The sortilin cytoplasmic tail conveys Golgi-endosome transport and binds the VHS domain of the GGA2 sorting protein
2001; Springer Nature; Volume: 20; Issue: 9 Linguagem: Inglês
10.1093/emboj/20.9.2180
ISSN1460-2075
Autores Tópico(s)Microtubule and mitosis dynamics
ResumoArticle1 May 2001free access The sortilin cytoplasmic tail conveys Golgi–endosome transport and binds the VHS domain of the GGA2 sorting protein Morten S. Nielsen Corresponding Author Morten S. Nielsen Department of Medical Biochemistry, University of Aarhus, 8000 Aarhus C, Denmark Search for more papers by this author Peder Madsen Peder Madsen Department of Medical Biochemistry, University of Aarhus, 8000 Aarhus C, Denmark Search for more papers by this author Erik I. Christensen Erik I. Christensen Department of Cell Biology, University of Aarhus, 8000 Aarhus C, Denmark Search for more papers by this author Anders Nykjær Anders Nykjær Department of Medical Biochemistry, University of Aarhus, 8000 Aarhus C, Denmark Search for more papers by this author Jørgen Gliemann Jørgen Gliemann Department of Medical Biochemistry, University of Aarhus, 8000 Aarhus C, Denmark Search for more papers by this author Dagmar Kasper Dagmar Kasper Center for Molecular Neurobiology, University of Hamburg, Germany Search for more papers by this author Regina Pohlmann Regina Pohlmann Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, Germany Search for more papers by this author Claus M. Petersen Corresponding Author Claus M. Petersen Department of Medical Biochemistry, University of Aarhus, 8000 Aarhus C, Denmark Search for more papers by this author Morten S. Nielsen Corresponding Author Morten S. Nielsen Department of Medical Biochemistry, University of Aarhus, 8000 Aarhus C, Denmark Search for more papers by this author Peder Madsen Peder Madsen Department of Medical Biochemistry, University of Aarhus, 8000 Aarhus C, Denmark Search for more papers by this author Erik I. Christensen Erik I. Christensen Department of Cell Biology, University of Aarhus, 8000 Aarhus C, Denmark Search for more papers by this author Anders Nykjær Anders Nykjær Department of Medical Biochemistry, University of Aarhus, 8000 Aarhus C, Denmark Search for more papers by this author Jørgen Gliemann Jørgen Gliemann Department of Medical Biochemistry, University of Aarhus, 8000 Aarhus C, Denmark Search for more papers by this author Dagmar Kasper Dagmar Kasper Center for Molecular Neurobiology, University of Hamburg, Germany Search for more papers by this author Regina Pohlmann Regina Pohlmann Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, Germany Search for more papers by this author Claus M. Petersen Corresponding Author Claus M. Petersen Department of Medical Biochemistry, University of Aarhus, 8000 Aarhus C, Denmark Search for more papers by this author Author Information Morten S. Nielsen 1, Peder Madsen1, Erik I. Christensen2, Anders Nykjær1, Jørgen Gliemann1, Dagmar Kasper3, Regina Pohlmann4 and Claus M. Petersen 1 1Department of Medical Biochemistry, University of Aarhus, 8000 Aarhus C, Denmark 2Department of Cell Biology, University of Aarhus, 8000 Aarhus C, Denmark 3Center for Molecular Neurobiology, University of Hamburg, Germany 4Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, Germany *Corresponding authors. E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2001)20:2180-2190https://doi.org/10.1093/emboj/20.9.2180 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Sortilin belongs to a growing family of multiligand type-1 receptors with homology to the yeast receptor Vps10p. Based on structural features and sortilin's intracellular predominance, we have proposed it to be a sorting receptor for ligands in the synthetic pathway as well as on the cell membrane. To test this hypothesis we examine here the cellular trafficking of chimeric receptors containing constructs of the sortilin tail. We report that sorting signals conforming to YXXΦ and dileucine motifs mediate rapid endocytosis of sortilin chimeras, which subsequently travel to the trans-Golgi network, showing little or no recycling. Furthermore, we found that cation-independent mannose 6-phosphate receptor (MPR300)–sortilin chimeras, expressed in mannose 6-phosphate receptor knockout cells, were almost as efficient as MPR300 itself for transport of newly synthesized β-hexosaminidase and β-glucuronidase to lysosomes, and established that the sortilin tail contains potent signals for Golgi–endosome sorting. Finally, we provide evidence suggesting that sortilin is the first example of a mammalian receptor targeted by the recently described GGA family of cytosolic sorting proteins, which condition the Vps10p-mediated sorting of yeast carboxypeptidase Y. Introduction Sortilin (∼100 kDa) is a type-1 membrane receptor which is expressed in a number of tissues, notably brain, spinal cord, testis and skeletal muscle (Petersen et al., 1997; Hermans-Borgmeyer et al., 1999). Like the related receptor sorLA (250 kDa), sortilin binds the endoplasmic reticulum-resident receptor-associated protein (RAP), a putative chaperone for members of the low density lipoprotein receptor (LDLR) family (Bu and Schwartz, 1998; Willnow, 1998), and the two were originally co-purified from human brain by RAP affinity chromatography (Jacobsen et al., 1996; Petersen et al., 1997). Along with a third, recently described brain receptor designated sorCS (∼130 kDa) (Hermey et al., 1999) and at least two other proteins (DDBJ/EMBL/GenBank accession Nos AB028982 and AB037750) (Kikuno et al., 1999; Nagase et al., 2000), sortilin and sorLA constitute a new family of receptors sharing the characteristic structural feature of an ∼600-amino-acid N-terminal domain with a strong resemblance to each of the two related domains in the luminal portion of the yeast sorting receptor Vps10p (Marcusson et al., 1994). Information is still limited, but previous findings, in particular regarding sortilin and sorLA, suggest that the receptors are neuropeptide binding proteins with multiple and possibly overlapping functions (Yamazaki et al., 1996; Mazella et al., 1998; Hampe et al., 1999; Nielsen et al., 1999; Petersen et al., 1999). Unlike in sorLA and sorCS, which both incorporate additional domain types (Jacobsen et al., 1996; Hermey et al., 1999), the Vps10p domain makes up the entire luminal part of sortilin (Petersen et al., 1997). The domain contains two distinctive features: a C-terminal conserved segment of 10 cysteines (10CC) and a 44-amino-acid N-terminal propeptide. The role of the 10CC segment is unknown, but we have recently demonstrated that the propeptide plays an important part in the functional activation of sortilin by preventing ligands, in the early part of the synthetic pathway, from gaining access to the receptor binding site(s). Thus, the receptor is produced as an inert precursor which is converted to its mature binding-active form upon furin-mediated propeptide cleavage in the trans-Golgi network (TGN) (Petersen et al., 1999). Although its physiological role remains unclarified, sortilin is the best described of the Vps10p-domain receptors and different lines of evidence have given hints towards a possible function. First, sortilin binds a variety of unrelated ligands, including lipoprotein lipase (LpL) (Nielsen et al., 1999) and neurotensin (NT) (Mazella et al., 1998; Petersen et al., 1999). These ligands have two things in common: they each bind to one or more alternative receptor(s) and they are candidates for regulated transport not accounted for by these receptors (Braun and Severson, 1992; Barbero et al., 1998). Thus, members of the LDLR family that bind LpL are primarily endocytic receptors, which are also implicated in signalling (Gliemann, 1998; Cooper and Howell, 1999), and the two seven-transmembrane receptors for neurotensin (NTR-1 and -2) are signalling receptors with a capacity for endocytosis (Vincent, 1995). Therefore, even though sortilin may contribute to endocytosis and perhaps even signalling, it seems plausible that its main concern is functions not covered by the alternative receptors, e.g. regulated transport. Secondly, sortilin has striking (and indicative) structural similarities to receptors involved in intracellular sorting and transport. Thus, Vps10p is the sorting receptor for carboxypeptidase Y (CPY) in yeast (Marcusson et al., 1994) and the C-terminal segment in sortilin's cytoplasmic tail is closely related to the corresponding and functionally important segment of the cation-independent mannose 6-phosphate receptor (MPR300) (Johnson and Kornfeld, 1992a,b). Moreover, the sortilin cytoplasmic domain contains several potential signal sequences that conform to established consensus motifs, known to be involved in adaptor protein binding, endocytosis, basolateral targeting and Golgi–endosome sorting (Ktistakis and Roth, 1996). Thirdly, previous findings have demonstrated that sortilin predominates in intracellular compartments. Only a minor fraction of the receptors is expressed on the cell surface, whereas ∼90% are found in Golgi and vesicles, showing extensive co-localization with MPR300 (Petersen et al., 1997; Morris et al., 1998). It would appear from the above that sortilin is likely to serve functions inside the cell and that the combined sum of available evidence suggests it to be a candidate sorting receptor, targeted for transport by ligands in the synthetic pathway as well as on the surface membrane. In the present study we have pursued this concept. We have probed for interactions between sortilin and cytosolic sorting proteins, and we have examined the cellular trafficking of chimeric receptors to determine and characterize active sorting signals in sortilin's cyto plasmic domain. In particular, sortilin's capacity for Golgi–endosome sorting was tested by examining the transport of lysosomal enzymes in mannose 6-phosphate receptor knockout (mpr−) cells expressing MPR300–sortilin chimeras. Our findings provide evidence that sortilin has the potential for intracellular sorting and may well be involved in a hitherto unrecognized type of Golgi–endosome transport. Results Expression of chimeric receptors For selection of chimeric receptors likely to hold information on sortilin trafficking, we first examined the sortilin cytoplasmic domain (sortilin-cd) to identify segments conforming to known sorting motifs. The primary sequence of the 53-amino-acid domain and the chosen segments are shown in Figure 1A. We next generated a series of tail constructs in which potentially critical residues in each of the selected sites were either deleted or replaced by alanine and, in the case of S47, by aspartate. The resulting mutant constructs (Figure 1B) and wild-type sortilin-cd were then combined with the extracellular and transmembrane segments of interleukin-2 receptor-α (IL2Rα) (CD25, Tac) and stably expressed as IL2R–sort chimeric receptors in CHO-K1 cells. Alternatively, the tail constructs were combined with the luminal and the transmembrane domains of MPR300 (MPR300–sort chimeras) and expressed in mouse fibroblasts deficient for both mannose 6-phosphate receptors. Figure 1.(A) Primary sequence of the sortilin cytoplasmic domain. Motifs that are known to constitute active sorting signals in other transmembrane proteins are underlined, putative key residues are boxed. (B) Schematic presentation of the chimeric receptors (upper panel) and the cytoplasmic domain constructs (lower panel) used in this study. The luminal domains of IL2Rα and of MPR300 are indicated. Download figure Download PowerPoint Endocytosis of chimeric receptors To examine the endocytic capability of the IL2R–sort chimeras, CHO transfectants were incubated with iodinated anti-Tac at 4°C. Following incubation, unbound antibody was removed and the cells were re-incubated in warm medium (37°C, zero time). At given times, reactions were stopped on ice and the degree of endocytosis was determined by incubation at pH 2.5, i.e. not internalized tracer was defined as the amount of cell-associated radioactivity that could be dissociated at low pH. Using this method, a maximum of ∼67% of the total amount of anti-Tac bound by IL2R–sort-wt was internalized within 2 h, which is similar to the amount (∼75%) of labelled anti-sortilin internalized by CHO cells expressing full-length sortilin (data not shown). Results obtained with the individual IL2R–sort chimeric constructs are summarized in Table I. The table shows the percentage of tracer not internalized (dissociable at pH 2.5) found on the respective transfectants at 30 min, i.e. at a time when >85% of maximum endocytosis had taken place. It appears that only alterations involving the tyrosine-based YXXΦ motif, near the membrane-spanning segment, and the C-terminal dileucine had any significant effect on internalization. The time course of [125I]anti-Tac internalization by IL2R–sort chimeras is delineated in Figure 2 and clearly designates Y14SVL17 as the main signal for endocytosis, being responsible for ∼60% of the activity, and the tyrosine as the single most important residue. Deletion of the dileucine L51L52 caused a relatively minor decrease in internalization, but in combination, disruption of the dileucine- and tyrosine-based signals accounted for an ∼80% reduction of the endocytosis conveyed by the sortilin wild-type tail. Figure 2.Time course of [125I]anti-Tac internalization in transfected CHO cells expressing IL2R–sort chimeras. Following incubation at 4°C, unbound tracer was removed by washing and the cells were re-incubated in warm medium (zero time). At the given times, incubation was stopped on ice and internalization was determined as the amount of cell-associated radioactivity that was not released upon incubation at pH 2.5. Each point represents a mean of triplicates and all values are shown relative to the maximal internalization (100% at 30 min) obtained in transfectants expressing IL2R–sort-wt. Wild-type tail, closed circles; L51L52 deleted, open triangles; Y14A, open circles; Y14/L17A, closed triangles; Y14A/L17A and L51L52 deleted, closed squares. Download figure Download PowerPoint Table 1. Internalization of ILR–sort chimeras Sortilin-tail constructs Surface-associated liganda Wild type 41.5 ± 6.3 F9A 47.5 ± 2.6 G42A/Y43A 37.9 ± 1.2 S47A 42.8 ± 0.2 Del H44DDSDED50 42.1 ± 4.7 Del E49DLLE53 53.6 ± 8.0 Del L51L52 52.3 ± 7.1 Y14A 66.4 ± 4.7 Y14A/L17A 76.5 ± 4.5 Y14A/L17A and Del H44DDSDED50 77.9 ± 1.0 Y14A/L17A and Del L51L52 90.5 ± 1.2 aPercentage ligand bound to CHO cells at 4°C (zero time) after 30 min at 37°C (mean ± SD, n = 3). Matching results were obtained by confocal microscopy (Figure 3). Thus, anti-Tac, bound at 4°C by CHO cells expressing IL2R–sort-wt, was translocated from the cell surface to intracellular vesicles within minutes of incubation in warm medium (Figure 3A). After 30 min, practically all staining for antibody was concentrated in perinuclear compartments, leaving little or no staining on the surface membrane. By comparison, translocation of anti-Tac by chimeras with a deleted dileucine (Figure 3B) or a disrupted YXXΦ motif (Figure 3C) was slow and incomplete, and in cells transfected with constructs carrying the combined defects (Figure 3D) anti-Tac was found exclusively on the cell surface, even after 2 h at 37°C. Figure 3.Internalization of IL2R–sort chimeras in transfected CHO cells (confocal microscopy). After binding of anti-Tac at 4°C, transfected CHO cells expressing chimeric receptors comprising (A) wild-type sortilin-cd or (B) cd constructs containing a deletion of L51L52, (C) a Y14A/L17A mutation or (D) both were washed and re-incubated in warm medium. At the given times, the cells were fixed, stained by Alexa 488-conjugated goat anti-mouse Ig and analysed by confocal microscopy. Download figure Download PowerPoint This suggested that chimeras with an impaired endocytic function accumulated on the cell surface. We therefore assessed whether the IL2R–sort constructs in question were relatively overexpressed on the plasma membrane. Surface-associated and intracellular receptors were separated from biolabelled CHO transfectants by stepwise immunoprecipitation and quantitated, after SDS–PAGE, by autoradiography and densitometry. Using this approach, we found that the fraction of mutant receptors exposed on the plasma membrane was 1.3- (L51L52 deleted), 2.8- (Y14A/L17A) and 3.8- (the two combined) fold higher than that of IL2R–sort-wt. Another set-up, based on biotinylation of surface proteins on whole cells and subsequent separation of the two receptor pools by streptavidin beads, gave similar results, confirming that a decrease in endocytic capacity was accompanied by an increased expression of receptors on the cell membrane (data not shown). Internalized IL2R–sort-wt chimeras are directed to the TGN without recycling As depicted in Figure 3A, internalized IL2R–sort-wt in complex with anti-Tac rapidly accumulated in perinuclear compartments. Even after prolonged incubation, staining seemed restricted to intracellular vesicles, suggesting insignificant recycling of the antibody–receptor complexes. Accordingly, during a 4 h chase at 37°C, little or no internalized 125I-labelled anti-Tac returned to the surface in a pH 2.5-releasable form. The routing of chimeric receptors in complex with anti-Tac was examined further by electron microscopy. The results are shown in Figure 4. It appears that within 15–20 min, the anti-Tac–receptor complexes, initially scattered on the plasma membrane or assembled in coated pits (Figure 4, panel 1), became localized in coated vesicles (Figure 4, panel 2, inserts) and in Lamp-1-negative early endosomes (Figure 4, panel 2). At 60 min, staining predominated in vesiculotubular structures in close proximity to the nucleus, and double staining demonstrated that the antibody–receptor complexes to a large extent co-localized with TGN38 (Figure 4, panel 3). At this point (60 min), minor staining was also seen in uncoated vesicles, some resembling multivesicular bodies, and in a few Lamp-1-positive structures (Figure 4, panel 4). It is remarkable that co-localization with Lamp-1 was scarce and only seen upon prolonged incubation (>45 min). Figure 4.Endocytosis of IL2R–sort-wt and co-localization with TGN38 and Lamp-1. After binding of anti-Tac at 4°C and removal of unbound antibody, CHO transfectants expressing IL2R–sort-wt were re-incubated at 37°C. At the given times, the incubation was stopped on ice and the cells were fixed. Staining was performed using gold beads coupled to goat anti-mouse antibody. For double staining, fixed cells were incubated with rabbit anti-Lamp-1 or rabbit anti-TGN38 prior to staining with gold beads coupled to goat anti-rabbit antibody. Staining: panel 1 (zero time), IL2R–sort-wt; panel 2 (20 min), IL2R–sort-wt (black arrowheads) and Lamp-1 (black arrows); panel 2 insets, IL2R–sort-wt; panel 3 (60 min), IL2R–sort-wt (black arrowheads) and TGN38 (black arrows); panel 4 (60 min), IL2R–sort-wt (black arrowheads) and Lamp-1 (black arrows). Magnification in all panels ×114 000. Download figure Download PowerPoint It can be concluded from the above that sortilin-cd mediates endocytosis of IL2R–sort-wt in clathrin-coated vesicles, avoids recycling and directs the receptors to the TGN via Lamp-1-negative endosomes. Sortilin-cd contains an acidic cluster (H44DDSDED50), which shows great similarity to clusters that are known to play an important part in the surface-to-Golgi sorting of other transmembrane proteins, e.g. furin. Separate experiments were therefore performed to determine the involvement of the sortilin acidic cluster. It was established that S47 is readily phosphorylated by casein kinase II in vitro (Figure 5) and small amounts of phosphorylated full-length sortilin could be immunoprecipitated from CHO transfectants treated with the phosphatase inhibitor calyculin, but not from untreated cells (data not shown). This suggested that sortilin may be a substrate for CKII in vivo but as described above (Table I), the acidic cluster does not influence endocytosis, and various measures, including deletion of the entire segment and substituting aspartate for S47 (to mimic permanent phosphorylation), failed to induce any change in the apparent lack of IL2R–sort-wt recycling. In contrast, confocal microscopy did leave the overall impression that conditions mimicking or favouring a state of phosphorylation (S47D, or the presence of calyculin) promoted the TGN localization of internalized antibody–receptor complexes. However, the cell-to-cell variation was not insignificant and further evidence is needed before final conclusions can be drawn. Figure 5.In vitro phosphorylation of the sortilin cytoplasmic domain. Constructs of the sortilin-cd were expressed with a GST tag and purified. The purified fusion proteins and GST were incubated at 2 μM in MOPS buffer pH 7.0 containing 0.5 nM casein kinase II. Phosphorylation was started by addition of 20 μM [γ-32P]ATP, stopped after 45 min in 5% sample buffer, and analysed by SDS–PAGE and autoradiography. Lane 1, GST; lane 2, mutant construct S47A; lane 3, wild-type sortilin tail. Download figure Download PowerPoint Sortilin's cytoplasmic tail conveys Golgi–endosome transport Information on direct sorting from the synthetic pathway to endo- and lysosomal compartments was obtained in a previously described model, using mouse embryonic fibroblasts deficient in both mannose 6-phosphate receptors (Pohlmann et al., 1995). Owing to their receptor deficiency, these (mpr−) cells are unable to transfer newly synthesized lysosomal enzymes, normally targeted by the mannose 6-phosphate receptors, from the secretory pathway to the late endosomes. The cells consequently release the ligands into the medium and are left with abnormal lysosomes. We transfected mpr− cells with chimeric receptor constructs containing the sortilin tail, mutant or wild type, combined with the luminal (ligand binding) and transmembrane parts of MPR300. Since these chimeras (MPR300–sort) bind the mannose 6-phosphate moieties of proenzymes via the MPR300 luminal domain, we were then able to determine their sorting by monitoring the fate and cellular release of ligands, i.e. β-hexosaminidase. The results shown in Figure 6 (left panel) demonstrate that the levels of β-hexosaminidase were found to be very similar in the culture medium of transfected mpr− cells expressing comparable amounts of either MPR300–sort-wt or wild-type MPR300. At the optimal level of receptor expression (≥1.0), the medium from transfected cultures contained ∼80% less β-hexosaminidase than that of untransfected cells and in cultures supplemented with mannose 6-phosphate, <40% of the enzyme was found in the medium. Since mannose 6-phosphate inhibits ligand binding at the cell surface and thereby prevents uptake from the medium, it follows that the chimeric receptor, similarly to MPR300 itself, downregulates the cellular secretion of β-hexosaminidase. Figure 6.Sorting of lysosomal enzymes by MPR300–sortilin chimeras. The left panel shows the percentage of newly synthesized β-hexosaminidase that could be detected in the culture medium of untransfected mpr− cells (black circles, at expression level zero) and of mpr− cells transfected with wild-type MPR300 (black circles, full line) or with the MPR300–sort-wt chimera (circle with cross). Corresponding data obtained in cultures supplemented with mannose 6-phosphate (5 mM), which inhibits uptake of ligand from the medium, are indicated by a stippled line and arrowheads. The points represent results with individual clones exhibiting different levels of receptor expression. The inset shows an SDS–PAGE analysis (autoradiography) of samples of biolabelled β-glucuronidase immunoprecipitated from the medium (right lane) and lysate (left lane) of mpr− cells expressing MPR300–sort-wt and cultured in the presence of mannose 6-phosphate. The positions of the mature converted enzyme (69 kDa) and that of its proform (72 kDa) are indicated. The right panel shows the amount of β-hexosaminidase found in the medium of mpr−-cells transfected with MPR300–sort chimeras containing the wild-type or various mutated constructs of sortilin-cd. Arrowheads indicate values obtained in cultures containing mannose 6-phosphate. Download figure Download PowerPoint Receptors likewise regulate the secretion of β-glucuronidase (data not shown), a lysosomal enzyme that is converted upon arrival in the late endosomes/lysosomes from its 72 kDa proform to its mature cleaved 69 kDa form. SDS–PAGE analysis (Figure 6, left panel inset) demonstrates that while β-glucuronidase was released into the medium in its proform, the cells contained only the converted mature enzyme, providing evidence that retained (not secreted) enzyme had been directed to the lysosomes. Moreover, as transfection with MPR300–sort-wt chimeras, as well as with wild-type MPR300, normalized the appearance of lysosomes in the mpr− mouse fibroblasts (Figure 7), it can be concluded that the sortilin cytoplasmic domain conveys transport between the Golgi and lysosomes. Figure 7.Morphology of late endosomes/lysosomes in normal mouse embryonic fibroblasts (MEF), in mpr− (i.e. MEF cells deficient in mannose 6-phosphate receptors) and in mpr− transfected with MPR300–sortilin-cd chimeras or the wild-type MPR300. Following fixation, the cells were permeabilized with 0.5% saponin. The distribution of receptors carrying the MPR300 luminal domain and of Lamp-1, a marker of late endosomes and lysosomes, was detected by immunofluorescence using conjugated secondary antibodies. Download figure Download PowerPoint The distribution of β-hexosaminidase was also determined in mpr− cultures expressing MPR300–sort chimeras containing mutations or deletions at selected sites in sortilin-cd. Figure 6 (right panel) shows representative experiments with the different constructs. It appears that only alterations addressing the YXXΦ motif (Y14A/L17A) and the C-terminal dileucine (L51A/L52A) had any significant effect on enzyme sorting. The importance of these sites was confirmed by repetitive experiments using different clones expressing the same constructs at comparative and near optimal levels (expression level 1.0–2.0, Table II). From findings obtained in the absence and presence of mannose 6-phosphate, it can further be deduced that the dileucine almost only contributes to the direct intracellular transport, whereas residues of the YXXΦ motif promote transport by endocytosis as well as by sorting from the Golgi (Figure 6, right panel; Table II). This is in good agreement with the data on IL2R–sort internalization in CHO cells (Table I) and therefore not surprising. However, it should be noted that chimeras carrying Y14A as a single mutation showed no signs of missorting. In contrast, missorting by double mutants (Y14A/L17A) was profound even at high receptor expression levels. It follows that in terms of sorting from the synthetic pathway, L17 may be a key residue, but tyrosine and the YXXΦ motif as such are not functionally significant. Table 2. Sorting by MPR–sort chimeras in mpr− cells Sortilin-tail constructsa Level of expression Extracellular β-hexosaminidaseb (mean % ± SD of total) − M6P + M6P Non-transfected (n = 2) – 89.5 88.5 Wt-tail (n = 9, cl = 6) 0.98 ± 0.2 25.34 ± 7.32 36.13 ± 7.75 Y14A/L17A (n = 3, cl = 2) 1.61 ± 0.38 58.2 ± 3.38 63.0 ± 1.11 L51A/L52A (n = 4, cl = 3) 1.07 ± 0.12 32.6 ± 6.15 57.18 ± 5.72 a Indicating the numbers of experiments and numbers of different clones used (cl). b In culture medium without (−) and with (+) 5 mM mannose 6-phosphate (M6P). The findings establish that sortilin-cd has the capacity for Golgi–endosome sorting and the evidence is that this function relies on sorting motifs other than those governing endocytosis. Sortilin binds the cytosolic sorting protein GGA2 For the purpose of finding cytosolic proteins partaking in sortilin trafficking, a two-hybrid screen was set up using sortilin-cd as bait. Among the resulting confirmed positive clones, which did not include any adaptor protein (μ) subunits, one was found to be a differentially spliced version of human GGA2 cDNA (DDBJ/EMBL/GenBank accession No. AF323754). This was determined by alignment with the human GGA2 genomic sequence (DDBJ/EMBL/GenBank accession No. AC002400). The GGA2 two-hybrid clone contains an additional exon positioned between exons 6 and 7 and spanning the genomic positions 117 165–117 304. This exon encodes 29 amino acids followed by an in-frame stop codon at position 117 252. The translated product of the isolated clone (GGA2s) therefore represents a truncated form of GGA2, which contains the first 193 N-terminal residues of the published GGA2 sequence, including the entire VHS domain, followed by the amino acid sequence LFLSASEPGPIHFPSTMNSPNRYSLDISI. In control experiments, both full-length GGA2 and GGA2s induced a positive response with sortilin-cd as bait (data not shown). To elaborate on this finding, various GGA2 constructs (Figure 8A) were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. Full-length GGA2 could not be purified as a uniform protein, but truncated constructs covering the 183 N-terminal residues (VHS) and the 100 C-terminal residues [(containing the γ-adaptin ear homology domain (GAEH)] were obtained as uniform soluble proteins. The purified constructs were then
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