The Targeting of Cystinosin to the Lysosomal Membrane Requires a Tyrosine-based Signal and a Novel Sorting Motif*

Stéphanie Cherqui, Vasiliki Kalatzis, Germain TrugnanDagger , and Corinne Antignac§

From the INSERM U423, Rein en Développement et Néphropathies Hereditaires, Hôpital Necker-Enfants Malades, 149 rue de Sèvres, 75015 Paris, France and Dagger   INSERM U538, Trafic Membranaire et Signalisation dans les Cellules Epithéliales, Centre Hospitalier Universitaire-St Antoine, 27 rue de Chaligny, 75012 Paris, France

Received for publication, November 22, 2000, and in revised form, January 6, 2001



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cystinosis is a lysosomal transport disorder characterized by an accumulation of intra-lysosomal cystine. Biochemical studies showed that the lysosomal cystine transporter was distinct from the plasma membrane cystine transporters and that it exclusively transported cystine. The gene underlying cystinosis, CTNS, encodes a predicted seven-transmembrane domain protein called cystinosin, which is highly glycosylated at the N-terminal end and carries a GY-XX-Phi (where Phi  is a hydrophobic residue) lysosomal-targeting motif in its carboxyl tail. We constructed cystinosin-green fluorescent protein fusion proteins to determine the subcellular localization of cystinosin in transfected cell lines and showed that cystinosin-green fluorescent protein colocalizes with lysosomal-associated membrane protein 2 (LAMP-2) to lysosomes. Deletion of the GY-XX-Phi motif resulted in a partial redirection to the plasma membrane as well as sorting to lysosomes, demonstrating that this motif is only partially responsible for the lysosomal targeting of cystinosin and suggesting the existence of a second sorting signal. A complete relocalization of cystinosin to the plasma membrane was obtained after deletion of half of the third cytoplasmic loop (amino acids 280-288) coupled with the deletion of the GY-DQ-L motif, demonstrating the presence of the second signal within this loop. Using site-directed mutagenesis studies we identified a novel conformational lysosomal-sorting motif, the core of which was delineated to YFPQA (amino acids 281-285).



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Lysosomes are a principal site of intracellular digestion in mammalian cells. Extracellular biological materials destined for degradation in the lysosomes are endocytosed and shuttled to the lysosomes via early and late endosomes (1). In contrast, endogenous cellular structures which are to be degraded in the lysosome are either selectively (chaperon-mediated) or nonselectively (macro- or micro-) transported independently of the endosomes via a process called autophagy (2, 3). The lysosomal membrane, due to the presence of a H+ pump, is responsible for acidification of the interior and for sequestration of the lysosomal enzymes responsible for the degradation process (4). Furthermore, the lysosomal membrane contains transport proteins that mediate the transport of degradation products such as amino acids, sugars, and nucleotides from the lysosomal lumen to the cytosol, where they are excreted or reused by the cell. A defect in either the degradation or transport process can result in an accumulation of the undegraded substrate or the degradation product within the lysosomes, impairing the physiology of the cell and leading to a lysosomal storage disorder.

Several such disorders have been described and are usually classified according to the molecule that has accumulated intra-lysosomally (5). Most of these disorders are due to a defect in one of the lysosomal hydrolases; however, a subset exists that arises from a defect in one of the membrane transporters (6). One such example is the autosomal recessive disorder cystinosis (MIM 21980), which is characterized by an accumulation of intra-lysosomal cystine (7). Various biochemical studies over the years show that cystine transport across the lysosomal membrane is carrier-mediated, that the carrier is located in the membrane itself, and that it is distinct from the plasma membrane cystine transporters (8), which in contrast do not exclusively transport cystine (9, 10). Moreover, the lysosomal cystine transporter is stimulated by the acid pH of the lysosome, which is ATP-dependent (11). Cystine is the disulfide of the amino acid cysteine, and cysteine has been found to exit the lysosome freely in cystinotic cells, implying that the cysteine transporter is independent of the cystine transporter (12). This finding has been exploited for the treatment of cystinosis as the currently used drug, cysteamine, is an aminothiol that reacts with cystine to form cysteine and a cystein-cysteamine mixed disulfide that can also readily exit the cystinotic lysosome (13). Thus, cysteamine, if used early and in high doses, delays the progression of cystinosis in affected individuals by reducing intra-lysosomal cystine levels (14).

We recently identified the gene underlying cystinosis, CTNS, (15) which is composed of 12 exons and extends over 23 kilobases of genomic sequence. The identification of mutations in the CTNS-coding sequence of affected individuals, the most common of which is a 57-kilobase deletion (15, 16), confirmed this gene as underlying cystinosis. Three allelic clinical forms have been defined based on the severity of symptoms and age of onset (11); the infantile form is the most severe and is characterized initially by the appearance of a proximal renal tubule dysfunction at 6-8 months, leading to end-stage renal failure by 10 years of age, and, eventually, by damage to most tissues due to widespread cystine accumulation. The juvenile form is less severe and characterized by a glomerular renal impairment appearing at a later age. Finally, the ocular nonnephropathic form is characterized by the presence of corneal cystine-crystal deposits without renal anomalies.

The 2.6-kilobase CTNS transcript encodes a novel 367 amino acid protein named cystinosin, which shows homology with a 55.5-kDa Caenorhabditis elegans protein (C41C4.7) and a yeast transmembrane protein, ERS1 (15). Computer-aided sequence analysis of cystinosin predicts that it is composed of seven transmembrane domains, suggesting that it also is a membrane protein. These domains are preceded by seven potential glycosylation sites at the N-terminal end, characteristic of lysosomal membrane proteins that are highly glycosylated to help protect them from the lysosomal proteases in the lumen (17). Finally, following the seventh transmembrane domain is a 10-residue C-terminal tail. Within this tail lies a 5-residue sequence, GY-DQ-L. This sequence is reminiscent of the tyrosine-based sorting motif, GY-XX-Phi (where Phi  is a hydrophobic residue), which was identified in the 10-20 amino acid carboxyl tails of lysosomal-associated membrane protein (LAMP)1 -1, -2, and -3 and in lysosomal acid phosphatase and shown to be responsible for their correct targeting to the lysosome (for review see Ref. 18). Taken together these results suggest that cystinosin is a lysosomal membrane protein. We constructed cystinosin-green fluorescent protein (GFP) fusion proteins and cystinosin-c-Myc epitope-tagged proteins to study the subcellular localization of cystinosin in vitro by transient and stable transfections of various cell lines. This study allowed the determination of the subcellular localization of cystinosin and resulted in the identification of a novel motif required for lysosomal sorting.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein Fusion Constructs-- To create a fusion construct with the CTNS sequence at the 5' end of and in-frame with the GFP sequence, the CTNS cDNA sequence was polymerase chain reaction-amplified (Advantage cDNA polymerase mix, CLONTECH) using a forward primer situated in the 5'-non-coding region (5'-AGT CTA GCC GGG CAG GGG AA-3') and a reverse primer (5'-GGG GTA CCC CGT TCA GCT GGT CAT ACC C-3') situated at the end of the coding region, which deleted the stop codon and added a KpnI restriction site. The amplified product was digested by XhoI (situated 250 base pairs upstream of the ATG initiator codon) and KpnI and inserted into the XhoI/KpnI-digested pEGFP-N1 (CLONTECH) (creating the construct pCTNS-EGFP). Clones containing the CTNS insert were checked by sequencing (Applied Biosystems 373XL). To generate a fusion construct containing the CTNS sequence at the 3' end of the GFP sequence, the CTNS-coding sequence was polymerase chain reaction-amplified using a forward primer (5'-CCG CTC GAG GGA TAA GGA ATT GGC TGA CTA T-3'), which added an XhoI site and deleted the ATG start codon, and a reverse primer situated in the arm of the lambda gt10 phage clone containing the CTNS cDNA. The amplified cDNA insert was digested by XhoI and HindIII (situated 303 base pairs downstream of the stop codon) and inserted into the XhoI/HindIII-digested pEGFP-C1 (creating pEGFP-CTNS). As a first step toward fusion of the c-Myc tag (EQKLISEEDL) to the 3' end of the CTNS sequence, the CTNS cDNA-coding region was amplified using the BamHI-containing forward primer 5'-GGG GAT CCT GAG CTC TGC CTC TTC C-3' and the XbaI-containing reverse primer 5'-GCT CTA GAG CCA GCC CTG TGT GCC AG-3', restriction enzyme-digested, and subcloned into the expression vector pcDNA 3.1 Zeo+ (Invitrogen). Site-directed mutagenesis (QuikChange Site-Directed Mutagenesis Kit, Stratagene) to introduce the c-Myc tag was then carried out on this construct using the primer 5'-TAT GAC CAG CTG AAC GAA CAA AAA CTT ATT TCT GAA GAA GAT CTG TAG CAC CCA GGG ACC-3' coupled with its reverse complement (creating pCTNS-cMyc).

Mutagenesis of the pCTNS-EGFP Construct-- All the modifications of pCTNS-EGFP listed under "Results" were carried out using the Stratagene QuikChange Site-Directed Mutagenesis Kit according to the manufacturer's recommendations and verified by sequencing the region surrounding the introduced mutation. Primers used are not listed here but are available upon request.

Cell Culture and Transfection-- MDCK and HeLa cells were grown in minimal essential medium (MEM; Life Technologies, Inc.) supplemented with 10% fetal calf serum (Vabliotek), 100 units/ml penicillin/streptomycin (Life Technologies, Inc.), and 2 mM L-glutamine (Life Technologies, Inc.). Confluent cells were passaged the day before transient transfection, and 2 × 105 cells were distributed into 35-mm wells containing 3 sterile glass coverslips. Once cells reached 60% confluency, the supplemented MEM media was replaced by 1 ml of OPTIMEM (Life Technologies, Inc.), and cells were incubated at 37 °C for 30 min. Transfections were carried out with 5 µg of DNA using 12.5 µg of DAC 30 (Eurogentec) in 1 ml of OPTIMEM, and the cells were incubated with the DNA-liposome complex for a minimum of 6 h. Transfected cells were then washed with phosphate-buffered saline (PBS), overlaid with supplemented MEM, and left to incubate for 48 h. For the stable transfections, cells were distributed into 35-mm wells without coverslips and treated as above. After the 48-h incubation, the culture medium was supplemented with 2 mg/ml G418 (Invitrogen).

Immunofluorescence-- Transfected cells were washed with PBS and fixed with 2% paraformaldehyde (Merck) for 20 min at room temperature. After washing with PBS, cells were blocked with 10 mM NH4Cl for 10 min, washed, and permeabilized using 0.5% Triton X-100 (Sigma) for 10 min. Coverslips were then either RNase-treated for 10 min and the cell nuclei stained with 0.5 µg/ml propidium iodide (Sigma) for 3 min or incubated with the first antibody for 1 h, washed in PBS, and incubated with the second antibody for 1 h. Finally, coverslips were washed in PBS, incubated with 100 mg/ml diazabicyclo[2.2.2]octane (Sigma) for 10 min to maintain the fluorescence, and mounted using Glycergel (DAKO). Stained coverslips were visualized using a Zeiss Axiovert 100M confocal microscope equipped with LSM 510 version 2.5 software and Ar/Kr (458 and 488 nm) and 2× He/Ne (543 and 633 nm) lasers with a 100× oil immersion objective. All images correspond to a 1-µm cell section. Photographs were processed with Adobe Photoshop 5.5. For the transferrin internalization studies, transiently transfected HeLa cells were washed three times in PBS and incubated for 30 min at 37 °C with 4 µg/ml Texas Red-conjugated human transferrin (Molecular Probes) in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 20 mM HEPES (pH 7.4) and 2 mg/ml bovine serum albumin. Cells were washed three times with PBS and fixed, mounted, and visualized as above.

Antibodies-- The mouse monoclonal antibodies used were as follows: AC17, directed against canine LAMP-2 (19), was kindly provided by A. Le Bivic (Faculté des Sciences, Luminy, Marseille, France) and used at a dilution of 1:500; H4B4, directed against human LAMP-2, was purchased from the Developmental Studies Hybridoma Bank and used at a dilution of 1:100; anti-GFP was purchased from Roche Molecular Biochemicals and used at a dilution of 1:250; 9E10, directed against the c-Myc epitope, was obtained from Santa Cruz Biotechnology and used at a dilution of 1:100; and EMA, directed against epithelial membrane antigen, was obtained from DAKO and used at a dilution of 1:50. Secondary donkey anti-mouse antibodies for the immunofluorescence studies conjugated to either fluorescein isothiocyanate or tetramethylrhodamine B isothiocyanate were purchased from Jackson Laboratories and used at a dilution of 1:200.

Western Blot Analysis-- Western blots were prepared from whole cell lysates. 293 cells, non-transfected or transiently transfected with the construct pCTNS-EGFP, were scraped in PBS, centrifuged for 5 min at 2500 rpm at 4 °C, and resuspended in lysis buffer (150 mM NaCl, 1% Nonidet P-40, 100 mM Tris HCl (pH 8), 0.02% sodium azide, 1 mM phenylmethylsufonyl fluoride, 10 µg/ml pepstatin A, 10 µg/ml aprotinin, and 10 µg/ml leupeptine). Cells were lysed for 20 min on ice and centrifuged for 2 min at 12,000 rpm at 4 °C. Cell lysates were mixed with Laemmli's sample buffer and loaded directly without boiling onto an 8% SDS-polyacrylamide electrophoresis gel. The separated proteins were electro-transferred to nitrocellulose filters (Bio-Rad) and, after blocking for 1 h in 0.05% Tween/PBS + 1% polyvinylpyrolidane (Sigma), the membrane was incubated for 1 h at room temperature with a 1/1000 dilution of the anti-GFP antibody. After three washes in 0.05% Tween/PBS, the filter was incubated for 1 h at room temperature with 1/12000 horseradish peroxidase-conjugated sheep anti-mouse antibody (Amersham Pharmacia Biotech). The final detection was performed using the Amersham Pharmacia Biotech ECL reagents according to the manufacturer's recommendations.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Subcellular Localization of Wild-type Cystinosin-- The CTNS-coding region was cloned 5' to the GFP cDNA in the plasmid pEGFP-N1, generating pCTNS-EGFP. In this orientation, the GFP end of the fusion protein was extra-lysosomal (Fig. 1). pCTNS-EGFP was transiently transfected into MDCK and HeLa cells, and the GFP fluorescence signal was observed 48 h post-transfection. In both cell types, cystinosin-GFP was localized to small, discrete intracellular vesicles and occasionally to large vesicular structures (Fig. 2A). No signal could be detected at the plasma membrane or in the nucleus. The same results were obtained upon stable transfection of MDCK cells (data not shown). Immunofluorescence studies using the anti-GFP antibody confirmed that the fluorescent signal did indeed correspond to cystinosin-GFP (data not shown). Moreover, western blot analysis was used to confirm the production of the cystinosin-GFP fusion protein. A major band with an apparent size of ~80 kDa (Fig. 3) was detected that was higher than the expected size of 67 kDa (cystinosin was predicted to be 40 kDa) and could be explained by the glycosylation of cystinosin via the 7 putative N-terminal glycosylation sites; a smaller band with an apparent size of ~70 kDa could also be seen that could presumably correspond to the unglycosylated form of the protein. Alternatively, this band could arise from proteolytic cleavage of cystinosin, although no known cleavage sites were detected. These same bands were not detected in nontransfected 293 cells (Fig. 3).


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Fig. 1.   Predicted structure of cystinosin and representation of the deletions/mutations concerning the third cytoplasmic loop. A, schematic representation of the predicted cystinosin structure. Blue indicates the N-terminal uncleavable signal peptide, orange indicates the seven potential N-glycosylation sites, black indicates the third cytoplasmic loop, and red indicates the C-terminal GY-DQ-L lysosomal-targeting signal. The yellow and green boxes indicate the positions of the c-Myc and GFP tags, respectively. B, the amino acid sequence of the third cytoplasmic loop is indicated. The two large deletions (Delta A and Delta B) and the smaller deletions (Delta a and Delta b) are represented by dashes. The amino acid residues mutated to alanine residues (µa and µb) are underlined.


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Fig. 2.   Subcellular localization of wild-type cystinosin. A, 48 h after transient transfection of MDCK cells with the pCTNS-EGFP construct, cells were fixed, permeabilized, and incubated with propidium iodide. The fluorescent signal from the cystinosin-GFP fusion protein can be seen to be localized to small discrete intracellular vesicles and to large vesicular structures. B, MDCK cells transiently transfected with pCTNS-cMyc were treated as above and then incubated with mouse monoclonal antibody directed against the c-Myc epitope followed by a fluorescein isothiocyanate-conjugated donkey anti-mouse secondary antibody. The same fluorescence labeling of intracellular vesicles can be seen. Scale bar, 10 µm for all figures.


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Fig. 3.   Western blot analysis. Non-transfected 293 cells (lane 1) and 293 cells transiently transfected with the pCTNS-EGFP construct (lane 2) were lysed, and the cleared lysate was loaded on a 8% SDS-polyacrylamide electrophoresis gel. After electro-transfer, the filter was incubated with a mouse monoclonal antibody directed against the GFP tag and subsequently with a horseradish peroxidase-conjugated sheep anti-mouse antibody. After immunodetection of the filter and a 10-s exposure to autoradiographic film, two bands could be observed in lane 2, a major band with an apparent size of ~80 kDa (large arrow), probably corresponding to the glycosylated form of cystinosin, and a smaller band with an apparent size of ~70 kDa (small arrow). Neither of these bands is detected in lane 1.

To verify that the fusion of the GFP to the C terminus of cystinosin did not interfere with its native localization, two additional constructs were made. First, the CTNS-coding region was subcloned downstream of the GFP gene in the plasmid pEGFP-C1 (pEGFP-CTNS) such that the GFP of the fusion protein would be predicted to lie intra-lysosomal. However, no fluorescence could be observed upon transfection of this construct presumably due to its quenching by the acidic lysosomal pH (20). Second, by site-directed mutagenesis, a c-Myc tag was added to the 3' end of CTNS in the plasmid pcDNA 3.1 Zeo+ (pCTNS-cMyc), and immunofluorescence analysis with the anti-epitope c-Myc antibody, 9E10, showed the same labeling of intracellular vesicles (Fig. 2B) as the construct pCTNS-EGFP.

To determine whether the labeled vesicles were indeed lysosomes, transfected cells were immunofluorescently labeled using antibodies directed against the late endosomal and lysosomal membrane protein LAMP-2. As seen in Fig. 4, in both MDCK (Fig. 4, A-C) and HeLa (Fig. 4, D-F) cells, the cystinosin-GFP signal colocalized with that of AC17, directed against canine LAMP-2, and H4B4, directed against human LAMP-2, respectively. The exact overlap between the LAMP-2 and cystinosin localization patterns demonstrates that cystinosin is a lysosomal protein. Interestingly, the above-mentioned large vesicular structures were also labeled by antibodies directed to LAMP-2 in cystinosin-overexpressing cells (Fig. 4, A and B), indicating that these large vesicles are also lysosomal structures; immunostaining of nontransfected cells with the same antibodies did not reveal the presence of these structures (data not shown). Finally, colocalization studies using fluorescently labeled transferrin uptake ruled out the possibility that cystinosin also localizes to the early endosomes (Fig. 4, G-I).


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Fig. 4.   Colocalization studies of wild-type cystinosin. A and B, MDCK cells transiently transfected with the pCTNS-EGFP construct (visualized in green) were fixed, permeabilized, and incubated with the mouse monoclonal antibody directed against canine LAMP-2 (AC17) followed by a tetramethylrhodamine B isothiocyanate-conjugated donkey anti-mouse secondary antibody (visualized in red). The large labeled vesicles and their lumen are visible (arrowhead). C, a superimposition of A and B shows a colocalization between cystinosin and LAMP-2 in canine cells. D and E, HeLa cells transiently transfected with the pCTNS-EGFP construct (visualized in green) were incubated with the mouse monoclonal antibody directed against human LAMP-2 (H4B4) followed by a tetramethylrhodamine B isothiocyanate-conjugated donkey anti-mouse secondary antibody (visualized in red). F, a superimposition of D and E shows that cystinosin also localizes to lysosomes in human cells. G and H, HeLa cells transiently transfected with the pCTNS-EGFP construct (visualized in green) were incubated 30 min at 37 °C with Texas Red-conjugated human transferrin (visualized in red) and then fixed. I, a superimposition of G and H shows that cystinosin is not localized to early endosomes.

Deletion and Mutagenesis of the Lysosomal-targeting Signal-- Previous studies on other lysosomal membrane glycoproteins have delineated one of the lysosomal-targeting signals as GY-XX-Phi (21). Such a five-residue targeting sequence, GY-DQ-L, exists in the C-terminal cytoplasmic tail of cystinosin (amino acids 362-366), and it is followed by an N residue. To confirm that the lysosomal targeting of cystinosin is indeed encoded by this signal, we deleted this motif (Delta GY-DQ-L) from the pCTNS-EGFP construct. Although transfection of this construct still resulted in the sorting of cystinosin-GFP to the lysosomes, both to small discrete vesicles as well as to abundant large vesicular structures, as shown by colocalization studies using AC-17 (Fig. 5, A-C), a significant signal was also found at the plasma membrane that colocalized with that of the epithelial membrane antigen antibody, EMA (Fig. 5, D-F). These results confirm that the GY-DQ-L motif is involved in targeting cystinosin to the lysosomes.


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Fig. 5.   Subcellular localization of cystinosin following deletion of the C-terminal GY-DQ-L signal. A, when MDCK cells were transiently transfected with the Delta GY-DQ-L construct, a signal from the cystinosin-GFP fusion protein could be seen at the plasma membrane as well as in small and abundant large intracellular vesicles (visualized in green). B, after incubation of these cells with AC17, a signal could be seen only in intracellular vesicles (visualized in red). C, a superimposition of A and B identifies the intracellular vesicles seen in A as the lysosomes. D, upon transient transfection of HeLa cells with the Delta GY-DQ-L construct, the same fluorescent pattern as that in panel A could be seen (visualized in green). E, after incubation of these cells with a mouse monoclonal antibody directed against EMA followed by a tetramethylrhodamine B isothiocyanate-conjugated donkey anti-mouse secondary antibody, a labeling of the plasma membrane can be seen (visualized in red). F, a superimposition of D and E confirms that the cystinosin-GFP fusion protein, upon deletion of the GY-DQ-L signal, is directed to the plasma membrane as well as to lysosomes.

By site-directed mutagenesis, each codon of the lysosomal-targeting sequence was individually mutated to an A residue to determine the critical residues of this sequence. Substitution of the Gly, Asp, or Gln residues for an Ala residue did not alter the lysosomal localization of cystinosin (Fig. 6, A, C, and D). In contrast, mutation of the Tyr or Leu residues resulted in the same localization profile as that obtained when the entire lysosomal-targeting sequence was deleted (Fig. 6, B and E). The Asn residue after the lysosomal-targeting motif was also mutated to Ala to ensure that this amino acid was not part of the targeting sequence. This substitution resulted in the same localization pattern as that seen with the wild type (Fig. 6F). Taken together these results demonstrate that it is the Tyr and Leu residues of the tyrosine-based sorting motif that are critical for the targeting of cystinosin to the lysosomes.


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Fig. 6.   Subcellular localization of cystinosin after site-directed mutagenesis of the GY-DQ-L-targeting signal. By site-directed mutagenesis, each residue of the GY-DQ-L-sorting motif in the construct pCTNS-EGFP was sequentially changed to an alanine residue. MDCK cells transiently transfected with the construct containing a mutation of the Gly (A), Asp (C), or Gln (D) residue results in a GFP fluorescence pattern identical to that of the wild-type cystinosin-GFP fusion protein. Transient transfection with the construct containing a mutation of the Tyr (B) or Leu (E) residue results in a fluorescent signal in intracellular vesicles but also in a redirection of the mutated cystinosin-GFP fusion protein to the plasma membrane. Mutation of the Asn residue (F) following the targeting motif resulted in the same subcellular localization pattern as the wild-type cystinosin-GFP protein.

Existence of a Second Lysosomal-targeting Signal-- Our mutation analysis of the GY-DQ-L motif demonstrated that it is only partially responsible for the targeting of cystinosin. These data strongly suggested the presence of a second lysosomal-targeting signal. In the first instance, to determine whether the remainder of the cytoplasmic tail, which is not included in the defined targeting signal, may also play a role in lysosomal targeting, the entire cytoplasmic tail was deleted (amino acids 358-367) from the pCTNS-EGFP construct. This resulted in the same subcellular localization of cystinosin-GFP as obtained after deletion of the lysosomal-targeting signal (data not shown).

A sequence analysis of the other three cytoplasmic domains was then performed to identify a possible motif resembling one of the documented lysosomal-targeting signals. The third cytoplasmic loop, located between the fifth and sixth potential transmembrane domains, is tyrosine-rich and, hence, seemed to be a good candidate for containing a lysosomal-targeting signal. In the first instance, we deleted the first nine amino acids (amino acids 280-288) of this 19-residue cytoplasmic domain (Delta A) from the construct pCTNS-EGFP and observed the same cystinosin-GFP localization pattern as when the GY-DQ-L motif was deleted alone, with the exception that no large labeled vesicles were seen (data not shown). Subsequently, the remaining 10 amino acids (amino acids 289-298) were deleted (Delta B) from pCTNS-EGFP, resulting in a localization of cystinosin-GFP to the lysosomes but, again, not to the large lysosomal structures without an obvious signal at the plasma membrane (data not shown). Each of these deletions, Delta A and Delta B, was then included with Delta GY-DQ-L in pCTNS-EGFP. Transfection of the Delta A/Delta GY-DQ-L construct resulted in the exclusive localization of cystinosin-GFP to the plasma membrane (Fig. 7A). In contrast, transfection of the Delta B/Delta GY-DQ-L construct gave a similar pattern (Fig. 7B) to that obtained using Delta GY-DQ-L alone, with the exception of the large labeled vesicles. In MDCK and HeLa cells transfected with the construct containing Delta A/Delta GY-DQ-L, there was no longer a colocalization between cystinosin-GFP and LAMP-2 (Fig. 8, A-C), whereas there was a colocalization between cystinosin-GFP and epithelial membrane antigen (Fig. 8, D-F). In contrast, in cells transfected with the construct containing Delta B/Delta GY-DQ-L, the cystinosin-GFP signal colocalized with both that of AC17 and epithelial membrane antigen (EMA), demonstrating that this double deletion still directed the fusion protein to the lysosomes (data not shown). Taken together, these results suggest that the third cytoplasmic loop, in particular the first half, plays a role in the targeting of cystinosin to the lysosome.


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Fig. 7.   Subcellular localization of cystinosin after deletion of the third cytoplasmic loop and the GY-DQ-L motif. A, in MDCK cells transiently transfected with the pCTNS-EGFP construct carrying the deletion Delta A and Delta GY-DQ-L, the deleted cystinosin-GFP is localized entirely to the plasma membrane. B, after transient transfection with the pCTNS-EGFP construct carrying the deletion Delta B and Delta GY-DQ-L, the GFP signal is divided between the plasma membrane and small intracellular vesicles.


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Fig. 8.   Colocalization studies of cystinosin carrying the deletion of the first nine amino acids of the third cytoplasmic loop and of the GY-DQ-L motif. A and B, MDCK cells transiently transfected with the pCTNS-EGFP construct carrying the deletions Delta A and Delta GY-DQ-L (visualized in green) were fixed, permeabilized, and incubated with AC17 (visualized in red). C, a superimposition of A and B shows that the deleted cystinosin-GFP fusion protein and LAMP-2 do not colocalize in canine cells. D and E, HeLa cells transiently transfected with the same construct (visualized in green) were incubated with EMA (visualized in red). F, a superimposition of D and E shows that the deleted cystinosin-GFP fusion protein colocalizes with the epithelial membrane antigen, demonstrating that this double deletion entirely directs the fusion protein to the plasma membrane.

As seen here and reported by others (22, 23) that a Tyr plays a critical role in lysosomal targeting, we individually replaced the two Tyr residues in the first half of the third cytoplasmic domain by an Ala residue using site-directed mutagenesis of pCTNS-EGFP. Each of these mutations coupled with Delta GY-DQ-L resulted in the same localization pattern (data not shown) as that observed with Delta GY-DQ-L alone (Fig. 2A), indicating that this loop does not play a role in lysosomal targeting via a tyrosine-based motif. Subsequently, blocks of four and five amino acids beginning at each of the aforementioned tyrosine residues were either deleted (designated Delta a and Delta b, respectively, see Fig. 1) or mutated (µa and µb, respectively) and assayed with Delta GY-DQ-L. Transfection of the Delta a/Delta GY-DQ-L construct resulted in the same plasma membrane localization of cystinosin-GFP as seen with the Delta A/Delta GY-DQ-L construct (Fig. 9A), whereas transfection of the µa/Delta GY-DQ-L construct resulted in the appearance of a distinct labeling of the lysosomes with no large lysosomal structures seen (Fig. 9B). In contrast, transfection of both the Delta b/Delta GY-DQ-L and µb/Delta GY-DQ-L constructs resulted in the same localization pattern as obtained with µa/Delta GY-DQ-L (data not shown). These results indicate that the beginning of the third cytoplasmic loop, from the Tyr to the Ala residue, is crucial for the targeting of cystinosin to lysosomes and that the rest of the loop also plays a minor role.


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Fig. 9.   Subcellular localization of cystinosin after deletion and mutagenesis of the YFPQA motif coupled with the deletion of the GY-DQ-L motif. A, in MDCK cells transiently transfected with the pCTNS-EGFP construct carrying the deletion Delta a and Delta GY-DQ-L, the deleted cystinosin-GFP is localized entirely to the plasma membrane. B, after transient transfection with the pCTNS-EGFP construct carrying the mutation µa and Delta GY-DQ-L, the GFP signal is divided between the plasma membrane and small intracellular vesicles.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cystinosin is a novel integral membrane protein that, when defective, is responsible for the lysosomal transport disorder cystinosis, characterized by an accumulation of intralysosomal cystine (15). The disease phenotype led to the prediction that cystinosin is a cystine transporter localized in the lysosomal membrane (8). The construction of a cystinosin-GFP fusion protein has now allowed us to confirm the predicted subcellular localization due to the colocalization of this fusion protein with an antibody directed against the lysosomal membrane protein, LAMP-2. The targeting of lysosomal membrane proteins has been largely studied, and two main classes of signals have so far been identified, a tyrosine-based GY-XX-Phi or Y-XX-Phi (where Phi  is a hydrophobic residue), and a di-leucine (or isoleucine)-sorting motif, both typically found in the short C-terminal cystoplasmic tails of glycoproteins that cross the membrane one or more times (18). Sequence analysis of the 10-amino acid cytoplasmic tail of cystinosin permitted the identification of a tyrosine-based targeting signal, GY-DQ-L, which when deleted, results in a redirection of cystinosin to the plasma membrane. By site-directed mutagenesis we have determined that the critical elements for this redirection are the Tyr and Leu residues, consistent with the results obtained for LAMP-1 (22, 23). Indeed, lysosomal membrane proteins can be delivered to the lysosome directly, from the trans-Golgi network or indirectly, by first appearing at the cell surface and subsequently being internalized via endocytosis, and the Tyr and Phi  residues have been shown to be involved in both of these pathways (21). In contrast, mutation of the Gly residue did not alter the lysosomal localization of cystinosin, again consistent with that seen for LAMP-1, although it has been shown that this residue does have a role in the direct targeting of this latter protein from the trans-Golgi network to the lysosome (21).

Interestingly, mutagenesis of the Tyr or Leu residues of GY-DQ-L or deletion of this entire pentapeptide still results in a localization of cystinosin-GFP to the lysosomes. This is in contrast to the situation for LAMP-1, where it has been shown that mutagenesis of the Tyr or Phi  residues from its GY-QT-I signal results in the exclusive detection of LAMP-1 on the cell surface and not in intracellular vesicles (21). These results imply that there may be a second sorting signal at play for cystinosin. Such a situation has been described for tyrosinase, which contains both a di-leucine motif as well as a tyrosine-based motif in its cytoplasmic tail, and mutagenesis studies show that it is the di-leucine motif that is necessary for the efficient sorting of this protein to late endosomes and lysosomes, whereas the Y-XX-Phi motif seems to be part of a weak secondary sorting signal (24). Along this line, we deleted the entire cytoplasmic tail from the cystinosin-GFP fusion protein to determine whether, as is the situation for tyrosinase, a second targeting signal resided in the tail even though a di-leucine motif was not detected. However, transfection of this construct resulted in the same localization pattern as obtained when just the GY-DQ-L signal was deleted. Because a second signal was not detected in the cytoplasmic tail of cystinosin, we focused our attention on the three predicted cytoplasmic loops, which would be the most susceptible to an interaction with adaptor proteins involved in the sorting of proteins to their fated location. None of these loops contain a di-leucine motif, but the third cytoplasmic loop, composed of 19 amino acids, was tyrosine-rich. Deletion of the first nine amino acids of this loop (amino acids 280-288) coupled with the deletion of the defined carboxyl GY-DQ-L motif, resulted in the complete relocalization of cystinosin-GFP to the plasma membrane without a signal remaining in the lysosomes. This redirection was due to a novel sorting motif, the core of which was defined as YFPQA (amino acids 281-285) by mutagenesis studies.

This novel sorting motif does not resemble any of the lysosomal sorting motifs so far defined. In addition, serial deletions of the remainder of the third cytoplasmic loop, although they do not provoke as drastic an effect, do seem to indicate that this region also has a minor role in lysosomal targeting. This is reminiscent of the situation involving the epidermal growth factor receptor. Epidermal growth factor receptor is a transmembrane domain protein with three lysosomal sorting signals in its carboxyl tail: a tyrosine-based motif (YLVI), a tyrosine kinase domain, and an uncharacterized region situated between amino acids 1022 and 1123 (25-27). Within this third 1022-1123 amino acid-targeting domain of epidermal growth factor receptor, it has been shown that the region 1022-1063 appears to contribute more significantly to sorting than residues 1063-1123 (27). Moreover, it is noteworthy, that mutating the core of the second sorting motif in cystinosin to alanine residues does not have as a dramatic effect on the relocalization of cystinosin as compared with the complete deletion of this pentapeptide. Thus, the YFPQA region of cystinosin seems to act as a conformational motif that is recognized as part of a particular secondary structure formed by the rest of the third cytoplasmic loop.

Other proteins that have a nonclassical lysosomal-sorting signal have been described. For example, P-selectin does not contain a classic lysosomal-targeting signal but rather a novel sequence, KCPL, in its C-terminal tail that has been shown to be responsible for its lysosomal targeting (28). The one striking difference between cystinosin and other proteins targeted to the lysosome is that, for the latter, the lysosomal-targeting signal(s) has been found to be situated in the carboxyl tail, whereas the second putative lysosomal-targeting signal for cystinosin is localized in one of its cytoplasmic loops. This seems to be a unique situation, although a recent study has shown that the late endosomal/lysosomal targeting of MLN64, a four transmembrane domain cholesterol-binding protein, is mediated via a region situated between amino acids 47 and 171, which comprises the transmembrane domains (29). However, the sorting motif and its exact localization within this region have not yet been defined.

The targeting of proteins carrying a Y-XX-Phi to the lysosome has been extensively studied and found to require a direct interaction with the adaptor proteins AP-1, AP-2, and AP-3 (30). AP-1 is responsible for the delivery of proteins from the trans-Golgi network to the endosomal-lysosomal system (31), AP-2 mediates the internalization at the plasma membrane and subsequent delivery to the lysosome (32), and AP-3 is involved in an alternative pathway of transport from the trans-Golgi network or endosomes to the lysosome (33). Because we have shown that the GY-XX-Phi motif is active in cystinosin, one of the pathways by which this protein is sorted to the lysosome must involve one or more of these three adaptor proteins. On the other hand, we do not know which pathway the second lysosomal sorting motif uses or with which adaptor proteins it interacts to bring cystinosin to the lysosome. It cannot be ignored, however, that when the third cytoplasmic loop is altered we no longer see the labeling of any large lysosomal structures but just small discrete lysosomes. Thus, it is tempting to speculate that this second sorting motif may act to bring cystinosin to the lysosome via the intermediate of these large structures. Additional support for this hypothesis arises from the fact that in cells transfected with Delta GY-DQ-L, these large vesicles seem to become more abundant than in cells transfected with wild-type cystinosin-GFP. These results could be correlated with the fact that removal of the tyrosine-based sorting motif would require an increase in activity of the second sorting motif and, thereby, in the number of large vesicles. Hence, the pathway used by this second and novel sorting motif may represent a previously uncharacterized pathway for the sorting of lyosomal proteins to their final destination.

Cystinosin has been shown to be homologous to a 55.5-kDa protein of C. elegans (C41C4.7) as well as to a yeast transmembrane protein ERS1 (15). Surprisingly the C-terminal lysosomal-targeting signal of cystinosin, GY-DQ-L, is not found in either of these two other organisms, whereas the YFPQA motif is conserved in both C. elegans (YFPQV) and yeast (YIPQV), suggesting an important role for this motif. Moreover, there has been a splice site mutation (IVS11+2T>C) reported as associated with the less severe juvenile cystinosis, which has been predicted to lead to a truncated protein missing exon 11. This splice site mutation alters the reading frame, producing a missense mutation after amino acid residue 284 and introducing a new termination codon at position 289 (34). Although the encoded product lacks the GY-DQ-L motif, a CTNS-GFP fusion construct carrying the IVS11+2T->C mutation transfected into CTNS-/- cells results in a partial rescue of the cellular phenotype, implying the truncated cystinosin protein is still targeted to lysosomes (34). These observations are consistent with our results demonstrating the existence of a second lysosomal sorting signal for cystinosin. Infantile cystinosis is a multisystemic disease that can lead to death at 10 years of age due to the widespread accumulation of intralysosomal cystine, highlighting the crucial role of cystinosin in the normal metabolic functions of the body. Due to this indispensable role of cystinosin in the lysosomes of all cells, it is thus not surprising to find that this protein has a rescue pathway by which it can be sorted to the lysosome. In conclusion, the identification of this novel lysosomal-targeting motif not only provides an insight into the way cystinosin functions but also represents an addition to the ever-growing list of targeting motifs required for the correct sorting of proteins to their fated cellular location.

    ACKNOWLEDGEMENTS

We are extremely grateful to Jean-Louis Delaunay for invaluable help and advice. We thank Tounsia Aït Slimane for help with the initial immunofluorescence experiments, Andre LeBivic for the kind and indispensable gift of the AC-17 antibody, Fabien Alpy for providing the Western blot protocol, Yann Goureau for technical assistance with the confocal microscope, Yves Deris for photographic assistance, and George Patterson for help with computer programming. Finally, we thank Patrice Codogno and Fred Dice for helpful discussions.

    FOOTNOTES

* This work was supported by Vaincre les Maladies Lysosomales, the Association Française contre les Myopathies, and the Association pour l'Utilisation du Rein Artificiel.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 33 1 44 49 50 98; Fax: 33 1 44 49 02 90; E-mail: antignac@necker.fr.

Published, JBC Papers in Press, January 9, 2001, DOI 10.1074/jbc.M010562200

    ABBREVIATIONS

The abbreviations used are: LAMP, lysosomal-associated membrane protein; MDCK, Madin-Darby canine kidney; MEM, minimal essential medium; PBS, phosphate-buffered saline; GFP, green fluorescent protein; EMA, epithelial membrane antigen.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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