From the Institute of Physiology, University of Zurich, Zurich 8057, Switzerland
Received for publication, January 8, 2003 , and in revised form, April 14, 2003.
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ABSTRACT |
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INTRODUCTION |
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The cation-dependent mannose 6-phosphate receptor (CD-MPR) is a type I integral membrane protein with a C-terminal cytoplasmic tail of 67 residues. Within this cytoplasmic tail, several signals have been identified that direct the intracellular trafficking of the receptor. Internalization from the plasma membrane is mediated by 3 independent signals, a pair of phenylalanine residues (Phe13-X-X-X-X-Phe18), a classical tyrosine motif (Tyr45-X-X-Val48), and as indicated by in vitro data by a di-leucine based motif (Leu64-Leu65) (5, 6). The di-leucine motif has been shown in addition to mediate the transport of the CD-MPR from the TGN to the endosomes by interaction with Golgi-localized, -ear-containing, adenosine diphosphate ribosylation factor-binding protein 1 and adaptor protein complex 1 (AP1) and possibly also a sorting event within endosomes (7, 8). An acidic cluster as part of a casein kinase II site was implicated to be important for sorting in endosomes (9). Furthermore, overlapping with one of the internalization motifs (Phe13-X-X-X-X-Phe18) is a di-aromatic motif (Phe18-Trp19), which is required for the sorting of the receptor from late endosomes back to the TGN and therefore preventing degradation of the receptor in lysosomes (10). The sorting efficiency of the di-aromatic motif seems to be regulated by a post-translational modification, the reversible palmitoylation of Cys34 (10, 11).
Sorting of the receptor requires the interaction of the signals in the cytoplasmic tail with components of the cytoplasmic sorting machinery at sites of vesicle formation. Two such factors are the clathrin-associated adaptor complexes AP1 and adaptor protein complex 2 (AP2). AP1 mediates the sorting step from the TGN to endosomes and in addition recent data suggest that AP1 might also play a role in recycling MPRs from early endosomes to the TGN (12), whereas AP2 is required for rapid internalization of the receptor from the plasma membrane (13). Another protein, phosphofurin acidic cluster sorting protein 1 (PACS-1), was shown to interact with the receptor tail and AP1 in vitro suggesting that PACS-1 and AP1 may cooperate to sort MPRs (14). Most recently, the novel family of Golgi-localized, -ear-containing, adenosine diphosphate ribosylation factor-binding proteins (GGA) have been shown to mediate the sorting of MPRs from the TGN to endosomes (15, 16). Yet another sorting event, from late endosomes back to the TGN, is mediated by tail interacting protein of 47 kDa (Tip47), which interacts with the di-aromatic motif (Phe18-Trp19) of the CD-MPR (17). Optimal Tip47 function depends on binding to Rab9, a late endosomal GTPase previously implicated in MPR retrieval, further confirming the role of Tip47 in this particular sorting event (18). Interestingly Tip47 binds to both MPRs, the CD-MPR and the cation-independent mannose 6-phosphate receptor (CI-MPR) but the CI-MPR does not contain a di-aromatic motif. A mutational analysis revealed that Tip47 seems to bind a putative loop generated by the sequence Pro49-Pro-Ala-Pro-Arg-Pro-Gly55 and other hydrophobic residues in the cytoplasmic tail of the CI-MPR (19). This raises the question of whether the binding of Tip47 to the CD-MPR is restricted to a di-aromatic motif or if other residues could replace the aromatic residues.
In this study we have analyzed the effect of mutations that change the key residue of the di-aromatic motif (Trp19) to bulky hydrophobic residues, non-polar residues, polar residues, or charged residues. Initial screening for the intracellular localization of CD-MPR constructs containing those mutations by immunofluorescence revealed some differences in the extent of co-localization with Lamp1, a lysosomal marker. To obtain a more quantitative measure for the effect of various mutations we tested their ability to interact with N-terminal His-tagged Tip47 in an in vitro pull-down assay. Constructs with Trp19 replaced by hydrophobic residues such as leucine and valine exhibited a modest impairment of binding to Tip47. However, correct endosomal sorting of the full-length CD-MPR in vivo was only achieved by wild-type receptors whereas the mutants showed an increased missorting to lysosomes by a factor of 5 for replacements of Trp19 by hydrophobic residues and up to a factor of 12 for replacements by charged residues.
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EXPERIMENTAL PROCEDURES |
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Recombinant DNAAll basic DNA procedures were as described (20). The PCR procedure of Ho et al. (21) was used to generate the mutant constructs with pBSK-MPR TMD/tail (22) serving as a template together with bp 170193 and 12601341 of pBSK as the down- and upstream primers, respectively. Appropriate partial complementary pairs of oligonucleotides in which the desired amino acid replacement had been incorporated were chosen as internal primers. The final PCR products were subcloned into pSFFV-neo as described (22). Human Tip47 was cloned by PCR from a Marathon Human Placental cDNA library (Clontech, Palo Alto, CA) using Tip47.down (5'-TTTGAATTCGCGGCCGCTGTTCCTGGGACG-3') and Tip47.up (5'-GAGACGCGTCCCGCTGAGTCCTCTCCTCTCCCC-3') oligos into the pcDNA3.1(+) vector (Invitrogen). His6-Tip47 was created using this pcDNA3.1(+)-Tip47 vector as a template for a PCR with the oligos BamHIpQE31-TIP47.-down (5'-CACGGATCCATGTCTGCCGAC-3') and SacIpQE31-TIP47.up (5'-GCGGAGCTCCCCCTATTTCTTCTCCTCCGGGGC-3'). The PCR product was cut with BamHI and SacI and ligated into pQE31 (Qiagen AG, Basel). All coding sequences created by PCR were verified by sequencing.
Cell Culture and TransfectionHeLa cells were grown in -minimal essential medium to 30% confluency before transfection with 1 µg of DNA using FuGENE according to the manufacturer's directions. The cells were incubated with 100 µM each of pepstatin A and leupeptin 24 h before the experiment and analyzed 48 h post-transfection by confocal microscopy.
A mannose-6-P/insulin-like growth factor-II receptor-deficient mouse L cell line designated D9 (LRec) was maintained in -minimal essential medium. The cells were transfected with XbaI-linearized DNA with Lipofectin according to the manufacturer's directions. Selection for resistance to neomycin (G418) was carried out using 500 µg/ml G418 as the final concentration. Resistant colonies were picked individually and screened for the expression of bovine CD-MPR by immunoblotting. Clones expressing similar amounts of receptor were expanded for further study and maintained in selective medium.
Confocal Immunofluorescence Microscopy and DeconvolutionConfocal immunofluorescence microscopy was carried out as described (23). Paraformaldehyde-fixed and saponin-permeabilized cells were incubated with a 1:200 dilution of monoclonal antibody 22D4 and a 1:400 dilution of Ab 931A. The secondary antibodies were goat anti-rabbit Alexa 488 antibodies and goat anti-mouse Alexa 568 conjugates, diluted 1:1000. The coverslips were mounted on glass slides with ProLong Antifade (Molecular Probes) for viewing with an Olympus Fluoview FV500 confocal laser-scanning microscope. Serial sections in the z axis through the entire cells were taken, and the resulting stacks of images were processed on a multiprocessor SGI UNIX computer using the Huygens program (Scientific Volume Imaging, Hilversum, The Netherlands). A maximum likelihood estimation-based algorithm was used for image reconstruction. Z-stacks were exported as Tiff files and analyzed with the use of the Imaris program (Bitplane AG, Zürich, Switzerland).
In Vitro Pull-down ExperimentsN-terminal His-tagged Tip47 was expressed in Escherichia coli strain M15. A saturated overnight culture was diluted 1:10 in 200 ml of growth medium and incubated for 1 h at 37 °C before induction with 1.0 mM isopropyl-1-thio--D-galactopyranoside for 2 h. The cells were then harvested by centrifugation, washed with ice-cold phosphate-buffered saline, and lysed by sonication in 20 ml of binding buffer (20 mM NaH2PO4·H2O, pH 7.8, 500 mM NaCl) containing a 1:500 dilution of a protease inhibitor mixture (5 mg/ml benzamidine, and 1 µg/ml each of pepstatin A, leupeptin, antipain, and chymostatin in 40% dimethyl sulfoxide, 60% ethanol) (PIC) and phenylmethylsulfonyl fluoride (40 µg/ml) (final concentration). Insoluble material was removed by centrifugation at 12,000 rpm for 10 min at 4 °C in a Sorvall GSA centrifuge. 4 ml of the supernatant was incubated for 30 min at room temperature on a rotating shaker with 400 µl of Ni-CAM HC resin (50% suspension) (Sigma) that was prewashed with binding buffer and blocked with binding buffer containing 0.1% bovine serum albumin. The beads were washed 2 times with wash buffer (binding buffer at pH 6.0) and once with assay buffer (50 mM HEPES, 150 mM KCl, 8 mM imidazole, 1 mM MgCl2, pH 7.5) by spinning them at 1000 rpm for 1 min in a Heraeus centrifuge. Extracts from mouse D9 (LRec) cells expressing wild-type or mutant CD-MPR constructs were prepared from cells grown on 15-cm Falcon tissue culture dishes in the following way. Confluent 15-cm dishes of cells were put on ice and washed once with 10 ml of ice-cold phosphate-buffered saline and scraped in 5 ml of assay buffer containing PIC and phenylmethylsulfonyl fluoride. The cells were pelleted at 1000 rpm for 5 min at 4 °C in a Heraeus centrifuge and resuspended in 1 ml of assay buffer containing PIC and phenylmethylsulfonyl fluoride. The cells were homogenized in a ball bearing homogenizer of 51.2 µm clearance using 12 strokes and spun at 700 x g at 4 °C for 10 min. The supernatant was collected and centrifugation was repeated until no pellet appeared. The protein concentration of the post-nuclear supernatant (PNS) was measured using a Bio-Rad protein assay. The appropriate amounts of PNS from each of the mutant cell lines and wild-type cell line expressing the same amount of CD-MPR were determined by Western blotting with anti-CD-MPR monoclonal antibody 22D4 followed by densitometric scanning and quantification using ImageQuant 5.0 software (Amersham Biosciences). These PNS amounts adjusted for expression levels were used for the assay.
Beads incubated with His6-Tip47 were resuspended in 200 µl of assay buffer containing PIC and phenylmethylsulfonyl fluoride. 50 µlof resuspended beads were incubated with 200 µg of PNS from the wild-type cell line or an appropriate amount of PNS from mutant cell lines according to the expression level adjustment described above for 2 h at 4 °C on a rotating shaker. Beads were spun at 2500 rpm for 4 min at room temperature in an Eppendorf tabletop centrifuge. The supernatant was collected and stored. The beads were washed three times with assay buffer. Sample buffer was added to the beads to a total volume of 40 µl, boiled, and analyzed by SDS-PAGE and Western blotting using anti-CD-MPR monoclonal antibody 22D4. An aliquot of each of the supernatants was also analyzed similarly to determine the amount of unbound receptor. The amount of CD-MPR bound to His6-Tip47 was determined by densitometric scanning using ImageQuant 5.0 software.
Percoll Gradient FractionationConfluent D9 (LRec) cells stably expressing wild-type or mutant forms of the CD-MPR were grown in a 100-mm Petri dish and incubated for 24 h in growth medium supplemented with 100 µM each of pepstatin A and leupeptin. The cells were then harvested, ruptured with a ball bearing homogenizer, and fractionated on 17.5% Percoll density gradients as described previously (22). The specificity of the lysosomal fractions was tested using a -hexosaminidase assay as described previously (23) (data not shown).
SDS-PAGE and ImmunoblottingProteins were separated on 10% SDS-polyacrylamide minigels (Bio-Rad) by using the Laemmli system (24). After electrophoresis gels were transferred onto nitrocellulose membranes according to the method of Towbin et al. (25). The immunoblotting was performed as previously described (22). The autoradiographs were quantitated by using a personal densitometer (Amersham Biosciences).
Assays and Miscellaneous Methods-Hexosaminidase activity was determined as described (22). Protein concentration was determined with the Bio-Rad protein assay kit by using protein standard I.
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RESULTS |
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These results demonstrate that the mutant receptors are not correctly sorted from the late endosomes back to the TGN and therefore accumulate in lysosomes. It also seems that the extent of missorting to lysosomes depends on the properties of the amino acid replacing Trp19 but this has to be characterized in more detail using a biochemical assay.
Hydrophobic Residues Poorly Replace Aromatic Residues at Position 19 in the CD-MPR Cytoplasmic Tail to Bind Tip47 in VitroThe immunofluorescence experiments suggested that the physical properties of the amino acid replacing Trp19 determines the sorting efficiency of the CD-MPR mutants in late endosomes. Tip47 was shown to interact specifically with the di-aromatic motif of the MPRs and to mediate the transport from endosomes back to the TGN (17). Therefore we wanted to test the interaction of various CD-MPR mutants with Tip47 using an in vitro binding assay. Because of the potential effect of post-translational modifications we used the full-length receptor expressed in mammalian cells in contrast to previously published assays with truncated fusion proteins produced in E. coli. For this purpose we created a series of receptor constructs that consist of the full-length CD-MPR with various mutations of the residue Trp19 (Fig. 2). Besides the replacements of Trp19 with the bulky hydrophobic residues (Val, Ile, and Leu) we also used a less hydrophobic amino acid with a nonpolar side chain (Ala) as well as an amino acid with an uncharged polar side chain (Ser) or charged polar side chains (Arg and Asp). Post-nuclear supernatant from wild-type and mutant receptors expressed in mouse D9 (LRec) cells were incubated with His-tagged Tip47 bound to nickel beads and bound receptor was subsequently detected by SDS-PAGE and Western blotting. The mouse monoclonal antibody 22D4 used to detect the bound CD-MPR does not recognize the endogenous receptor in mouse D9 cells and reacts only with transfected bovine receptor (23). The antibody recognizes two bands of the CD-MPR, the upper one at 67 kDa being a dimeric form of the receptor as shown previously (23). Tip47 bound the wild-type receptor with an affinity greater than all the mutant constructs not containing a di-aromatic motif (Fig. 3A). The di-aromatic constructs MPR-FW-YF and MPR-W19Y bound Tip47 as well as the wild-type receptor. There was a modest decrease in binding of Tip47 to constructs MPR-W19V, MPR-W19I, and MPR-W19L indicating that hydrophobic residues replacing Trp19 do not completely abolish the interaction with Tip47. Binding barely above background was seen when Trp19 was replaced either by alanine (MPR-W19A) or serine (MPR-W19S). Similarly, binding barely above background was detected when charged residues like aspartic acid or arginine were used to replace Trp19. These data indicate that the hydrophobic residues, leucine, isoleucine, and valine alone are partially able to replace Trp19 in an in vitro Tip47 binding assay.
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Replacement of Trp19 by Hydrophobic Residues Prevents Moderately the Delivery of the Mutant Receptors to Lysosomes Because the Tip47 binding assay was performed in vitro with Tip47 in the context of a His-tagged fusion protein isolated from E. coli, an additional assay was performed to analyze the efficiency of mutant constructs to avoid lysosomal delivery in vivo. Mouse L cells stably expressing the wild-type and mutant receptors were preincubated for 24 h in the presence of pepstatin A and leupeptin to inhibit degradation of receptors that had entered lysosomes (22). The cells were then harvested, homogenized, and the lysosomes were separated on 17.5% isoosmotic Percoll density gradients (22, 26). Under these conditions, dense lysosomes are recovered at the bottom of the gradient (pool I), whereas low density membranes including endosomes, Golgi apparatus, plasma membrane, and endoplasmic reticulum are found near the top of the gradient (pool III). Intermediate density membranes are recovered in pool II. The distribution of the receptor in the three pools was determined by SDS-PAGE followed by Western blotting (Fig. 4 and Table I for quantitation of multiple experiments). As reported previously, the wild-type CD-MPR was almost completely excluded from dense lysosomes (5% recovered in pool I) and replacement of the di-aromatic motif (Phe18-Trp19) with other aromatic residues did not affect the distribution significantly (MPR-FF, 5%; MPR-FY, 4%; MPR-YF, 8%; and MPR YW, 3%) (10). Substitution of Trp19 with bulky hydrophobic residues led to an accumulation of the mutant receptors in dense lysosomes to about 30% (MPR-W19V, 27%; MPR-W19I, 27%; and MPR-W19L, 29%). A slightly larger accumulation of mutant receptors in lysosomes was found with the mutants that contained less hydrophobic, nonpolar side chains as replacements for Trp19 (MPR-W19A, 38%; and MPR-W19M, 40%). To our surprise we found that the mutant MPR-W19C with the polar side chain of a cysteine residue accumulated only to about 34% in dense lysosomes. However, according to Kyte and Doolittle (27) the cysteine residue with its polar side chain is more hydrophobic than an alanine or a methionine residue. We next examined the effect of mutating Trp19 to a hydrophilic residue (MPR-W19S) or even charged residues (MPR-W19R and MPR-W19D). All three mutant receptors accumulated more than 50% in dense lysosomes (MPR-W19S, 52%; W19R, 56%; and MPR-W19D, 59%) compatible with the hypothesis that only hydrophobic residues can replace to a small extent an aromatic residue of the di-aromatic motif.
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DISCUSSION |
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To accomplish their biological function, sorting of lysosomal enzymes from the TGN to endosomes, it is essential that the MPRs avoid delivery to lysosomes where they would be degraded. Therefore the sorting step in late endosomes, which is mediated by the di-aromatic motif (10), is crucial and needs to be well characterized. Diaz and Pfeffer (17) first demonstrated that Tip47 interacts with the CD-MPR through the di-aromatic motif and with the CI-MPR that is lacking a di-aromatic motif and subsequently identified the sequence PPAPRP within the cytoplasmic tail of the CI-MPR as part of a complex three-dimensional binding domain for Tip47 (19). This raised the question if the interaction between Tip47 and the CD-MPR is strictly limited to aromatic residues or if other amino acids can replace Trp19, the key residue of the di-aromatic motif.
Using immunofluorescence we found that there is an increasing colocalization of mutant receptors with the lysosomal membrane protein Lamp1 upon decreasing hydrophobicity of the amino acid replacing Trp19. Interestingly, there was even less overlap than expected between the wild-type CD-MPR and Lamp1 as both proteins are present to some extent in late endosomes (28). This might indicate that Lamp1 and the CD-MPR are in different subdomains within late endosomes. It would make perfect sense that proteins destined for the lysosomes would be separated from proteins that recycle back to the Golgi thus increasing the sorting efficiency of such a complex structure as the multivesicular late endosomes. In fact, it has been observed that even the CD-MPR and the CI-MPR are localized in separate subdomains within late endosomes (29). Mutant receptors that were delivered partially to lysosomes like MPR-W19A (38% in lysosomes) and MPR-W19D (59% in lysosomes) did show an increasingly overlapping staining with Lamp1, validating this method as a screening procedure to test for the sorting efficiency of the mutant receptors. Intriguingly, there seemed to be subdomains of lysosomes that were labeled with either one or both proteins. Actually the majority of the structures that were colabeled for CD-MPR mutants and Lamp1 did not show a uniform yellow labeling that would indicate an equal distribution of the proteins but rather an overlap with separate labeling. Such a pattern of segregation of proteins indicates the existence of subdomains on the lysosomal membrane. These subdomains may be specialized for degradation as in the case of CD-MPR mutants or for export as in the case of molecules such as Lamp1 (3033). It has been shown by Traub et al. (36) that AP-2 and clathrin could be recruited to lysosomes in permeabilized cells in vitro indicating that clathrin-coated vesicles mediate this transport step. Contrary to the existing notion of lysosomes as terminal organelles in the secretory pathway our observation suggests in addition that a functional specialization of its subdomains might exist. To follow up on this would require a method with a higher resolution such as immunoelectron microscopy.
To determine the sorting efficiency in a more quantitative way we first analyzed the ability of CD-MPR to bind His-tagged Tip47 in an in vitro assay. As with the immunofluorescence experiments the binding efficiency decreased if Trp19 was replaced by decreasing hydrophobic residues. However, even the mutants with Trp19 substituted by bulky hydrophobic residues such as leucine, valine, and isoleucine were moderately reduced in their capacity to bind Tip47. Mutants with a less hydrophobic residue at position 19, like MPR-W19A, displayed only marginal Tip47 binding whereas charged residues prevented binding almost completely. Binding was almost not affected when the di-aromatic mutants MPR-FW-YF and MPR-W19Y were tested in the assay. This clearly demonstrates that optimal interaction of the CD-MPR cytoplasmic tail with Tip47 depends on the di-aromatic motif with only limited possibilities for variations. The strict requirement for individual amino acids is further emphasized by the fact that the cytosolic tail of CD-MPR is 100% identical in all mammalian species cloned so far with only two homologous substitutions (Val5-Ile5 and Asp55-Glu55) in lower vertebrates such as chicken (4, 34).
When we analyzed the mutants in vivo in the context of the full-length receptor for their efficiency to avoid delivery to lysosomes we found that there were roughly three categories. Substitution of Trp19 with either one of the bulky hydrophobic residues resulted in mislocalization of the mutant construct to lysosomes just below 30%. This number was slightly increased (3440%) when less hydrophobic residues like cysteine, alanine, and methionine were used to replace Trp19. Hydrophilic (serine) or charged residues (arginine and aspartic acid) had the greatest effect on endosomal sorting with more than 50% of the mutants accumulating in dense lysosomes (Fig. 4, Pools III). It is important to note that sorting of the mutant CD-MPRs in endosomes was not good even with the bulky hydrophobic residues as substitutions for Trp19 when compared with mutants with aromatic residues as replacements for Trp19 (Table I) that were sorted like wild-type receptor.
It is interesting that the CD-MPR has a clearly defined signal, the di-aromatic motif that is required for the interaction with Tip47 and the sorting in late endosomes, which seems to be in contrast to the CI-MPR that relies on a domain comprising residues 4875 in its cytoplasmic tail, with additional residues required for the proper presentation (19). Nevertheless, proper presentation of the di-aromatic signal is also required as mutations that prevent the reversible palmitoylation of the CD-MPR lead to a missorting to lysosomes (11). Clearly, further studies are required to determine whether both receptors have the same binding site on Tip47 or if there are two different binding sites. However, these experiments will be technically demanding because the receptors are membrane proteins that undergo important post-translational modifications such as palmitoylation.
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FOOTNOTES |
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Both authors contributed equally to this paper.
To whom correspondence should be addressed: Institute of Physiology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland. Tel.: 41-01-6355137; Fax: 41-01-6356814; E-mail: rohrer.jack{at}access.unizh.ch.
1 The abbreviations used are: MPR, mannose 6-phosphate receptor; CD-MPR, cation-dependent mannose 6-phosphate receptor; CI-MPR, cation-independent mannose 6-phosphate receptor; TGN, trans-Golgi network; AP, adaptor protein complex; PNS, post-nuclear supernatant; Tip47, tail interacting protein of 47 kDa.
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ACKNOWLEDGMENTS |
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REFERENCES |
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