(Received for publication, August 25, 1995; and in revised form, November 14, 1995)
From the
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.
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 40 soluble hydrolases is recognized in the late
secretory pathway by the mannose 6-phosphate receptors (MPRs), (
)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.
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).
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.
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 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).
Figure 2:
AP-1
binding in MPR-negative fibroblasts re-expressing the MPRs. A,
mock-transfected MPR-negative fibroblasts (), MPR-negative
fibroblasts re-expressing the wild type CD-MPR (
), 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 GTP
S. 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
75% of their newly synthesized cathepsin D while control
fibroblasts expressing the two MPRs secrete only a minor (
18%)
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
50% 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). (
)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.
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 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 115 nM, an affinity
3-fold
lower when compared with membranes of cells expressing the wild type
CD-MPR. Since the cytosolic concentration of AP-1 is
200 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
20 nM) when compared with cells
expressing the wild type CD-MPR (K
40
nM). This K
value was very similar to
that determined for cells expressing the wild type Man-6-P/IGF II
receptor (K
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
85 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.
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.
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.