©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Invariant Chain Induces a Delayed Transport from Early to Late Endosomes (*)

(Received for publication, September 12, 1994 )

Jean-Pierre Gorvel (1)(§) Jean-Michel Escola (1)(¶) Espen Stang (2)(**) Oddmund Bakke (2)(§§)

From the  (1)Centre d'Immunologie INSERM-CNRS de Marseille Luminy, Case 906, 13288 Marseille Cedex 9, France and the (2)Division of Molecular Cell Biology, Department of Biology, University of Oslo, N 0316 Oslo, Norway

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Invariant chain associated with class II molecules is proteolytically processed in several distinct intermediates during its transport through the endocytic pathway. Using subcellular fractionation, early and late endosomal compartments were separated in human fibroblasts transfected with HLA-DR (4N5 cells) and supertransfected with invariant chain (4N5Ii cells) or invariant chain lacking most of the cytoplasmic tail (4N5Delta20Ii cells). Early and late endosome membrane fractions were characterized by morphology and by analyzing the presence of the Rab5 and Rab7 GTPases as markers of early and late endosomes, respectively. The transfer of endocytosed horseradish peroxidase from early to late endosomes proceeded relatively rapid both in 4N5 and 4N5Delta20Ii cells (t = 25 min), whereas this transfer was significantly delayed (t = 2 h) in 4N5Ii cells. Pulse-chase experiments showed that invariant chain and its degradation products were first observed in early endosomes and thereafter in late endosomes. Our results strongly suggest that invariant chain induces a retention mechanism in the endocytic pathway.


INTRODUCTION

The major histocompatibility complex (MHC) (^1)class II molecules are heterodimers (alpha and beta 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(2)-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 alphabetaIi complexes is believed to occur by the sequential addition of alphabeta 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, alphabetaIi complexes traverse the Golgi apparatus before reaching the endosomes. It has been shown that the endosomal sorting signal for newly synthesized alphabetaIi 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 alphabetaIi 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 (Delta20Ii) 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 Delta20Ii expressed large amounts of surface Ii (4N5Delta20Ii 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-alpha and -beta 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 times g at 4 °C in an Airfuge (Beckman), post-fixation of membrane pellets was performed using 2% OsO(4) and 1.5% potassium ferrocyanide in H(2)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 [alpha-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 times 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), 3 mM Na(2)CO(3), pH 9.8). Then, filters were rinsed twice for 10 min in 50 mM NaH(2)PO(4), pH 7.5, 10 µM MgCl(2), 2 mM dithiothreitol, 4 µM ATP, and 0.3% Tween 20 (binding buffer) and incubated with 1-2 µCi/ml [alpha-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(2) and 0.1 mM CaCl(2) (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(4)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 times 75-cm^2 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 4N5Delta20Ii 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 4N5Delta20Ii 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 4N5Delta20Ii 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 4N5Delta20Ii (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 4N5Delta20Ii 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 4N5Delta20Ii 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 Delta20Ii. 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 4N5Delta20Ii 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 4N5Delta20Ii 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 4N5Delta20Ii 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, 4N5Delta20Ii.



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 alphabetaIi 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 Delta20Ii deletion prevents Ii to be targeted to endosomes (Bakke and Dobberstein, 1990). By comparing results obtained from 4N5Ii and 4N5Delta20Ii 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 alphabeta 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.


FOOTNOTES

*
This work was financed by grants from the Norwegian Research Council, the Norwegian Cancer Society, the Association de la Recherche contre le Cancer (ARC), and the Ligue Française Nationale contre le Cancer (LFNCC). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a fellowship from the Norwegian Research Council.

Supported by a fellowship from the Ministère de la Recherche et de la Technologie.

**
Supported by a fellowship from the Norwegian Cancer Society.

§§
To whom correspondence should be addressed. Tel.: 47-22855787; Fax: 47-22854605; obakke{at}bio.uio.no.

(^1)
The abbreviations used are: MHC, major histocompatibility complex; FCS, fetal calf serum; HRP, horseradish peroxidase; PNS, post-nuclear supernatants; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline.


ACKNOWLEDGEMENTS

We thank Hege Hardersen for expert technical assistance and A. Le Bivic and T. Nordeng for helpful discussions.


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