrin-coated vesicles from the TGN does not. HeLa cells
stably transfected with a dominant negative form of dynamin (K44A)
expressed under the control of a tightly regulated inducible promoter
have previously been well characterized (23). Expression of the K44A
dynamin blocks receptor-mediated endocytosis from the cell surface,
apparently without disturbing the vesicular transport from the TGN to
endosomes. Delivery of the lysosomal protease cathepsin D is normal in
these cells and the distributions of cation-independent mannose
6-phosphate receptor and -adaptin (which is part of the TGN-specific
AP1 complex) have normal distributions (Ref. 23 and not shown).
Using transiently transfected K44A-expressing HeLa cells we found that
Ii internalization from the cell surface was dependent on the function
of normal dynamin and that most Ii molecules as well as Ii-class II
complexes were delivered to the cell surface instead of the endosomal
system. Pulse-chase experiments with K44A-expressing cells transfected
with DR and Ii showed that the rate of invariant chain dissociation
from DR molecules was very slow, with 80% of the transported Ii
chains still associated with DR molecules after 8 h of chase. Ii
is very sensitive to proteolytic degradation in the endosomal system
and the slow rate of Ii dissociation suggested that the majority of
Ii-class II complexes did not reach this system within 8 h of
chase. Pulse-chase experiments with leupeptin-treated cells confirmed
that under these conditions <20% of Ii-class II complexes did enter
endosomal compartments where Ii degradation could occur. Analysis of
cell surface molecules in the same transfectants showed that most DR molecules at the plasma membrane were indeed associated with Ii. The
fraction of Ii-class II complexes that did reach the endosomal system
may represent material directly transported from the TGN to endosomes.
Alternatively, endocytosis of Ii-class II complexes from the cell
surface was not totally blocked, either because the mutant dynamin may
not completely block the formation of clathrin-coated vesicles or
because some Ii-class II complexes may be internalized in
non-clathrin-coated vesicles. Clathrin-independent pinocytosis of fluid
phase markers is increased in cells where dynamin function is inhibited
(40).
Although the intracellular transport of transfected Ii-class II complexes, as well as Ii dissociation and final subcellular distribution of class II molecules, is similar in HeLa cells to what has been reported for both B lymphoblastoid cell lines and different types of transfected cell lines (1, 34, 41), HeLa cells are not normally antigen presenting cells and we cannot conclude that a majority of Ii-class II complexes reach endosomal and lysosomal compartments via the cell surface also in professional antigen presenting cells. It is apparent, however, that difficulty to detect a substantial amount of Ii-class II complexes at the plasma membrane does not allow the conclusion that a majority of these complexes reach the endosomal system via a direct pathway from the TGN.
The route of transport to the endosomal system may matter in the case of class II molecules since both lysosomal compartments and lighter compartments containing early endosome markers have been indicated in peptide loading (8, 9, 42, 43). Internalization of Ii-class II complexes together with antigenic proteins from the cell surface may allow the most extensive contact between class II molecules and internalized antigens, thus increasing the chances that class II molecules will be able to bind and present some epitope from a given protein.
In conclusion, we have used a new experimental approach to analyze the intracellular transport of Ii-class II complexes. We find that in transfected HeLa cells a majority of Ii-class II complexes, as well as of free Ii reach the endosomal system via the plasma membrane. This approach shows that the ability to specifically control different steps in membrane trafficking using other dominant negative molecules (for example, Rabs) is potentially useful for elucidating where class II molecules acquire peptides and how peptide-loaded class II molecules return to the plasma membrane.
We thank H. Damke and S. Schmid for providing the K44A transfected cells, L. Pond for the Iip31(AA-AA) construct, D. Uranowski for secretarial assistance, G. Klier for help with confocal microscopy, R. Castaño, M. Jackson, T. Kuwana, M. Liljedahl, R. Teasdale, and O. Winqvist for discussions and suggestions.