©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Mannose 6-Phosphate Receptors and ADP-ribosylation Factors Cooperate for High Affinity Interaction of the AP-1 Golgi Assembly Proteins with Membranes (*)

(Received for publication, August 25, 1995; and in revised form, November 14, 1995)

Roland Le Borgne Gareth Griffiths Bernard Hoflack (§)

From the European Molecular Biology Laboratory, Postfach 10-2209, Meyerhofstrasse 1, 69012 Heidelberg, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Clathrin coat assembly in the trans-Golgi network, leading to the sequestration of the mannose 6-phosphate receptors (MPRs) into nascent vesicles, requires the ARF-1-dependent translocation of the cytosolic AP-1 Golgi assembly proteins onto the membranes of this organelle. The mechanistic role of the MPRs, i.e. the cargo molecules, in coat assembly is at present unclear. Using a GTP-dependent, brefeldin A-sensitive in vitro AP-1 binding assay, we have determined here the parameters of the AP-1 binding reaction. We demonstrate that, in addition of ARF-1, the MPRs contribute to create high affinity AP-1 binding sites (K approx 25 nM), since their number correlates the number of MPR molecules expressed in MPR-negative cells. The quantitative electron microscopy shows that these high affinity binding sites are present on trans-Golgi network membranes, as expected, and to some extent on early endosomes. The high affinity binding sites are lost when the MPRs or ARF-1 become rate-limiting components. Conversely, GTPS (guanosine 5`-O-(3-thiotriphosphate)), which increases the amount of membrane-bound ARF-1, mostly uncovers low affinity AP-1 binding sites (K approx 150 nM) on trans-Golgi network membranes, normally not detected in its absence. Collectively, these results argue that MPR sorting is highly coupled to the first step of coat assembly and that the MPRs, ARF-1, and possibly other proteins cooperate for high affinity interactions of AP-1.


INTRODUCTION

In eukaryotic cells, vesicular transport from the trans-Golgi network (TGN) (^1)and from the plasma membrane to endosomes involves the formation of clathrin-coated vesicles as transport intermediates (Kornfeld and Mellman, 1989; Pearse and Robinson, 1990). Although the plasma membrane- and the Golgi-derived vesicles share clathrin as one of the major coat components, their coat can be distinguished by the nature of the underlying assembly proteins (APs). Immunofluorescence localization studies indicate that AP-1 is restricted to Golgi-derived vesicles, whereas AP-2 is associated with plasma membrane-derived vesicles (Robinson, 1987; Ahle et al., 1988). Although it is still not understood how the APs specifically interact with their target membranes (Robinson, 1992, 1994), these coat proteins are now well characterized biochemically (Morris et al. 1989; Pearse and Robinson, 1990), and the cDNAs encoding their different subunits have been isolated (Robinson, 1989; Kirchhausen, 1989; Robinson, 1990; Ponnanbalam, 1990). AP-1 is a heterotetrameric complex composed of two approx100-kDa subunits (the and beta` adaptins) associated with a 47-kDa and a 19-kDa polypeptide. AP-2 has a similar protein composition also made of two large subunits of approx100 kDa (the alpha and beta adaptins) associated with a 50-kDa and a 17-kDa polypeptide.

Due to their topological position, APs are likely to play a major role in coat assembly by interacting with both clathrin triskelions and the appropriate membrane. Their heterotetrameric structure is probably a reflection of these multiple functions. In vitro reconstitution studies indicate that purified APs can bind to reconstituted clathrin cages (Pearse and Robinson, 1984). It has been proposed that the beta and beta` adaptins, two closely related polypeptides (Ahle et al., 1988), mediate this interaction, because these purified adaptins (Ahle et al., 1988; Ahle and Ungewickell, 1989) or the recombinant beta and beta` adaptins (Gallusser and Kirchhausen, 1993) also bind to reconstituted clathrin cages via their NH(2)-terminal, trunk domains. In vitro assays also indicate that, besides interacting with clathrin, APs can bind to cytoplasmic domains of receptors, thereby mediating their clustering into plasma membrane- or Golgi-derived clathrin-coated vesicles (Pearse and Robinson, 1990). Accordingly, purified AP-2 binds to immobilized cytoplasmic domains of receptors recycling via the plasma membrane, including the low density lipoprotein receptor, the mannose 6-phosphate/IGF II receptor (Pearse, 1988), the asialoglycoprotein receptor (Beltzer and Spiess, 1991) and the lysosomal acid phosphatase (Sosa et al., 1993). In all cases, this binding is dependent on specific endocytosis motifs in the cytoplasmic tail of these proteins. In contrast, purified AP-1 has, until now, only been found to bind to the immobilized cytoplasmic tail of mannose 6-phosphate/IGF II receptor (Glickman et al., 1989) which sorts lysosomal enzymes in the TGN (Kornfeld, 1992). The alpha and adaptins, the two most divergent polypeptides of APs (Robinson, 1990), are suspected to play a role in these interactions (Pearse and Robinson, 1990).

Recent studies have shown that the small GTPase ADP-ribosylation factor (ARF) not only regulates coatomer assembly in the early secretory pathway (Donaldson et al., 1992a; Palmer et al., 1993), but also acts as a potent regulator of clathrin coat assembly in the TGN. The evidence comes from in vivo studies showing that the fungal metabolite brefeldin A inhibits binding of AP-1 on Golgi membranes (Robinson and Kreis, 1992; Wong and Brodsky, 1992) as was previously observed for beta-COP (Orci et al., 1991), a subunit of the coatomer required for vesicle budding in the early secretory pathway (Rothman and Orci, 1992). It is now known that this drug prevents the exchange of GDP for GTP on the small GTPase ARF which is required for its membrane insertion via its myristoylated moiety (Donaldson et al., 1992b: Helms and Rothman, 1992). Several members of the ARF family have now been identified in mammalian cells (Tsuchiya et al., 1991; Kahn et al., 1991), but their function remains elusive. It has been shown that binding of AP-1 to membranes present in Golgi-enriched fractions is stimulated upon addition of recombinant, myristoylated ARF-1 (Stamnes and Rothman, 1993; Traub et al., 1993). ARF-1 has been involved in several transport reactions occurring along the secretory (Balch et al., 1992; Taylor et al., 1992) and the endocytic pathways (Lenhard et al., 1992) leading to the proposal that ARF-1 may act as a common molecular switch for coat assembly. Recent data suggest that ARF-6 regulates the process of endocytosis of transferrin receptor (D'Souza-Schorey et al., 1995). ARF-6 localizes to the plasma membrane and endosomes and overexpression of GTP hydrolysis mutants results in extensive plasma membrane invaginations as well as a depletion of endosomes (Peters et al., 1995).

It is currently believed that cargo proteins are segregated into transport vesicles when the first steps of coat assembly have occurred. According to this view, the mannose 6-phosphate receptors (MPRs), two trans-membrane proteins sorted in the TGN (Kornfeld, 1992; von Figura, 1991) are clustered into nascent vesicles when AP-1 has already been recruited onto TGN membranes (Pearse and Robinson, 1990; Robinson, 1994; Traub et al., 1995). Our ealier studies have suggested that the MPRs may play a role in the recruitment of AP-1, because AP-1 binding is drastically reduced in MPR-deficient cells and the addition of the soluble cytoplasmic domain of the mannose 6-phosphate/IGF II receptor inhibits AP-1 binding almost completely (Le Borgne et al., 1993). In order to get more insight on the role of the MPRs in AP-1 recruitment, we have determined the parameters of the AP-1 binding reaction under conditions in which the concentration of the MPRs or ARF would vary in membranes. We report that AP-1 binds with high affinity to membranes in a MPR-dependent manner when ARF-1 is also present, strongly suggesting that protein sorting in the TGN is highly coupled to the first step of clathrin coat assembly. These results suggest that the first step of clathrin coat assembly on TGN membranes, namely the recruitment of AP-1, requires some cooperation between MPRs, i.e. cargo proteins and ARF-1.


EXPERIMENTAL PROCEDURES

Materials

All reagents were of analytical grade. Brefeldin A (stored as a 5 mM stock solution in methanol), ATP were from Sigma. GTPS was from Boehringer Mannheim. [alpha-P]GTP (3,000 Ci/mmol), ECL reagents were from Amersham Corp. Streptolysin O was from Welcome Laboratory (Murex diagnostica). Superose 6, DEAE-cellulose were from Pharmacia Biotech Inc.

Cells

NRK cells and SA-48 HeLa cells were grown in complete alpha-minimal essential medium containing 5% fetal calf serum. Primary cultures of mannose 6-phosphate receptor-deficient embryonic fibroblasts were obtained as described in Ludwig et al. (1994) after crossing cation-dependent mannose 6-phosphate receptor-negative mice (Ludwig et al., 1993) and T mice. Fibroblasts lacking both the cation-dependent and -independent mannose 6-phosphate/IGF II receptor were selected for further studies and grown in complete alpha-minimal essential medium containing 5% fetal calf serum. Immortal clones of these cells were obtained after expression of the large and middle T antigen of the SV40 and will be described elsewhere (Mauxion et al., 1996). Stable expression of the mouse cation-dependent mannose 6-phosphate receptor and of the bovine cation-independent mannose 6-phosphate/IGF II receptor in MPR-negative embryonic fibroblasts were obtained after transfection with the corresponding cDNAs inserted into the pSVneo vector using calcium phosphate precipitation.

Preparation and Fractionation of Cytosol

Bovine brains obtained from the local slaughterhouse were processed within 2 h of slaughter. Gray matter is crudely dissected to remove brain stem and the larger portion of white matter. Tissue from five brains were homogenized in 2.5 volumes of KOAc buffer, pH 7.0 (115 mM KOAc, 25 mM Hepes, 2.5 mM MgCl(2)) in a Waring blender (3 burst of 20 s). The homogenate was centrifuged at 20,000 times g for 50 min. The supernatant was then spun at 100,000 times g for 1 h. The resulting supernatant was spun again at 100,000 times g for 1 h to remove the remaining membranes. The resulting cytosol (20 mg/ml) was then aliquoted and stored at -80 °C.

The bovine brain cytosol (16 mg of protein) was separated on a preparative Superose 6 column (1.6 times 50 cm) equilibrated in KOAc buffer, pH 7. The column was eluted at 0.4 ml/min with the same buffer, and 2-ml fractions were collected. AP-1 present in the different fractions were detected by Western blotting using the mAb 100/3 anti--adaptin monoclonal antibody. The elution profiles of ARF proteins was determined by GTP overlay and Western blotting after SDS-gel electrophoresis using the 1D9 anti-ARF monoclonal antibody. AP-1 containing fractions were pooled, dialyzed overnight against 50 mM Tris/HCl, pH 7.5, 0.1 M NaCl, 1 mM phenylmethylsulfonyl fluoride, and applied onto a 1-ml DEAE-cellulose column equilibrated in the same buffer. AP-1 was eluted with 0.2 M Tris-HCl, pH 7.5, 0.1 M NaCl, 1 mM phenylmethylsulfonyl fluoride and dialyzed overnight against KOAc buffer, pH 7. AP-1 and ARF containing fractions were concentrated in order to reach the same concentration as that found in cytosol.

Quantitation of AP-1 in Cytosol

Clathrin-coated vesicles were prepared from fresh bovine brains and AP-1 and AP-2 were purified as described by Méresse et al.(1990). Briefly, coat proteins were extracted with 1 M Tris, pH 7.5, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride and separated from the vesicles by high speed centrifugation. The coat proteins were fractionated by gel filtration, DEAE-cellulose, and hydroxylapatite column. Several independent preparations of APs were used for the quantitations. If AP-2 is pure on SDS-gel stained with Coomassie Blue, AP-1 contains few contaminants. The concentration of AP-1 in these preparations was estimated after densitometric analysis of SDS-gels stained with Coomassie Blue. The cytosolic concentration of AP-1 was determined by quantitative Western blotting using different amounts of cytosolic proteins and a standard curve generated with purified AP-1. The quantitative Western blotting of the cytosolic AP-1 was performed with the anti--adaptin mAb 100/3 monoclonal antibody and the anti-betabeta` mAb 100/1 monoclonal antibody (Sigma ImmunoChemicals). Both antibodies gave similar values. The concentration of the cytosolic AP-1 was estimated to be 200 nM.

We also determined the cytosolic concentration of AP-2. For the quantitative Western blotting, several antibodies against AP-2 were used: the mAb AP-6 (a generous gift from Dr. F. Brodsky) against the alpha-subunit and the mAb 100/2 (Sigma ImmunoChemicals) against the beta-subunit and two immunopurified polyclonal antibodies raised against two synthetic peptides corresponding to the A and C forms of the bovine alpha-adaptin (these latter recognized by Western blot a single protein with the expected molecular weight). Similar values were obtained with three antibodies (the two polyclonal antipeptide antibodies and the mAb 100/1), the mAb AP-6 which recognizes an epitope on the carboxyl-terminal region giving lower values. The cytosolic AP-2 was estimated to be 450 nM.

GTP Overlay and Immunoblotting

After SDS-polyacrylamide gel electrophoresis electrophoresis, the proteins were transferred onto nitrocellulose. The detection of small GTPases by GTP overlay was as described by Serafini et al.(1991). After transfer, the nitrocellulose was incubated for 1.5 h in 200 ml of blocking buffer (1 times PBS, 0.1% gelatin, 0.05% Tween 20). The nitrocellulose was then incubated for 1 h in binding buffer (20 mM Tris-HCl, pH 8.0, 10 mM MgCl(2), 2 mM dithiothreitol, 0.1% (w/v) Triton X-100, 0.5% (w/v) gelatin) containing 2 µCi/ml [alpha-P]GTP.

Immunodetection of -adaptins and ARF-1 was performed as followed. Immediately after transfer, the nitrocellulose was incubated for 2 h in blocking buffer (1 times PBS, 0.05% Tween 20, 0.1% gelatin, 5% (w/w) defatted milk). The filter was then incubated overnight at 4 °C with the appropriate antibody (mAb 100/3 anti--adaptin monoclonal antibody and 1D9 anti ARF-1 monoclonal antibody) diluted in blocking buffer. Filters were washed 10 min in 1 times PBS, two times for 10 min with 1 times PBS, 0.05% Nonidet P-40, and 10 min in 1 times PBS. Filters were then incubated for 1 h with a rabbit anti-mouse IgG coupled to horseradish peroxidase in blocking buffer. After washing, the immunocomplexes were detected using the ECL detection kit.

AP-1 Binding Assay

The AP-1 binding assay was as described previously (Le Borgne et al., 1993). Briefly, the cells were grown on 24 multiwell plates and permeabilized with the bacterial toxin streptolysin O (SLO). They were then incubated with bovine brain cytosol (11 mg/ml) or partially purified AP-1 for 10 min at 37 °C in the absence or the presence of 400 µM GTPS. The cells were then washed three times for 10 min on ice with KOAc buffer and fixed with 3% paraformaldehyde. Newly bound bovine AP-1 was detected using the monoclonal mAb 100/3 anti--adaptin antibody and then quantitated using an horseradish peroxidase-based ELISA as described previously.

To deplete acceptor membranes of endogenous ARF proteins, the cells were incubated for 10 min at 37 °C with 100 µg/ml BFA prior to permeabilization.

The number of moles of AP-1 bound to membranes was determined by ELISA as described above using as a standard curve several concentrations of bovine brain cytosol immobilized on plastic. This determination was also performed by Western blotting after incubation of the permeabilized cells with cytosol or semi purified AP-1 and solubilization of membranes with 1% Triton X-100 using as a standard curve bovine brain cytosol or purified AP-1.

Production of Recombinant Mannose 6-Phosphate/IGF II Receptor Tails

The full-length bovine mannose 6-phosphate/IGF II cytoplasmic domain was expressed as a glutathione S-transferase fusion protein in Escherichia coli as described previously (Méresse et al., 1990). Two major products are obtained after purification: the approx45-kDa full-length fusion protein and a approx30-kDa proteolytic breakdown product mostly made of the glutathione S-transferase. The relative amount of full-length fusion protein present in the preparation was quantitated by Western blotting using a polyclonal antibody raised against the glutathione S-transferase. The full-length fusion protein was also detected with a polyclonal antibody raised against a synthetic peptide corresponding to the 12 last amino acids of the receptor cytoplasmic domain. Quantitative Western blotting indicates that the full-length protein is stable when incubated for 10 min at 37 °C with a bovine brain cytosol. The fusion protein was phosphorylated in vitro by a casein kinase type II enzyme purified from clathrin-coated vesicles as described (Méresse et al., 1990). The phosphorylation mixture was then directly added to either the cytosol or partially purified AP-1, and the amount of bound AP-1 was quantitated by ELISA. As shown previously (Le Borgne et al., 1993), the phosphorylation mixture alone (kinase and poly-L-lysine) or the addition of glutathione S-transferase did not significantly modify AP-1 binding.

Fluorescence Microscopy

HeLa cells stably expressing a VSV-G-tagged sialyltransferase were first incubated in culture medium with fluorescein isothiocyanate-transferrin (25 µg/ml) at 37 °C for 1.5 h. To determine the distribution of the endogenous AP-1, the cells were first extracted with 0.05% saponin in PBS at 4 °C for 10 min and then fixed with PBS containing 3% paraformaldehyde and 0.03 M sucrose for 30 min. After a post-extraction with 0.1% Triton X-100, the cells were incubated with the mAb 100/3 anti--adaptin antibody and the rabbit anti-VSV-G tag polyclonal antibody. The bound antibodies were detected with fluorescently labeled goat anti-mouse and anti-rabbit second antibodies. To determine the distribution of the AP-1 in permeabilized cells, HeLa cells were first permeabilized with SLO as for the quantitative in vitro assay. After incubation with bovine brain cytosol in the presence or the absence of GTPS and washing, the cells were fixed with 3% paraformaldehyde and processed as above.

Electron Microscopy

HeLa cells were first incubated at 37 °C overnight with 16 nm BSA-gold and chased for 2 h in order to label late endocytic compartments. The early endocytic compartments were labeled with 5 nm BSA-gold internalized for 7 min at 37 °C. The cells were then directly processed for electron microscopy or permeabilized, incubated with cytosol at 37 °C for 10 min in the absence or in the presence of GTPS and processed for electron microscopy. Cryosectioning and labeling was done according to Griffiths (1993). Double labeling was done according to the method of Slot et al.(1991) with the anti--adaptin monoclonal antibody mAb 100/3 and a polyclonal antibody against the VSV-G tag. For quantifying the number of gold particles per linear length of membrane, systematically sampled micrographs were taken at a primary magnification of 28,000, and the density of label was estimated by relating the number of gold particles to the number of intersections with the lines of a test grid (Griffiths, 1993).


RESULTS

In order to understand the contribution of the MPRs in the recruitment of AP-1 on membranes, we have re-expressed the MPRs in MPR-negative fibroblasts and calculated the parameters of the AP-1 binding reaction (K(d) and number of binding sites) using the in vitro assay that we have previously described (Le Borgne et al., 1993). This assay relies on the incubation of SLO-permeabilized rodent cells (NRK or mouse embryonic fibroblasts) with a bovine brain cytosol followed by the detection of the newly bound bovine AP-1 using a species-specific monoclonal antibody against its -subunit. These determinations were performed in the presence of GTPS, as this slowly hydrolyzable analogue of GTP prevents the uncoating of COP-coated vesicles thereby blocking vesicular transport in the secretory pathway (Rothman and Orci, 1992; Tanigawa et al., 1993). GTPS also blocks the late stages of clathrin coat assembly at the plasma membrane (Carter et al., 1992).

AP-1 Binding and ARF Proteins

Previous in vitro studies have demonstrated that the GTPase ARF-1 regulates AP-1 binding onto Golgi-enriched fractions (Stamnes and Rothman, 1993; Traub et al., 1993). It could be expected from our in vitro GTPS and BFA-sensitive AP-1 binding assay in SLO-permeabilized cells (Le Borgne et al., 1993) that this GTPase is also a critical component in our system. The cytosolic AP-1 was fractionated from the cytosolic ARF proteins on a sizing column (see ``Experimental Procedures''). These semi-purified AP-1 bound to membranes of SLO-permeabilized NRK cells but to a lower extent than when present as a component of the cytosol (Fig. 1). Full binding of semi-purified AP-1 could be restored upon addition of low molecular weight, ARF-enriched fractions or of the recombinant myristoylated ARF-1, suggesting that ARF-1 supports most of the activity of the low molecular weight, ARF-enriched fractions. The binding of semi-purified AP-1 was dependent on the concentration of recombinant myristoylated ARF-1 or ARF-enriched fractions (not shown).


Figure 1: AP-1 binding and ARF-1. NRK cells were permeabilized and incubated for 10 min at 37 °C with cytosol or with partially purified AP-1 in the absence (AP-1) or the presence of 2 µM recombinant myristoylated ARF-1 (AP-1+mARF-1) or with an excess of cytosolic ARF-enriched fractions (AP-1+ARF FRACTION). NRK cells were also pretreated for 10 min with 100 µg/ml BFA, permeabilized in the absence (BFA I) or in the presence (BFA II) of 100 µg/ml BFA, and finally incubated for 10 min at 37 °C with cytosol in the presence of 100 µg/ml BFA. The amount of bound AP-1 was then quantitated by ELISA.



AP-1 recruitment becomes temperature-independent when the acceptor membranes have been primed at 37 °C with ARF-enriched fractions. To show this, SLO-permeabilized NRK cells, treated with BFA to remove endogenous ARFs (Donaldson et al., 1992b; Helms and Rothman, 1992), were first incubated with ARF-enriched fractions and GTPS at 37 °C to allow efficient membrane insertion of these GTPases. They were then washed and subsequently incubated at 4 or 37 °C with semi-purified AP-1. Fig. 2shows that AP-1 binds to membranes whether this subsequent incubation is performed at 4 or 37 °C and with the same efficiency as when ARF proteins and AP-1 are simultaneously incubated with membranes at 37 °C. Therefore, AP-1 behaves as the coatomer which is also recruited in a temperature-independent manner, provided that the membranes have been primed at 37 °C in order to incorporate ARF (Palmer et al., 1993).


Figure 2: Effect of the temperature on AP-1 binding. NRK cells were permeabilized with SLO, preincubated at 37 °C as indicated. They were re-incubated at 4 or 37 °C in the presence or the absence of GTPS with cytosol or with ARF-enriched fractions (ARF) and semi-purified AP-1 (AP-1) as indicated. The amount of membrane-bound AP-1 was then determined. The values are the means ± S.E. of two independent experiments performed in duplicates.



Parameters of the AP-1 Binding Reaction

Brefeldin A impairs the recruitment of both ARF-1 (Donaldson et al., 1992b; Helms and Rothman, 1992) and AP-1 onto membranes (Robinson and Kreis, 1992; Wong and Brodski, 1992). As shown in Fig. 1, AP-1 binding is reduced by 90% when NRK cells are pretreated with BFA, permeabilized in the presence of BFA, and incubated with cytosol and BFA. However, some residual binding can still be detected (approx30% of the control) when brefeldin A is not included during the permeabilization, suggesting that the membrane-bound ARFs have become limiting in the assay. These latter conditions were used for the subsequent binding study. NRK cells were treated with brefeldin A, permeabilized with SLO, and incubated with increasing concentrations of partially purified AP-1 in the presence of GTPS (Fig. 3A). AP-1 bound to membranes but to a lower extent than when the incubation medium was supplemented with an excess of ARF-enriched fractions or recombinant myristoylated ARF-1. Under these conditions, two distinct binding sites are measured after linearization of the concentration curves (Fig. 3B and Table 1): the high affinity binding sites with an apparent K(d) approx 25 nM and the low affinity sites with an apparent K(d) approx 150 nM. When ARF proteins are limiting (Fig. 3B and Table 1), the high affinity binding sites for AP-1 are lost and the number of low affinity binding sites with an apparent K(d) approx 125 nM were reduced by 50%. These results show that, in the presence of GTPS, two types of interactions (high and low affinities) contribute to the in vitro recruitment of AP-1 on membranes and that ARF proteins are, as expected, required to promote the high affinity binding of AP-1 onto membranes.


Figure 3: Effect of GTPS on AP-1 binding. A, NRK cells were pretreated for 10 min at 37 °C with Brefeldin A (100 µg/ml), permeabilized with SLO in the absence of Brefeldin A, and incubated in the presence of GTPS with increasing amounts of partially purified AP-1 in the absence (bullet) or the presence of an excess of ARF-enriched fractions (up triangle) or 2 µM recombinant myristoylated ARF-1 (). For comparison, BFA-treated NRK cells were incubated with increasing amounts of cytosol in the presence (box) or the absence of GTPS (). The amount of bound AP-1 was then quantitated, and the concentration curves were linearized according to the Scatchard method (B and C). The indicated values are means ± S.E. of three independent experiments performed in duplicate.





Effect of GTPS on AP-1 Binding

The addition of GTPS to in vitro systems increases the amount of both ARF-1 (Palmer et al., 1993) and AP-1 (Robinson and Kreis, 1992; Stamnes and Rothman, 1993; Traub et al., 1993; Le Borgne et al., 1993) bound to Golgi membranes. Scatchard analyzes were performed in SLO-permeabilized cells treated with cytosol in the presence or in the absence of GTPS ( Fig. 3and Table 1). The concentration curve obtained with a cytosol was indistinguishable from that obtained with semi-purified AP-1 and an excess of ARF-containing fractions or recombinant myristoylated ARF-1 (Fig. 3A), indicating that the cytosolic ARFs are not the limiting factors when the AP-1 binding reaction is performed at low concentrations of cytosol. High (K(d) approx 25 nM) and low (K(d) approx 150 nM) affinity binding sites are also detected when SLO-permeabilized cells are incubated with cytosol and GTPS (Fig. 3C, Table 1). However, the number of the high affinity binding sites is only slightly decreased (approx30%), and the high number of low affinity binding sites are no longer detectable when GTPS is omitted (Fig. 3C, Table 1). Thus, these results show that the addition of GTPS, which increases the amount of membrane-bound ARF, mostly uncovers low affinity AP-1 binding sites on membranes.

Effect of GTPS on the Specificity of Interactions

GTPS induces a mistargeting of the plasma membrane adaptors AP-2 onto endocytic compartments of freeze-thawed NRK cells incubated with cytosol (Seaman et al., 1993). Therefore, it was important to determine whether these low affinity binding sites for AP-1 detected upon the addition of GTPS were present on Golgi structures or on other intracellular compartments. Because of the lack of reactivity of the anti--adaptin antibody for the endogenous rodent protein as well as the unavailability of antibodies against bona fide TGN markers of NRK cells, these experiments (Fig. 4) were performed on HeLa cells expressing a VSV-G epitope-tagged sialyltransferase as acceptor membranes (Rabouille et al., 1995). SLO-permeabilized HeLa cells exhibit the same GTPS and brefeldin A-sensitive AP-1 binding properties as NRK cells (not shown). In untreated HeLa cells, the anti--adaptin antibody exhibited by immunofluorescence a strong perinuclear staining as well as some punctuate staining previously observed by others (Robinson and Kreis, 1992). In double immunofluorescence, a large proportion of the perinuclear -adaptin staining colocalized with the VSV-G-tagged sialyltransferase. The peripheral staining as well as some of the perinuclear -adaptins colocalized with fluorescently labeled transferrin endocytosed to label predominantly early endocytic structures. The same distribution was observed in SLO-permeabilized HeLa cells incubated with cytosol in the absence or the presence of GTPS.


Figure 4: Effect of GTPS on AP-1 distribution in HeLa cells. HeLa cells expressing a VSV-G-tagged sialyltransferase were first incubated with fluorescein isothiocyanate-transferrin for 90 min at 37 °C. Then, they were either permeabilized with saponin in the cold and fixed (A, B, E, F) or permeabilized with SLO, incubated with bovine brain cytosol in the presence of GTPS (C, D, G, H), and then fixed. The different samples were processed for double immunofluorescence confocal microscopy. A, B, C, and D are focal planes of the Golgi region, and E, F, G, and H are focal planes of the bottom of the cells close to the substratum. A, C, E, and G, AP-1 staining; B and D, sialyltransferase staining; F and H, internalized transferrin. The processing of the images does not take into account the differences in the intensity of fluorescence associated with the -adaptin as observed between the nonpermeabilized and the permeabilized cells incubated with cytosol and GTPS. The same AP-1 distribution was obtained in permeabilized cells incubated with cytosol alone (not shown).



The electron microscopy confirmed and extended these results. Both in vivo and in permeabilized cells systems, the anti--adaptin antibody decorated two distinct kinds of structures (Fig. 5). First, it labeled membrane structures in the vicinity of the Golgi stack that also contained the sialyltransferase. Second, it labeled some early endosomal structures identified by the presence of endocytosed BSA-gold. Both in vivo and in vitro, the late endocytic structures as well as other intracellular organelles remained unlabeled, thereby confirming the specificity of the in vitro interactions. Table 2shows the quantitation of these experiments. While the permeabilization allows an apparent better detection of the membrane-bound AP-1, it does not appear to modify its distribution on membranes when compared with nonpermeabilized HeLa cells. In permeabilized cell systems, GTPS induced a strong, 3-4-fold increase in the density of AP-1 labeling on the membranes of the sialyltransferase-rich compartment and a more modest 2-fold increase on membranes of early endosomes. This quantitation is consistent with the biochemical experiments showing that GTPS induces an overall 2-3-fold increase of AP-1 binding. Thus, the results show that the low affinity AP-1 binding sites generated by the addition of GTPS are localized to the same compartments as the high affinity binding sites.


Figure 5: Distribution of AP-1 on cryosections of HeLa cells. Hela cells expressing a VSV-G tagged sialyltransferase were first incubated with BSA-gold under conditions to label early endosomes (EE, 5 nm gold) (C) or late endocytic structures (LE, 16 nm gold) (B and C). The cells were permeabilized, incubated with cytosol in the presence of GTPS, and then processed for electron microscopy. In A and B, the sections were double-labeled with an anti-VSV-G tag polyclonal antibody (5 nm gold, small arrows) which identifies the TGN (T) on one side of the Golgi stacks (G) and the 100/3 anti--adaptin monoclonal antibody (10 nm gold, arrowheads). In C, single labeling with the 100/3 anti--adaptin monoclonal antibody was also performed for localization on endosomes. A and B, examples of AP-1 labeling over the Golgi region. Most of the AP-1 labeling is distributed in a nonclustered fashion on the TGN membrane (small arrowheads). Some coated buds (large arrowheads) are also labeled. C shows an example of -adaptin-labeled buds on early endosome elements (arrowheads) identified by 5 nm BSA-gold (small arrows). Late endosomes identified by 16 nm BSA-gold do not label for -adaptin (B, C). Bars: 100 nm. The same distribution was observed in nonpermeabilized cells or in permeabilized cells incubated with cytosol. The quantitation of these experiments is shown in Table 2.





AP-1 Binding and Cargo Proteins

We have reported previously that membranes of MPR-negative mouse embryonic fibroblasts (Ludwig et al., 1994) bind only 30% of the total amount of AP-1 that binds under the same conditions to membranes of the corresponding wild type cells (Le Borgne et al., 1993). It is extremely unlikely that the amount of membrane-bound ARF-1 is affected in MPR-negative cells (see ``Discussion''). If the MPRs are critical components for AP-1 recruitment on membranes, it can be expected that the low AP-1 binding activity of membranes of MPR-negative cells could be rescued upon the simple re-expression of the MPRs. To test this hypothesis, the MPR-negative cells were immortalized, each MPR stably re-expressed, and the AP-1 binding activity of their membranes was determined. Fig. 6A shows that, as for the primary cells (Le Borgne et al., 1993), the membranes of the immortalized MPR-negative fibroblasts exhibit a much lower AP-1 binding activity than the corresponding immortalized, wild type embryonic fibroblasts expressing the two MPRs. Strikingly, the level of AP-1 binding activity seen in control cells is restored in MPR-negative fibroblasts that stably re-express either the cation-dependent or the cation-independent mannose 6-phosphate/IGF II receptors.


Figure 6: AP-1 binding in MPR-positive and -negative cells. A, wild type fibroblasts, MPR-negative fibroblasts (vector alone), or MPR-negative fibroblasts stably re-expressing either the cation-independent mannose 6-phosphate/IGF II receptor (CI-MPR) or the cation-dependent (CD-MPR) mannose 6-phosphate receptor were permeabilized with streptolysin O, incubated with bovine brain cytosol in the presence of GTPS, and the membrane-bound AP-1 was then quantitated as described under ``Experimental Procedures.'' The values represent the means ± S.D. of two experiments performed on independent clones re-expressing physiological levels of cation-dependent MPR or cation-independent MPR (approx1.5- and approx1.9-fold the endogenous level, respectively). B, MPR-negative fibroblasts () or the corresponding wild type fibroblasts () were permeabilized with streptolysin O, incubated with increasing amounts of cytosol in the presence of GTPS, and the membrane-bound AP-1 was then quantitated. The indicated values are means ± S.E. of three independent experiments performed in duplicate. The concentration curves were linearized according to the Scatchard method (C).



Scatchard analyses were performed on these MPR-positive and -negative fibroblasts in the presence of GTPS (Fig. 6, B and C). Linearization of the concentration curves shows that permeabilized MPR-positive fibroblasts also exhibit two types of binding sites for AP-1 in the presence of GTPS (Table 1): high affinity binding sites with an apparent K(d) approx 22 nM and low affinity binding sites with an apparent K(d) approx 150 nM. These two types of binding sites could still be detected in MPR-negative embryonic fibroblasts. However, the number of high affinity binding sites was drastically reduced to 25% of the control values and that of the low affinity binding sites to 35% (Fig. 6C and Table 1). We believe that these residual high affinity binding sites are due to additional proteins that are similarly sorted via this clathrin-dependent pathway. Scatchard analyses were also performed on MPR-negative fibroblasts expressing different amounts of the cation-dependent mannose 6-phosphate receptor and the parameters of the AP-1 binding reaction determined. Fig. 7shows that the number of high affinity binding sites for AP-1 that are detected depends on the concentration of MPR in membranes. These results clearly demonstrate that the MPRs, i.e. cargo proteins are key components for the interactions of AP-1 with membranes and that they are essential to create the high affinity binding sites for AP-1.


Figure 7: MPR expression and high affinity AP-1 binding sites. The mouse cation-dependent MPR was re-expressed in MPR-negative fibroblasts and clones expressing different levels of the cation-dependent MPR were selected. The level of expression refers to the endogenous cation-dependent MPR expressed in control fibroblasts expressing the two MPRs. The cells were permeabilized with SLO, incubated with increasing amounts of cytosol in the presence of GTPS. The concentration curves were linearized according to the Scatchard method and the number of high affinity binding sites calculated. The indicated values are means ± S.E. of two or three independent experiments performed in duplicate.



The results described above indicate that ARF proteins and the MPRs are simultaneously required in order to provide the high affinity binding sites for AP-1. We have previously observed that the addition of the soluble, phosphorylated mannose 6-phosphate/IGF II receptor cytoplasmic domain fused to the glutathione S-transferase could completely abolish AP-1 binding onto membranes (Le Borgne et al., 1993). Thus, one could anticipate that this soluble, phosphorylated cytoplasmic domain could become a better competitor of the AP-1 binding reaction when low affinity interactions of AP-1 with membranes are measured in ARF-limiting conditions. As shown before, the addition of the phosphorylated tail fused to the glutathione S-transferase completely abolished AP-1 binding in the ARF-complemented system with a 50% inhibition observed at approx1 µM, while the glutathione S-transferase alone or a 50 molar excess of phosphorylated or nonphosphorylated synthetic peptides corresponding to the last 13 highly charged amino acids of the MPR tail remained without any effect (Fig. 8). However, the fusion protein becomes a stronger inhibitor of AP-1 binding in the ARF-limiting system, and 50% inhibition was obtained with a much lower concentration of approx300 nM.


Figure 8: Competition of AP-1 binding in the absence or the presence of ARF. A fusion protein corresponding to the full-length soluble cytoplasmic domain of the mannose 6-phosphate/IGF II receptor fused to the glutathione S-transferase was first phosphorylated in vitro as described under ``Experimental Procedures.'' NRK cells pretreated with BFA before permeabilization were then incubated with partially purified AP-1 and increasing concentrations of the phosphorylated fusion protein in the absence (box) or the presence (circle) of an excess of ARF-enriched fractions. The bound AP-1 was then quantitated. As controls, 5 µM glutathione S-transferase (up triangle) and 50 µM peptide corresponding to the highly charged COOH terminus of the Man-6-P/IGF II receptor (AAATPISTFHDDSDEDLLHV) phosphorylated (bullet) or not phosphorylated () on the more distal serine (Méresse et al., 1990) were used. The indicated values represent the means ± S.E. of two independent experiments performed in duplicate.




DISCUSSION

The formation of clathrin-coated vesicles in the TGN requires the recruitment of the cytosolic Golgi-specific assembly protein AP-1 and clathrin on the membrane of this organelle. The interaction of AP-1 with its target membrane is regulated by the small GTPase ARF-1 (Stamnes and Rothman, 1993; Traub et al., 1993). During this process, the MPRs and their bound lysosomal enzymes are clustered into the nascent vesicles (Geuze et al., 1992). We show here that the high affinity binding of AP-1 to membranes also requires the presence MPRs, i.e. the cargo transmembrane proteins, strongly suggesting that MPR sorting in the TGN is highly coupled to the first step of clathrin coat assembly. These high affinity interactions are lost when either the MPRs or ARF proteins become rate-limiting. This suggests that these components cannot promote by themselves efficient recruitment of AP-1 and that they cooperate to trigger efficient coat assembly on TGN membranes.

MPRs and AP-1 Recruitment

The presented data demonstrate the importance of the cargo protein in coat recruitment. They show that the simple re-expression of MPRs in MPR-negative cells restore the low AP-1 binding activity of their membranes and that the number of high affinity AP-1 binding sites depends on the number of MPR molecules present in membranes. There are two possible ways of explaining these data. The MPRs could be directly involved in the high affinity interaction of AP-1 with membranes. Alternatively, the MPRs could somehow regulate the insertion of ARF-1 into membranes, thereby affecting AP-1 recruitment. Although this latter possibility would give to the MPRs an unsuspected function and place the molecules as critical components for both ARF and AP-1 recruitment, we favor the direct role of the MPRs in AP-1 binding. As the recruitment of AP-1 on Golgi membranes (Stamnes and Rothman, 1993; Traub et al., 1993; this study), vesicular transport in the early secretory pathway is regulated by ARF-1 (Balch et al., 1992; Taylor et al., 1992). Yet, MPR-negative fibroblasts re-expressing or not different levels of one MPR secrete the same amount of total proteins as control fibroblasts expressing the two MPRs (Ludwig et al., 1994; Mauxion et al., 1996), suggesting that the recruitment of ARF-1 is not affected. A stronger argument comes from subsequent studies in which the parameters of the AP-1 binding reaction were determined in MPR-negative cells re-expressing the cation-dependent mannose 6-phosphate receptor mutated on its cytoplasmic domain (Mauxion et al., 1996). These results show that simple mutations in MPR cytoplasmic domains can modulate the affinity of AP-1 for membranes. Thus, it seems extremely unlikely that the MPRs affects the recruitment of ARF-1 onto membranes.

GTPS Uncovers Low Affinity AP-1 Binding Sites

Our binding study argues that the MPRs or ARF-1 cannot provide by themselves high affinity binding sites for AP-1 as evidenced by the effect of brefeldin A or MPR expression on AP-1 recruitment. These two components must somehow cooperate in order to provide the high affinity binding sites for AP-1. Even though it is still unknown how ARF-1 functions in regulating coat assembly, further support for this notion could come from the effect of GTPS itself. The addition of this slowly hydrolyzable analogue increases the amount of membrane-bound ARF-1 (Palmer et al., 1993) which has been found in two distinct pools (Helms et al., 1993): one nonsaturable, extractable with phosphatidylcholine-containing liposomes, and another, saturable pool-resistant to such extraction. Concomitantly, GTPS produces a 2-3-fold (Stamnes and Rothman, 1993; Le Borgne et al., 1993) or a approx8-10-fold increase (Robinson and Kreis, 1992; Traub et al., 1993) in AP-1 binding to membranes. Our data clearly show that the addition of GTPS only slightly increases (1.4-fold) the number of high affinity binding sites for AP-1 and mainly uncovers a large number of low affinity binding sites, predominantly on TGN membranes. Thus, a large excess of active ARF-1 in membranes does not result in a large increase of high affinity AP-1 binding sites.

Specificity of AP-1 Binding

Both confocal and quantitative electron microscopy show that, both in vivo and in vitro, AP-1 is bound to membranes of a sialyltransferase-positive compartment, presumably the TGN as well as to membranes of early endocytic structures. These morphological observations show that the permeabilization procedure does not modify the specificity of interactions of AP-1 with membranes and further validates our in vitro system. The biological significance of AP-1 on early endosomes remains unclear at present. A possible interpretation is that it reflects a partial uncoating of the TGN-derived vesicles that have fused with this compartment. This view is consistent with the fact that AP-1 distributes both to membranes and associated buds of the sialyltransferase-positive compartment, while it appears to be more restricted to coated buds on the early endosomes. The fact that the membranes of the sialyltransferase-positive compartment become more labeled in the presence of GTPS than do those of the early endosomal compartment also argues that the TGN is the primary site of AP-1 binding. This further illustrates the functional differences between AP-1 and its counterpart AP-2, which is mistargeted to endocytic structures when permeabilized cells are incubated with cytosol and GTPS (Seaman et al., 1993).

Both in vivo and in vitro, AP-1 recognizes specific features of the binding sites provided by the TGN (or endosome?) membranes. ARF-1 probably acts as a general factor for coat assembly, since it regulates both AP-1 and coatomer binding onto enriched Golgi membranes (Stamnes and Rothman, 1993; Traub et al., 1993; Palmer et al., 1993), coatomer-mediated transport in the early secretory pathway (Balch et al., 1992; Taylor et al., 1992; Dasher and Balch, 1994; Zhang et al., 1994a) as well as in vitro endosome fusion (Lenhard et al., 1992). The MPRs could potentially provide some of the features required for a specific interaction of AP-1 with its target membrane. Although the MPRs are present in several compartments that do not recruit AP-1, the different pools of receptors are not totally equivalent owing to the phosphorylation of their cytoplasmic domains (Méresse et al., 1990; Méresse and Hoflack, 1993; Hemer et al., 1993). Indeed, our subsequent studies show that these phosphorylation sites are important for the high affinity interaction of AP-1 with membranes (Mauxion et al., 1996). It is likely that the high affinity binding sites for AP-1 also contain additional unknown proteins that may also contribute to the specificity of interaction. Some support to this view comes from our observation showing that the addition of GTPS, which largely uncovers low affinity AP-1 binding sites, does not affect the specificity of interaction of AP-1 with its target membranes. The putative ARF receptor (Helms et al., 1993; Traub et al., 1995) or other proteins similar to those described by Anderson and colleagues for the interaction of AP-2 with membranes (Mahafrey et al., 1990; Chang et al., 1993; Peeler et al., 1993; Zhang et al., 1994b) could potentially fulfill this function.

Clathrin Coat Assembly and Protein Sorting in the TGN

It is currently believed that MPR sorting and AP-1 binding are two uncoupled events (Pearse and Robinson, 1990; Robinson, 1994, Traub et al., 1995). According to the proposed model, ARF-1 is translocated from the cytosol onto TGN membranes and regulates the recruitment of AP-1 onto a putative membrane receptor. When AP-1 is bound to membranes, the MPRs can then be recruited into the nascent vesicle by interacting with one subunit of AP-1. This view, in which MPR sorting is subsequent to AP-1 binding (Traub et al., 1995), predicts that AP-1 binding is independent of MPR expression. This model is primarily based on the effect of GTPS, which increases the amount of ARF-1 in membranes and stimulates AP-1 binding without affecting the number of cargo proteins present. However, our data show that GTPS mostly uncovers low affinity binding sites for AP-1, normally not detected, and that the level of MPR expression determines the number of the high affinity AP-1 binding sites. In contrast to the above model, our study shows that MPR sorting is highly coupled to the first step of clathrin coat assembly and therefore strongly suggests that the number of vesicles formed depends to some extent on the number of cargo molecules to be sorted. Alternatively, AP-1 could bind to membranes with a low affinity in the absence of cargo proteins and their presence could stabilize these interactions. Although there is some evidence that ARF-1 can be included in COP- and clathrin-coated vesicles (Serafini et al., 1991; Lenhard et al., 1992), it remains largely unknown how this GTPase functions in regulating AP-1 binding. It will be important to determine whether ARF-1 functions as a stoichiometric component also included in TGN-derived clathrin-coated vesicles like the MPRs or as a catalytic factor regulating the activity of molecules involved in AP-1 binding. Coatomer assembly in the early secretory pathway and clathrin coat assembly in the TGN are increasingly thought of as mechanistically related processes. The efficient recruitment of coatomers may also require the presence of membrane proteins in addition of ARF-1. Interestingly, endoplasmic reticulum membrane proteins can bind to the coatomer via a KKXX retrieval motif (Cosson and Letourneur, 1994), and in vivo studies in yeast have shown that mutants unable to retain proteins in the endoplasmic reticulum are also defective in functional coatomers (Letourneur et al., 1994). This raises the interesting possibility that coatomer recruitment may also requires the cooperation of ARF-1 and membrane proteins, as proposed here for ARF-1 and the MPRs in the case of the Golgi assembly proteins AP-1.


FOOTNOTES

*
This work was supported in part by grants from ``Vaincre les Maladies Lysosomales'' and NATO(900226) and by the European communities (Grant B102-CT93-0205). 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.

§
To whom correspondence should be addressed. Tel.: 49-6221-387-285; Fax: 49-6221-387-306.

(^1)
The abbreviations used are: TGN, trans-Golgi network; IGF, insulin-like growth factor; MPR, mannose 6-phosphate receptor; GTPS, guanosine 5`-O-(3-thiotriphosphate); NRK, normal rat kidney; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; BFA, brefeldin A; VSV-G, vesicular stomatitis virus-G protein; BSA, bovine serum albumin; SLO, streptolysin O.


ACKNOWLEDGEMENTS

We gratefully acknowledge the expert EM assistance of Heins Horstmann and Anja Haberman. We also thank Dr. Ernst Ungewickell for the generous gift of the mAb 100/3 monoclonal antibody, Dr. Thomas Kreis for the polyclonal antibody against the VSV-G tag, and Dr. R. Kahn for the recombinant myristoylated ARF-1 and the 1D9 anti-ARF-1 monoclonal antibody. The SA 48 HeLa cells expressing the epitope-tagged sialyltransferase were kindly provided by Dr. Tommy Nilsson. Drs. R. Parton, K. Simons, and M. Zerial are warmly acknowledged for the critical reading of the manuscript.


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