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INTRODUCTION |
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-
(where
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.
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EXPERIMENTAL PROCEDURES |
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
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.
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RESULTS |
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 ( A and B)
and the smaller deletions ( a and 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.
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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.
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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-
(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 (
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 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
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.
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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.
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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 (
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 (
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,
A and
B, was
then included with
GY-DQ-L in pCTNS-EGFP. Transfection of the
A/
GY-DQ-L construct resulted in the exclusive localization of
cystinosin-GFP to the plasma membrane (Fig.
7A). In contrast, transfection
of the
B/
GY-DQ-L construct gave a similar pattern (Fig.
7B) to that obtained using
GY-DQ-L alone, with the
exception of the large labeled vesicles. In MDCK and HeLa cells
transfected with the construct containing
A/
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
B/
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 A and 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 B and 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 A and 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.
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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
GY-DQ-L resulted in the same
localization pattern (data not shown) as that observed with
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
a
and
b, respectively, see Fig. 1) or mutated (µa and µb,
respectively) and assayed with
GY-DQ-L. Transfection of the
a/
GY-DQ-L construct resulted in the same plasma membrane
localization of cystinosin-GFP as seen with the
A/
GY-DQ-L
construct (Fig. 9A), whereas
transfection of the µa/
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
b/
GY-DQ-L and µb/
GY-DQ-L constructs
resulted in the same localization pattern as obtained with
µa/
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 a
and 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 GY-DQ-L, the GFP
signal is divided between the plasma membrane and small intracellular
vesicles.
|
|
 |
DISCUSSION |
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-
or Y-XX-
(where
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
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
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-
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-
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-
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
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.