©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
A Casein Kinase II Phosphorylation Site in the Cytoplasmic Domain of the Cation-dependent Mannose 6-Phosphate Receptor Determines the 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)

Fabienne Mauxion (§) Roland Le Borgne Hélène Munier-Lehmann Bernard Hoflack (¶)

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

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The transport of proteins from the secretory to the endocytic pathway is mediated by carrier vesicles coated with the AP-1 Golgi assembly proteins and clathrin. The mannose 6-phosphate receptors (MPRs) are two major transmembrane proteins segregated into these transport vesicles. Together with the GTPase ARF-1, these cargo proteins are essential components for the efficient translocation of the cytosolic AP-1 onto membranes of the trans-Golgi network, the first step of clathrin coat assembly. MPR-negative fibroblasts have a low capacity of recruiting AP-1 which can be restored by re-expressing the MPRs in these cells. This property was used to identify the protein motif of the cation-dependent mannose 6-phosphate receptor (CD-MPR) cytoplasmic domain that is essential for these interactions. Thus, the affinity of AP-1 for membranes and in vivo transport of cathepsin D were measured for MPR-negative cells re-expressing various CD-MPR mutants. The results indicate that the targeting of lysosomal enzymes requires two distinct determinants at the carboxyl terminus of the CD-MPR cytoplasmic domain that are different from tyrosine-based endocytosis motifs. The first is a casein kinase II phosphorylation site (ESEER) that is essential for high affinity binding of AP-1 and therefore probably acts as a dominant determinant controlling CD-MPR sorting in the trans-Golgi network. The second is the adjacent di-leucine motif (HLLPM), which, by itself, is not critical for AP-1 binding, but is absolutely required for a downstream sorting event.


INTRODUCTION

The targeting of the newly synthesized hydrolases to lysosomes is essential for the efficient catabolism of macromolecules entering the endocytic pathway of mammalian cells. For this process, the common mannose 6-phosphate (Man-6-P) recognition marker present on the oligosaccharides of approx40 soluble hydrolases is recognized in the late secretory pathway by the mannose 6-phosphate receptors (MPRs), (^1)two related type I transmembrane proteins (Kornfeld and Mellman, 1989; Kornfeld, 1992). It is now well accepted that the trans-Golgi network (TGN) is the major site where proteins are sorted from the biosynthetic pathway for efficient delivery to endosomes (Griffiths and Simons, 1986). There, the MPRs and their bound ligands are segregated into nascent clathrin-coated vesicles (Geuze et al., 1985), probably together with other transmembrane proteins destined to the endosomes/lysosomes. After budding and uncoating, these Golgi-derived vesicles fuse with endosomal compartments where the MPRs discharge their bound ligands. While the soluble lysosomal enzymes are directed toward the lysosomes, the MPRs recycle back to the TGN (Duncan and Kornfeld, 1988) or to the cell surface where they are found in small amounts at steady state. At the plasma membrane, the MPRs undergo endocytosis via clathrin-coated pits like many other cell surface receptors.

As for many transmembrane proteins, the MPRs contain multiple molecular sorting signals in their cytoplasmic domains that mediate their intracellular traffic between distinct membrane-bound compartments (Lobel et al., 1989). The endocytosis of cell surface proteins is mediated by tyrosine-based (Trowbridge et al., 1993) or di-leucine-based motifs (Sandoval and Bakke, 1994). In the case of the mannose 6-phosphate/insulin-like growth factor II (Man-6-P/IGF II) receptor, its endocytosis requires a single YSKV sequence (Canfield et al., 1991; Jadot et al., 1992), while that of the other, cation-dependent mannose 6-phosphate receptor (CD-MPR), requires two distinct motifs: a phenylalanine-containing sequence (FPHLAF) and a YRGV sequence that function, respectively, as dominant and weak determinants (Johnson et al., 1990). The determinants that are essential for the sorting of the MPRs in the TGN or in endosomes have been more difficult to elucidate. However, the available data indicate that a di-leucine-based motif near the carboxyl terminus of the CD-MPR (HLLPM sequence) cytoplasmic domain is essential for efficient targeting of newly synthesized lysosomal enzymes (Johnson and Kornfeld, 1992a). The cytoplasmic domain of the Man-6-P/IGF II receptor has two signals for lysosomal enzyme sorting in the Golgi, a di-leucine-based motif (LLHV sequence) and the tyrosine-based endocytosis motif (YKYSKV sequence) (Johnson and Kornfeld, 1992b). Several other transmembrane proteins destined to the lysosomes also contain di-leucine-based motifs in their cytoplasmic domains that are essential for their proper delivery to lysosomes (Sandoval and Bakke, 1994). In the light of these different results, it has been proposed that di-leucine-based motifs mediate sorting of membrane proteins in the TGN. In both MPRs, the di-leucine motifs are flanked by casein kinase II phosphorylation sites that are phosphorylated in vivo (Méresse et al., 1990; Hemer et al., 1993). Such a post-translational modification occurs when the Man-6-P/IGF II receptor exits from the TGN and represents a major, albeit transient, modification (Méresse and Hoflack, 1993). Thus far, the functional importance of the phosphorylation sites in the Man-6-P/IGF II receptor trafficking has remained controversial (Johnson and Kornfeld, 1992b; Chen et al., 1993).

Membrane traffic from the TGN to the endosomes involves the formation of carrier vesicles coated with clathrin. The Golgi-derived and plasma membrane-derived clathrin-coated vesicles can be distinguished by the nature of their underlying assembly proteins AP-1 and AP-2, two related heterotetrameric complexes (Keen, 1990; Pearse and Robinson, 1990; Robinson, 1994). Localization studies are consistent with the notion that AP-1 is associated with TGN-derived vesicles, whereas AP-2 is found in plasma membrane-derived vesicles. In vitro studies have shown that the translocation of the cytosolic AP-1 onto membranes requires the ADP-ribosylation factor ARF-1 (Stamnes and Rothman, 1993; Traub et al., 1993), a small GTPase also involved in coatomer binding and vesicular transport in the early secretory pathway (Rothman, 1994; Donaldson and Klausner, 1994; Boman and Kahn, 1995).

The MPRs are also critical components for the recruitment of AP-1 onto membranes. The evidence comes from in vitro studies performed on fibroblasts devoid of the MPRs which secrete their newly synthesized lysosomal enzymes and as a consequence accumulate undigested substrates in their lysosomes (Ludwig et al., 1994). The membranes of these cells have a much lower capacity of recruiting AP-1 than those of control cells expressing the two MPRs (Le Borgne et al., 1993). Conversely, the re-expression of either MPR in MPR-negative cells creates high affinity AP-1 binding sites on their membranes (Le Borgne et al., 1996). Thus, ARF-1 and the MPRs must somehow cooperate for the efficient translocation of AP-1 onto membranes. In order to decipher the protein motifs of the MPR cytoplasmic domains that are involved in AP-1 recruitment, we have stably re-expressed CD-MPR mutants in MPR-negative fibroblasts and determined the ability of the corresponding clones to recruit AP-1 in vitro and to transport lysosomal enzymes in vivo. Altogether, our data demonstrate that the transport of lysosomal enzymes to lysosomes requires two distinct determinants in the CD-MPR carboxyl-terminal domain, a casein kinase II phosphorylation site critical for the efficient interaction of AP-1 with its target membranes and the adjacent di-leucine motif which appears more important for a post AP-1 binding step in the CD-MPR cycling pathway.


MATERIALS AND METHODS

Plasmid Construction and Mutagenesis

A full-length mouse CD-MPR cDNA clone (Ludwig et al., 1992) was digested with EcoRI-SspI restriction enzymes and the EcoRI-SspI fragment containing the entire CD-MPR coding sequence was cloned into the eukaryotic expression vector pSFFV6 (Chen et al., 1993) cut XbaI, filled in with Klenow and cut EcoRI.

For in vitro mutagenesis, the sequence encoding the transmembrane and cytoplasmic domains of CD-MPR was amplified by the polymerase chain reaction technique with the following primers: upstream primer: GGAAATTTCCGAAGACCGATCTTACTTGGTCATATTT, downstream primer: CCGGATCCAATATTAAAGGGCAAGGTGAG. The resulting polymerase chain reaction fragment was cloned into pBluescript II KS+ vector in EcoRI-BamHI restriction sites and used to generate single-stranded templates. Mutations were then introduced by the in vitro mutagenesis dutung technique (Kunkel, 1985) using the following primers: mutation *51, CCCAGCTGCTAATCTTCCCA; mutation A56A58A59, CATCCCTTGCTGCCCGACGCTTCCCCC; mutation A57D60, TGATCATCGTCTTCTTCCGCCTCTTCCC; mutation D60, TGATCATCGTCTTCTTCCG; mutation *63, CACATTGGTACTAGTTAATCATCCCCTT. For the internalization minus mutant, two in vitro mutagenesis reactions were performed with the following oligonucleotides: CCTGCCAGGCGGCCAGATGAGGAGCCTGCTCCA and CTCCACGAGCTGGGCTGCAG. Then a unique PstI restriction site present in the sequence encoding the CD-MPR cytoplasmic tail was used to join the two mutations.

To generate full-length CD-MPR mutants, a three-fragment ligation was performed with a EcoRI-PstI fragment encoding the luminal and transmembrane domains of CD-MPR, a PstI-XbaI fragment encoding the mutated D60 cytoplasmic tail, and a EcoRI-XbaI fragment coming from the eukaryotic expression vector pSFFV6. The other full-length mutants were generated by exchanging the mutated fragments using the unique PstI and StyI restriction sites present in the sequence encoding the CD-MPR cytoplasmic tail. All constructions obtained after polymerase chain reaction reactions or in vitro mutagenesis were verified by sequencing.

The construct encoding the Man-6-P/IGF II receptor was as described in Chen et al.(1993).

Cell Culture and Transfection

The parental double MPR-negative cell line was obtained after immortalization of primary culture of mouse fibroblasts lacking both MPRs (Ludwig et al., 1994) by transfection with the large T antigen of SV40. This cell line as well as all the transfected colonies were maintained in DMEM medium complemented with 10% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin.

For transfection, 20 µg of the pSFFV6 constructions expressing the different MPR mutants were linearized with FspI and cotransfected with 1 µg of BamHI-linearized pBHyg vector (which encodes the hygromycin resistance gene) in MPR-negative cells using the calcium phosphate precipitation technique. After 12-14 days of selection in medium containing 300 µg/ml hygromycin B (Boehringer Mannheim), 20-30 isolated colonies were picked for each constructions and tested for CD-MPR expression by indirect immunofluorescence. Colonies showing homogenous as well as various level of expression of CD-MPR by immunofluorescence and immunoprecipitation techniques were selected and maintained in complete DMEM medium with 150 µg/ml hygromycin B.

Indirect Immunofluorescence

After fixation with cold methanol, cells were processed for indirect immunofluorescence staining using 0.2% bovine serum albumin, 0.2% fish skin gelatin (Sigma) in solution in phosphate-buffered saline as blocking reagent. The mouse CD-MPR was detected with a mouse polyclonal antibody raised against the purified bovine CD-MPR.

Metabolic Labeling and Immunoprecipitation

For quantification of CD-MPR expression level, cells seeded on six-well plates were labeled overnight with 0.2 mCi/ml [S]methionine/cysteine (PRO-MIX(TM), Amersham Corp.) in methionine/cysteine-free MEM medium (ICN Biomedicals) supplemented with 10% dialyzed fetal calf serum, 10 mM Hepes, pH 7.2, 2 mM glutamine, and 10% normal complete DMEM medium. Alternatively, the cells were also pulsed for 30 min with 1 mCi/ml [S]methionine/cysteine in methionine/cysteine-free medium supplemented with 10% dialyzed fetal calf serum, 10 mM Hepes, pH 7.2, and 2 mM glutamine. Cells were then lysed in cold lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, and 1 mM benzamidine) and processed for immunoprecipitation with a mouse anti-bovine CD-MPR antiserum. The immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) followed by fluorography and autoradiography. The band intensities were quantified using a PhosphorImager and normalized to the total count incorporated as quantified by trichloroacetic acid precipitation. Transport of cathepsin D was assayed by pulse-labeling cells seeded on 24-well plates for 1 h with 1 mCi/ml [S]methionine/cysteine in methionine/cysteine-free MEM medium (ICN Biomedicals) supplemented with 10% dialyzed fetal calf serum, 10 mM Hepes, pH 7.2, and 10 mM mannose 6-phosphate (Man-6-P) when indicated. Cells were then chased for 4 h by the addition of 1 mML-methionine and 2.5 mML-cysteine. After the chase period, medium was kept and cells lysed with cold lysis buffer. Cathepsin D was immunoprecipitated from medium and cell lysates with a rabbit anti-mouse cathepsin D antiserum. Immunoprecipitates were analyzed by SDS-PAGE followed by fluorography and autoradiography. The band intensities were quantified with a PhosphorImager (Molecular Dynamics). Each determination was done in duplicate using as a positive control immortalized normal fibroblasts expressing both the CD-MPR and the Man-6-P/IGF II receptor and as negative control the mock-transfected (SFFV, vector alone) MPR-negative parental cell line.

AP-1 Binding Assay

The AP-1 binding assay was performed as described previously (Le Borgne et al., 1993; Le Borgne et al., 1996). Briefly, cells grown on 24-well plates were permeabilized with bacterial toxin streptolysin O and incubated with bovine brain cytosol 10 min at 37 °C in the presence of 400 µM GTPS (Sigma) to prevent uncoating linked to GTP hydrolysis. Cells were then extensively washed and fixed with 3% paraformaldehyde. Exogenously bound AP-1 was detected using the monoclonal anti--adaptin antibody 100/3 (kindly provided by Dr. E. Ungewickel) and quantified using an horseradish peroxidase-based enzyme-linked immunosorbent assay. Each determination for cells expressing the CD-MPR was done in duplicate using as the same time the parental, mock-transfected MPR-negative cells as negative control and the immortalized normal fibroblasts expressing both the CD-MPR and the Man-6-P/IGF II receptor as a positive control. The values obtained for the parental MPR-negative cell line were subtracted from those obtained for the transfected cells expressing the CD-MPR. These values reflecting the contribution of the CD-MPR alone were linearized according to the Scatchard method. The affinity of AP-1 was calculated after linear regression of the results. High and low affinity binding sites were detected. Only high affinity binding was taken into account. The low affinity binding of AP-1 is due to the presence of GTPS (Le Borgne et al., 1996).


RESULTS

Measurements of endocytosis rates of cell surface membrane proteins have been instrumental to identify the critical amino acids of their cytoplasmic domains that are essential for internalization through clathrin-coated pits (Trowbridge et al., 1993). Such precise determinations have not been possible for membrane proteins that exit the biosynthetic pathway through clathrin-coated buds for delivery to endocytic compartments. Thus far, the assays for such proteins have remained limited to the determination of the steady state distribution of protein mutants. However, the routes (direct or indirect via the plasma membrane) by which membrane proteins reach the lysosomes are still under some debate. In the case of the MPRs, transport of ligands has been used to monitor MPR sorting function (Lobel et al., 1989; Johnson and Kornfeld, 1992a, 1992b; Chen et al., 1993). Thus, the intracellular accumulation of the mature cathepsin D, a typical lysosomal enzyme, reflects delivery to the lysosomes while the secretion of cathepsin D precursor into the medium reflects a defect in MPR sorting function. The mannose 6-phosphate-dependent transport of lysosomal enzymes to lysosomes depends on several parameters, i.e. the number of MPRs present at steady state in the TGN, their efficient packaging in Golgi-derived vesicles, and their proper routing within the endosomal system.

We introduce here a new assay to measure MPR sorting function. This assay is based on the in vitro binding of the AP-1 Golgi-specific assembly proteins onto membranes (Le Borgne et al., 1993, 1996). We have shown previously that the membranes of MPR-negative cells have a low AP-1 binding activity, which can be rescued upon re-expression of the MPRs. It could be expected that mutations introduced in the MPR cytoplasmic domains affect the affinity of AP-1 for their target membranes. Thus, the K(d) values for AP-1 binding could provide a direct measurement for MPR sorting in the secretory pathway. Therefore, different mutations were introduced in the CD-MPR cytoplasmic domain (Fig. 1). They were designed to affect sequences previously shown to be important for its intracellular traffic: the endocytosis motif (mutant E), the carboxyl-terminal di-leucine motif (mutant *63). The casein kinase II phosphorylation site, adjacent to the di-leucine motif, was also mutated (mutants *51, A565859, D60, and A57D60). These CD-MPR mutants were stably re-expressed in mouse fibroblasts lacking the two MPRs (Ludwig et al., 1994). Colonies showing by immunofluorescence an homogenous expression of the CD-MPRs were selected and tested for in vitro AP-1 binding as well as in vivo cathepsin D sorting as described above.


Figure 1: Schematic representation of the CD-MPR mutants. The amino acids or the sequences shown previously to be important for CD-MPR trafficking are indicated. The point mutations are highlighted by asterisks under the mutagenized residues. The amino acid sequence of the two casein kinase II phosphorylation sites of the Man-6-P/IGF II receptor is shown for comparison. The numbers refer to the position of the amino acids in the murine CI-MPR cytoplasmic domain according to Chen et al.(1993) and in the CD-MPR cytoplasmic domain according to Johnson and Kornfeld (1990).



AP-1 Binding and Cathepsin D Sorting in Fibroblasts Expressing the Wild Type MPRs

Both the wild type CD-MPR and Man-6-P/IGF II receptor were first re-expressed in MPR-negative cells. These cells were permeabilized with streptolysin O, incubated with increasing concentrations of cytosol in the presence of GTPS to prevent uncoating reactions and the amount of newly bound AP-1 quantitated (see ``Materials and Methods''). As shown in Fig. 2A, MPR-negative fibroblasts stably re-expressing the wild type MPRs recruit on their membranes higher amounts of AP-1 than the mock-transfected (SFFV vector alone) MPR-negative fibroblasts at every concentration of cytosol tested. Thus, the differences observed between the clones stably re-expressing the MPRs and the parental MPR-negative fibroblasts provides a direct measurement for the contribution of the MPRs in AP-1 binding. The parameters of the AP-1 binding reaction were calculated after linearization of these curves according to the Scatchard method (Fig. 2B). As shown in Table 1, both the CD-MPR and the Man-6-P/IGF II receptor contribute almost identically for high affinity binding of AP-1 onto membranes (K(d) approx 40 and 25 nM, respectively). This determination was performed on several clones expressing different levels of CD-MPR. As shown in Table 1, the K(d) values measured were nearly identical, indicating that this parameter is, as expected, independent of the level of CD-MPR expression. We conclude from these results that the CD-MPR and the Man-6-P/IGF II receptor contribute almost identically in providing the high affinity binding sites for AP-1. Thus, it appears that the two MPRs are sorted along the AP-1-dependent pathway in a similar manner.


Figure 2: AP-1 binding in MPR-negative fibroblasts re-expressing the MPRs. A, mock-transfected MPR-negative fibroblasts (bullet), MPR-negative fibroblasts re-expressing the wild type CD-MPR (box), or the wild type Man-6-P/IGF II receptor () were permeabilized with streptolysin O and incubated with increasing amounts of cytosol in the presence of GTPS. The membrane bound AP-1 was then quantitated as described under ``Materials and Methods.'' The values represent the means ± S.E. of at least two experiments performed in duplicate. B, after subtraction of the contribution of the MPR-negative fibroblasts to AP-1 binding, the concentration curves representing the contribution of the CD-MPR or the Man-6-P/IGF II receptor to AP-1 binding were linearized according to the Scatchard methods (coefficients of correlation r > 0.99). The calculated K values of the high affinity AP-1 binding sites are summarized in Table 1.





The intracellular transport of cathepsin D was also monitored using typical pulse-chase experiments as shown in Fig. 3. Typically, fibroblasts devoid of the MPRs secrete approx75% of their newly synthesized cathepsin D while control fibroblasts expressing the two MPRs secrete only a minor (approx18%) proportion (Table 2). This corroborates our previous observations using primary cultures of mouse embryonic fibroblasts (Ludwig et al., 1994). The expression of the CD-MPR in the MPR-negative fibroblasts partially corrected the secretion of cathepsin D and the efficiency of cathepsin D sorting correlated with the level of expression of the CD-MPR (Table 2). Typically, cells expressing physiological levels of CD-MPR secreted approx50% of the newly synthesized cathepsin D. However, cells exhibiting higher levels of CD-MPR expression always secreted more of this lysosomal enzyme than do control fibroblasts expressing the two MPRs. The fraction of cathepsin D that was still secreted by these cells reflects the absence of the other Man-6-P/IGF II receptor (Ludwig et al., 1994). (^2)The transport of cathepsin D was intracellular since the addition of Man-6-P in the medium, which displaces ligands bound to the cell surface receptor, remained without any effect. Altogether, these results show that there is a direct correlation between the CD-MPR expression, high affinity binding of AP-1, and efficient intracellular transport of lysosomal enzymes.


Figure 3: Cathepsin D sorting in MPR-negative fibroblasts re-expressing various CD-MPR mutants. The cells were labeled for 1 h with [S]methionine and then chased for 4 h as described under ``Materials and Methods.'' Cathepsin D was immunoprecipitated from the medium (M) or from the cell lysates (C) and resolved by SDS-PAGE followed by autoradiography. The positions of the unprocessed procathepsin D (P) and the mature cathepsin D (M) are indicated. This figure shows typical immunoprecipitations obtained with immortalized mouse fibroblasts (normal fibroblasts) expressing both MPRs, mock-transfected MPR-negative fibroblasts (SFFV, vector alone), and MPR-negative fibroblasts re-expressing the wild type CD-MPR (clone 4) or mutated on its cytoplasmic domain (mutant *51, clone 14; mutant *63, clone 16; mutant A565859, clone 7. The clone numbers refer to individual colonies isolated after transfection). The quantitation of these experiments are shown in Table 2.





AP-1 Binding and Cathepsin D Sorting in Fibroblasts Expressing a CD-MPR Impaired in Endocytosis

The AP-1 Golgi-specific and the AP-2 plasma membrane-specific assembly proteins are two related heterotetrameric complexes which could function in a similar manner (Keen, 1990; Pearse and Robinson, 1990; Robinson, 1994). It has also been shown that the cytoplasmic domain of the Man-6-P/IGF II receptor contains two signals that are required for efficient lysosomal enzyme sorting in the Golgi: the tyrosine-based motif that also functions as internalization signal and the di-leucine based motif at the carboxyl terminus of the cytoplasmic tail (Johnson and Kornfeld, 1992b). Thus, it was of interest to investigate the possible contribution of endocytosis signals in the recruitment of AP-1 onto membranes. Previous in vivo re-expression studies in mouse L-cells have shown that the critical amino acids responsible for most of the endocytosis of the CD-MPR are Phe, Phe^18 in the sequence FPHLAF, and Tyr in the sequence YRGV contained in its 67-amino acid-long cytoplasmic domain (according to the numbering of Johnson and Kornfeld(1990)). A CD-MPR in which these amino acids are replaced by alanines is clearly impaired in endocytosis and tends to accumulate at the cell surface. Therefore, these critical amino acids in the CD-MPR tail were replaced by alanine residues (mutant E, Fig. 1), the CD-MPR mutant was stably re-expressed in MPR-negative fibroblasts and the parameters of the AP-1 binding reaction determined (Fig. 4A). Table 1shows that AP-1 bound to membranes of cells expressing this mutant in a similar fashion as to membranes of cells expressing the wild type CD-MPR. In both case, the K(d) was around 40 nM. Conversely, this CD-MPR mutant appeared almost normal in lysosomal enzyme transport since rather low levels of expression could clearly correct the secretion phenotype of MPR-negative fibroblasts, as efficiently as the wild type CD-MPR (Table 2). We conclude from these results that the protein motifs which determine the endocytosis of the CD-MPR do not significantly contribute by themselves to AP-1 binding and proper targeting of lysosomal enzymes.


Figure 4: AP-1 binding in MPR-negative fibroblasts expressing various CD-MPR mutants. The MPR-negative fibroblasts re-expressing the various CD-MPR mutants were permeabilized with streptolysin O and incubated with increasing amounts of cytosol in the presence of GTPS. The contribution of the MPR-negative fibroblasts to AP-1 binding was subtracted from the obtained values in order to determine the contribution of each CD-MPR mutant in AP-1 binding. The curves were linearized according to the Scatchard method. A, mutant E impaired in endocytosis (coefficient of correlation r = 0.97); B, mutant *63 lacking a di-leucine motif (coefficient of correlation r = 0.99); C, mutant *51 lacking the carboxyl-terminal domain; D, mutant A565859 (coefficient of correlation r = 0.99); E, mutant D60 (coefficient of correlation r = 0.99); F, mutant A57D60 (coefficient of correlation r = 0.96). The K values of high affinity AP-1 binding sites are summarized in Table 1.



The Carboxyl-terminal Di-leucine Motif Is Not Essential for AP-1 Binding but Is Necessary for Cathepsin D Sorting

Earlier studies have clearly illustrated that the carboxyl-terminal domain of the CD-MPR is important for efficient transport of lysosomal enzymes by this receptor (Johnson and Kornfeld, 1992a). The detailed mutational analysis of these latter studies led to the conclusion that a di-leucine-based motif contained in the sequence HLLPM acts as the dominant determinant for lysosomal enzyme sorting in the Golgi. Thus, we investigated the potential implication of this di-leucine-based motif in AP-1 binding. A CD-MPR mutant truncated from the 5 COOH-terminal amino acids which contains this di-leucine motif was stably re-expressed in MPR-negative fibroblasts (mutant *63, Fig. 1) and the parameters of the AP-1 binding reaction determined (Fig. 4B). Table 1shows that the deletion of this sequence in the CD-MPR tail does not significantly affect the high affinity of AP-1 for membranes. Typically, AP-1 bound to membranes of cells expressing this mutants with a K(d) approx 42 nM, a value not significantly different from that measured with cells re-expressing the wild type CD-MPR. As reported previously for this mutant expressed in mouse L-cells lacking the Man-6-P/IGF II receptor (Johnson and Kornfeld, 1992a), this mutant expressed in MPR-negative fibroblasts was found to be completely impaired in proper targeting of cathepsin D to lysosomes (Table 2). MPR-negative cells expressing such a mutant secreted cathepsin D in a similar manner as the mock-transfected MPR-negative cells. This secretion was independent of the presence of Man-6-P in the culture medium. This contrasts with what has been observed with mouse L-cells expressing similar mutants of the Man-6-P/IGF II receptor (Johnson and Kornfeld, 1992b; Chen et al., 1993). Those cells secreted cathepsin D only when Man-6-P was added to the culture medium. This suggests that the spontaneous release of the ligand from the CD-MPR is faster than its internalization, a notion consistent with the fact that the CD-MPR does not bind ligand at the cell surface (Stein et al., 1987). We conclude from these different results that the carboxyl-terminal domain of the CD-MPR containing the di-leucine motif is not by itself essential for the high affinity interaction of AP-1 with its target membranes although it is critical for proper targeting of lysosomal enzymes. Therefore, this di-leucine motif must be required for a post AP-1 binding event.

The Carboxyl-terminal Casein Kinase II Phosphorylation Site Is Essential for Both AP-1 Binding and Cathepsin D Targeting

The results described above indicate that mutations introduced in the tyrosine-based or the di-leucine-based motifs in the CD-MPR cytoplasmic domain are not sufficient to modify the affinity of AP-1 for membranes. In order to decipher the protein motif of the CD-MPR tail which contributes for the high affinity interaction of AP-1 with its membranes, a larger truncation was first introduced in its cytoplasmic domain. The 17 COOH-terminal amino acids containing both the casein kinase II phosphorylation site and the di-leucine motif were deleted from the CD-MPR (mutant *51, Fig. 1), and this mutant was stably re-expressed in MPR-negative fibroblasts. With respect to AP-1 binding, the membranes of these cells behaved as the mock-transfected MPR-negative cells (Fig. 4C). However, it was not clear whether the lack of AP-1 binding sites was due to the truncation per se or to the low stability of this particular CD-MPR mutant. Indeed, as shown in Table 1, all the selected clones contained only small amounts of the mature CD-MPR mutant at steady state (determined by an overnight labeling followed by immunoprecipitation). However, they all exhibited a 2-3-fold higher level of synthesis (determined by a 30-min pulse with [S]methionine followed by immunoprecipitation) of this CD-MPR mutant when compared with control cells expressing the two wild type MPRs. This result indicated that this particular CD-MPR mutant was probably rapidly degraded.

The low stability of the CD-MPR mutant with a large carboxyl-terminal truncation prompted us to introduce several point mutations in the casein kinase II phosphorylation site (Fig. 1), a protein motif also found twice in the cytoplasmic domain of the Man-6-P/IGF II receptor and highly conserved among species (Lobel et al., 1988; Morgan et al., 1987; Oshima et al., 1988; MacDonald et al., 1988). These mutants were re-expressed in MPR-negative cells and clones showing reasonable steady state levels of the CD-MPR mutants were isolated. Indeed, none of these point mutations affected the stability of the protein (not shown). We first replaced three negatively charged amino acids surrounding the serine at position 57 by alanine residues (sequence ESEER to ASAAR) in order to neutralize this phosphorylation site (mutant A565859; Fig. 1). When tested in vitro for the interaction of AP-1 (Fig. 4D), the membranes of the cells expressing this mutant were found to be impaired in AP-1 binding (Table 1). Typically, AP-1 bound to the membranes of these permeabilized cells with a K(d) 115 nM, an affinity approx3-fold lower when compared with membranes of cells expressing the wild type CD-MPR. Since the cytosolic concentration of AP-1 is approx200 nM (Le Borgne et al., 1996), it could be expected that this CD-MPR mutant is not efficiently segregated into AP-1-coated vesicles. Indeed, this CD-MPR mutant was impaired in transport of cathepsin D to lysosomes, since relatively high amounts of this mutant did not allow the transport of cathepsin D as efficiently as the wild type CD-MPR. The sorting capacity of this mutant was reduced by 65%. We then replaced the positively charged arginine at position +3 of the serine 57 in the CD-MPR tail by a aspartic acid, a negatively charged residue (mutant D60, Fig. 1). This mutation would render this sequence as a typical casein kinase II phosphorylation site, very similar to those found in the Man-6-P/IGF II receptor cytoplasmic domain. AP-1 bound to membranes of cells expressing such a mutant (Fig. 4E, Table 1) with a higher affinity (K(d) approx 20 nM) when compared with cells expressing the wild type CD-MPR (K(d) approx 40 nM). This K(d) value was very similar to that determined for cells expressing the wild type Man-6-P/IGF II receptor (K(d) approx 25 nM). As expected from the affinity of AP-1, this CD-MPR mutant transported cathepsin D as efficiently as the wild type CD-MPR provided that similar levels of expression were compared (Table 2). An additional mutation was further introduced in this CD-MPR mutant and the serine residue at position 57 was mutated into an alanine residue (mutant A57D60, Fig. 1). This mutation also affected the affinity of AP-1 for the membranes (Fig. 4F, Table 1). Indeed, AP-1 bound to membranes with a K(d)approx85 nM, a value four times lower when compared with the previous mutant (mutant D60). However, the cells expressing this mutant secreted similar amounts of cathepsin D as cells expressing similar amounts of wild type CD-MPR (Table 2), indicating that transport of cathepsin D was not affected. This result suggests that a minimum affinity of AP-1 for membranes is required for efficient packaging of the CD-MPR in transport vesicles (see ``Discussion''). Collectively, these results demonstrate that the casein kinase II phosphorylation site in the CD-MPR cytoplasmic domain acts as a critical determinant for both high affinity interaction of AP-1 for its target membranes and efficient transport of lysosomal enzymes.


DISCUSSION

The translocation of the cytosolic, AP-1 Golgi assembly proteins onto TGN membranes is the first step in the formation of TGN-derived vesicles in which membrane proteins destined to endosomes are segregated. Several in vitro studies have illustrated the functional importance of the GTPase ARF-1 in this process (Stamnes and Rothman, 1993; Traub et al., 1993). The MPRs also contribute to the high affinity interactions of AP-1 with its target membranes as shown by the re-expression of MPRs in MPR-negative fibroblasts (Le Borgne et al., 1993, 1996). We have made use of this observation to decipher the signal in the CD-MPR cytoplasmic domain that is critical for these interactions. We show here that the high affinity interaction of AP-1 with membranes, as the efficient targeting of cathepsin D, requires a casein kinase II phosphorylation site at the carboxyl terminus of the CD-MPR cytoplasmic domain. Surprisingly, the adjacent di-leucine based motif, also required for efficient targeting of cathepsin D, is not critical for these high affinity interactions. Thus, the CD-MPR-mediated transport of lysosomal enzymes requires two essential determinants, possibly involved in two different types of molecular interactions.

Di-leucine Motifs and CD-MPR Trafficking

Since the first study of Letourneur and Klausner(1992) showing that a di-leucine and a tyrosine-based motifs independently mediate lysosomal targeting and endocytosis of CD3 chains, several other studies have also illustrated the functional importance of di-leucine motifs in the delivery of membrane proteins to endocytic compartments, namely endosomes and lysosomes (for review, see Sandoval and Bakke(1994)). Simple mutations introduced in the di-leucine based motifs typically result in a drastic mislocalization of those proteins which, at steady state, are now predominantly found at the plasma membrane. Extensive mutational analyses have demonstrated that the efficient delivery of cathepsin D to endocytic compartments also relies on the presence of di-leucine based motifs in the cytoplasmic domains of both the Man-6-P/IGF II receptor (Johnson and Kornfeld, 1992b; Chen et al., 1993) and the CD-MPR (Johnson and Kornfeld, 1992a). It has been proposed from these results that di-leucine-containing sequences represent the motif which mediates the interaction of the MPRs with the Golgi assembly proteins and therefore their sorting in the TGN. Our in vivo studies of cathepsin D sorting are in good agreement with those previously reported by Johnson and Kornfeld (1992a) showing the importance of the di-leucine motif for the CD-MPR-mediated delivery of this lysosomal enzyme. It is clear from our in vitro AP-1 binding studies that this motif in the CD-MPR cytoplasmic domain is not by itself essential for the interaction of AP-1 with its target membranes, since its removal does not significantly affect AP-1 binding. However, this di-leucine motif must be required for another event important for the sorting function of the CD-MPR that occurs subsequently to AP-1 binding. It is difficult from our data to pinpoint this event in the CD-MPR recycling pathway. It is unlikely that it affects the quaternary structure of the molecule which might be critical for ligand binding (Waheed et al., 1990; Waheed and von Figura, 1990), since a similar mutation also affects the sorting function of the Man-6-P/IGF II receptor (Johnson and Kornfeld, 1992b), which has, however, a different quaternary structure. Several hypotheses could be envisaged. First, it is possible that di-leucine motifs could be involved in the late stages of vesicle formation or targeting and fusion of the vesicles with endosomes. According to this view, the mutation of the di-leucine motif would lead to the accumulation of the CD-MPR in the TGN or in transport vesicles without modifying AP-1 binding. This would result in the secretion of lysosomal enzymes via the constitutive pathway. However, localization studies performed at the fluorescence level do not favor this hypothesis. This CD-MPR mutant and the bound AP-1 exhibit the same distribution as in cells expressing the wild type CD-MPR (not shown). A second hypothesis is that the di-leucine motif controls the proper routing of the CD-MPR in the endosomal system. If a CD-MPR lacking its di-leucine motif were correctly packaged into Golgi-derived vesicles, which would then fuse with endosomes, how could this receptor mutant discharge its ligand in the extracellular medium? The precise routes taken by the MPRs at the exit of the TGN have not been clearly elucidated (Kornfeld and Mellman, 1989; Hoflack and Lobel, 1993). It is possible that the CD-MPR is first delivered to an endosome where the bound ligands do not completely dissociate. An early endosome could provide such conditions. From there, a CD-MPR lacking its di-leucine-based motif could escape more efficiently toward the cell surface where it would release its bound ligands. According to this scheme, the role of di-leucine motifs would be to prevent the access of the CD-MPR to the cell surface, an important feature for efficient delivery of lysosomal enzymes to lysosomes. However, the removal of the di-leucine motif from the CD-MPR tail does not drastically affect its recycling from the plasma membrane back to the TGN (Johnson and Kornfeld, 1992a). Clearly, additional work will be required to understand which aspect of the CD-MPR cycling pathway relies on the presence of this particular di-leucine motif.

CD-MPR Sorting in the TGN

Our in vitro AP-1 binding study strongly suggests that the entire casein kinase II phosphorylation site in the CD-MPR cytoplasmic domain is the critical motif that determines the interaction of AP-1 with membranes. Any mutation in this site affects the affinity of AP-1 for membranes. It is unlikely that these mutations affect the quaternary structure of the CD-MPR, since the substitution of an arginine by an aspartic residue (mutant D60) rather results in a 2-fold increased affinity of AP-1 for membranes and the substitution of a serine by an alanine (mutant A57D60) significantly reduces the affinity of AP-1 for membranes without drastically altering transport of cathepsin D. It is well accepted that most of the AP-1 is bound to the TGN membrane. Since the specificity of interaction is preserved in the in vitro assay used in this study (Le Borgne et al., 1996), our results argue that this casein kinase II phosphorylation site functions as a dominant TGN sorting determinant for efficient packaging of the CD-MPR in Golgi-derived vesicles. It is worth noting that the mutations which decreases the affinity of AP-1 for membranes have striking different effects on cathepsin D sorting. While the serine mutation (mutant A57D60) does not affect the targeting of this lysosomal enzyme, the mutation of the surrounding charged amino acids (mutant A565859) impairs its sorting almost completely. Since AP-1 binds to membranes of cells expressing these mutants with a K(d) approx 85 nM and a K(d) approx 115 nM, respectively, the simplest interpretation suggests that a minimal affinity of AP-1 for membranes is required for an efficient sorting function of the CD-MPR, the threshold being above 100 nM.

Similar CD-MPR mutants have been overexpressed in the background of the endogenous CD-MPR of mouse L-cells lacking the Man-6-P/IGF II receptor (Johnson and Kornfeld, 1992a). These authors could not detect an effect of these mutations on cathepsin D-sorting and, thus, concluded that the casein kinase II phosphorylation site is not important for CD-MPR sorting function. The reasons for these discrepancies are unclear at present. It is possible that they are due to the two different cell systems used. It is worth mentioning that the mutational analyses performed by these authors on the two casein kinase II phosphorylation sites of the Man-6-P/IGF II receptor could not detect their potential implication in cathepsin D targeting (Johnson and Kornfeld, 1992b). More recently, Chen et al.(1993) could demonstrate, however, the functional importance of the carboxyl-terminal casein kinase II phosphorylation site in the sorting function of this MPR using an in vivo lysosomal enzyme sorting assay. In the light of our results, it seems possible that this casein kinase II phosphorylation site of the Man-6-P/IGF II receptor could, as that of the CD-MPR, determine the high affinity binding of AP-1 and control the sorting of this MPR in the TGN.

The casein kinase site in the CD-MPR cytoplasmic domain may also be important in other aspects of the CD-MPR trafficking. If the deletion of a di-leucine motif or point mutations in the casein kinase II phosphorylation site do not affect the stability of the CD-MPR, a larger truncation removing the entire casein kinase II phosphorylation site reduces its half-life drastically (data not shown). The high turnover of this mutant (10 times faster than that of the wild type CD-MPR) is consistent with preliminary localization studies showing that, in contrast to the wild type CD-MPR, this mutant can be detected in lysosomes defined as Lamp-1-positive structures. This would suggest that this mutant with a large carboxyl-terminal truncation is also impaired in recycling back to the TGN and is transported to lysosomes by default. Thus, the casein kinase II phosphorylation site might also be an important feature controlling the recycling of the CD-MPR from endosomes in order to prevent its access to degradative compartments.

Function of the Casein Kinase II Site in CD-MPR Trafficking

Our study demonstrates that the casein kinase II phosphorylation site in the CD-MPR cytoplasmic domain determines the high affinity of AP-1 for membranes and that mutations introduced independently in the tyrosine-based or the di-leucine-based motifs are not sufficient to modify these interactions. However, it remains to determine whether this casein kinase II phosphorylation site functions directly in interactions with AP-1 or indirectly by uncovering another protein motif in the CD-MPR cytoplasmic domain which would then become accessible for interactions with AP-1. We have shown earlier that the casein kinase II phosphorylation sites of the Man-6-P/IGF II receptor cytoplasmic domain are phosphorylated when this receptor is present in, or leaves, the TGN (Méresse and Hoflack, 1993). Although a similar in vivo study has not been performed on the CD-MPR, this later is also found phosphorylated in vivo on the serine contained in its casein kinase II phosphorylation site (Hemer et al., 1993). It is very likely that the phosphorylation of both the Man-6-P/IGF II receptor and the CD-MPR takes place at the same time and involves the same machinery. Thus far, the functional relevance of this post-translational modification in MPR trafficking has remained unclear. A first possibility is that the phosphorylation of the casein kinase site in the CD-MPR cytoplasmic domain could increase the affinity of AP-1 for membranes by providing additional negative charges if this stretch of negatively charged amino acids were found to be directly involved in the interaction of AP-1 with membranes. A second possibility is that the phosphorylation of the casein kinase II site could change the conformation of the CD-MPR cytoplasmic domain and regulate the accessibility of additional sorting signals and their subsequent interactions with the AP-1-dependent sorting machinery. According to this view, phosphorylation could act as a switch to turn on sorting signals and regulate MPR sorting in the TGN. Our study does not preclude the possibility that the tyrosine-based and the di-leucine-based motifs in the CD-MPR tail are implicated in AP-1 binding. Our mutational analysis neutralizing one motif or the other could have underscored their contribution if the tyrosine-based and the di-leucine-based motifs in the CD-MPR cytoplasmic domain would contribute equally and independently to AP-1 binding onto membranes or if they would function in combination with another unknown protein motif of the CD-MPR tail important for AP-1 binding. It will be important to investigate whether each motif, taken individually, is able to determine the recruitment of AP-1 onto membranes.


FOOTNOTES

*
Part of this research was supported by the Association ``Vaincre les Maladies Lysosomales'' and the European Communities (Grant B102-CT93-02205). 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.

§
From the CNRS on leave from URA 1815.

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

(^1)
The abbreviations used are: MPR, mannose 6-phosphate receptor; CD-MPR, cation-dependent mannose 6-phosphate receptor; TGN, trans-Golgi network; IGF, insulin-like growth factor; MEM, minimum Eagle's medium; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; GTPS, guanosine 5`-O-(3-thiotriphosphate).

(^2)
H. Munier-Lehmann, F. Mauxion, U. Bauer, P. Lobel, and B. Hoflack, manuscript in preparation.


ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. E. Ungewickell for the generous gift of the mAb 100/3 monoclonal antibody used in this study. We also thank Dr. G. Griffiths, K. Simons, and M. Zerial for critical reading of the manuscript. We are indebted to Dr. P. Lobel for helpful discussions and communication of unpublished results.


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