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J. Biol. Chem., Vol. 275, Issue 32, 24367-24374, August 11, 2000
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From the Department of Human Genetics, Mount Sinai School of
Medicine, New York, New York 10029
Received for publication, March 15, 2000, and in revised form, April 26, 2000
The Niemann-Pick C1 (NPC1) protein is predicted
to be a polytopic glycoprotein, and it contains a region with
extensive homology to the sterol-sensing domains (SSD) of
3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-R) and sterol
regulatory element binding protein cleavage-activating protein (SCAP).
To aid the functional characterization of NPC1, a model of NPC1
topology was evaluated by expression of epitope-tagged NPC1 proteins
and investigation of epitope accessibility in selectively permeabilized
cells. These results were further confirmed by expression of NPC1 and
identification of glycosylated domains that are located in the lumen of
the endoplasmic reticulum. Our data indicate that this glycoprotein
contains 13 transmembrane domains, 3 large and 4 small luminal
loops, 6 small cytoplasmic loops, and a cytoplasmic tail. Furthermore,
our data show that the putative SSD of NPC1 is oriented in the same
manner as those of HMG-R and SCAP, providing strong evidence that this
domain is functionally important.
Niemann-Pick type C
(NPC)1 is a rare autosomal
recessive disorder that results in progressive neurodegeneration and
hepatosplenomegaly, leading to death during early childhood (1). The
most prominent biochemical feature is the accumulation of low density
lipoprotein-derived unesterified cholesterol in lysosomes (2, 3). In
addition, cholesterol accumulates in the trans-Golgi network
(TGN), and its relocation to and from the plasma membrane is delayed
(1). In fibroblasts, the defect in cholesterol exit from lysosomes is
accompanied by an attenuation in the down-regulation of two key
components of cholesterol homeostasis,
3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-R) and the low
density lipoprotein receptor (4, 5). Recently NPC1,
the gene for the major linkage and complementation group, which maps to
chromosome 18q11-12, was identified. It encodes an approximately
4.5-kilobase mRNA (6) that is predicted to produce a 1278-amino
acid protein. At least 36 mutations have been described in NPC1
patients (6-10), and although most are found throughout the gene and
do not reveal any functionally critical protein domains, a small
cluster are located in a the carboxyl-terminal third of the protein in
a region that possesses conserved cysteine residues (9). A minor group
of NPC patients does not map to the NPC1 locus, and complementation
studies (11, 12) have shown that these patients are defective for a
second gene, NPC2, which is currently unknown.
The effects of reduced or absent NPC1 in both humans (4, 13) and animal
models (1, 3) have been well studied, establishing this gene as an
essential component of intracellular cholesterol trafficking. In
addition, several Chinese hamster ovary cell mutants that display the
NPC1 phenotype have been characterized (14) and most have been shown to
be defective for the NPC1 gene (15). However, the precise function(s)
of the NPC1 protein is still unknown. NPC1 has been shown to be a
membrane glycoprotein that localizes to Lamp-positive organelles,
presumably endosomes and lysosomes (16-18). Further studies have
demonstrated that NPC1 resides primarily in late endosomes and only
secondarily in lysosomes and the TGN (18). This is an important
distinction in view of recent data suggesting that cholesterol
accumulation in NPC cells occurs primarily in endosomes (19), which are
sorting sites for various cellular components. In addition, NPC cells
appear to be defective in their efflux of endocytosed sucrose and in the sorting of the mannose-6 phosphate receptor, suggesting that the
retrograde movement of proteins and cargo from late endosomes to the
TGN is perturbed (16, 19).
Interestingly, although NPC1 is not closely related to any known
proteins, a cluster of five potential membrane-spanning sequences, from
residues 616 to 797, is homologous to the sterol-sensing domain (SSD)
found in two key regulators of cholesterol homeostasis, HMG-R and
sterol regulatory element binding protein cleavage-activating protein
(SCAP). In SCAP, a single point mutation in its SSD is sufficient to
render the protein sterol-insensitive, indicating the importance of
this region (20, 21), whereas in HMG-R the SSD mediates
sterol-regulated degradation of the protein (22, 23). The transmembrane
(TM) regions of NPC1, including the putative SSD, also share homology
with regions of Patched (PTC), a receptor for the
cholesterol-activated protein sonic hedgehog (24, 25). Together these data support the view that the SSD is functionally significant.
In these studies we describe and validate a topological model for the
structure of NPC1, showing that the orientation of the putative SSD is
identical to those of HMG-R and SCAP. These data should support efforts
in elucidating the precise function of this large membrane glycoprotein.
Materials--
Dulbecco's modified Eagle's medium was
purchased from Mediatech (Herndon, VA) and fetal bovine serum was from
HyClone (Logan, UT). LipofectAMINE, L-glutamine,
gentamicin, colcemid, and trypsin/EDTA were obtained from Life
Technologies, Inc. Fluoromount-G was purchased from Southern
Biotechnology Associates (Birmingham, AL). All enzymes were obtained
from New England BioLabs (Beverly, MA), except for N-glycosidase F, which was from Roche Molecular Biochemicals
(Indianapolis, IN). The goat anti-mouse IgG-fluorescein isothiocyanate
(FITC) and goat anti-rabbit IgG-FITC were also from Roche Molecular
Biochemicals, the anti-FLAG M2 monoclonal antibody was from
Eastman Kodak Co. and the goat anti-mouse IgG-rhodamine-X and
anti-rabbit IgG-rhodamine-X were from Jackson Immunoresearch
Laboratories Inc. (West Grove, PA). Filipin was purchased from
Polysciences, Inc. (Warrington, PA). Oligonucleotides were synthesized
using phosphoramidite chemistry on a 380B DNA synthesizer (Applied
Biosystems, Foster City, CA). All other reagents were obtained from
Sigma-Aldrich.
Creation of FLAG-tagged NPC1 Plasmids--
All plasmids were
constructed using the wild-type (wt) human NPC1 cDNA (6). For
analysis of the full-length NPC1 protein, hydrophilic loops A to E were
tagged with the FLAG peptide sequence DYKDDDDK by inserting an adapter
oligonucleotide at a unique restriction endonuclease site in the wt
NPC1 cDNA. For loops A to E, these sites were NdeI,
ClaI, BsiWI, BclI, and
Bsu36I and resulted in the insertion of the following
sequences at the indicated amino acid positions: 36-FLAG-Y, 306-FLAG-I,
519-FLAG-VR, 554-FLAG-DQ, and 992-FLAG-DQ, respectively (Fig.
1B). A second loop E tag
construct was created by placing the FLAG sequence at the
SphI site resulting in the sequence 886-FLAG-HA. Each tag
was verified by sequencing prior to cloning into the expression vector
pAsc9.
For analysis of the glycosylation pattern in NPC1, a number of
constructs were created. Truncated NPC1 cDNAs were generated by
polymerase chain reaction using the wt NPC1 cDNA as a template and
then cloned using standard procedures (26). Each cDNA extended from
the start codon of NPC1 to the 3'-end of loops A to E (Fig. 2), followed by at least one putative
membrane-spanning domain for correct protein anchorage and a
BamHI site for cloning. All cDNAs were verified by
sequencing on an automated ABI prism 377 DNA sequencer (Perkin-Elmer).
Each truncated cDNA was cloned into the expression vector pAsc9
(18) at the BamHI site, adjacent to a 3' in-frame FLAG-tag
linker sequence (DYKDDDDK-stop) for convenient detection. The plasmid
constructs were named according to the last loop and TM domain in their
cDNA sequences as follows: pLaTM1, pLbTM2, pLcTM3, pLdTM4, pLdTM6,
and pLeTM13; and these encoded amino acids 1-296, 375, 549, 644, 727, and 1164, respectively (Fig. 2). An additional slightly
truncated, FLAG-tagged protein was produced, as above, in which
the carboxyl terminus tail after TM15 was completely replaced with a
FLAG tag. This plasmid, pTM15tail, encoded NPC1 amino acids
1-1252.
Immunofluorescence Analysis of NPC1 and Filipin Staining--
To
assess the ability of the full-length FLAG-tagged NPC1 proteins to
correct the NPC1 phenotype, human NPC1 fibroblasts (GM03123), obtained
from Coriell cell repositories (Camden, NJ), were grown and transfected
with the appropriate constructs using LipofectAMINE Plus reagent as
described previously (18). At 24 h post-transfection, the medium
was replaced and the cells were grown for a further 24 h in
Dulbecco's modified Eagle's medium that contained 5%
lipoprotein-deficient serum. The cells were fixed with 3%
paraformaldehyde in phosphate-buffered saline (PBS) for 30 min, washed
with PBS, and then quenched with blocking solution (3% bovine serum
albumin (BSA), 50 µg/ml filipin in PBS) containing 1.5 mg/ml glycine
for 15 min. Cells were costained for cholesterol and the epitope-tagged
NPC1 proteins by incubating with blocking solution that contained 2 µg/ml M2 anti-FLAG antibody for 45 min, washing 3 × 5 min with
50 µg/ml Filipin in PBS, and then incubating with blocking solution
that contained 2 µg/ml anti-mouse IgG-rhodamine-X for 45 min. After
further washing the cells were mounted and visualized as above.
Selective Permeabilization of Plasma Membranes and
Immunofluorescence Microscopy--
The expression and epitope
accessibility of all FLAG-tagged proteins was assessed by transient
transfection followed by selective permeabilization and
immunofluorescence microscopy. COS-7 cells, obtained from the American
Type Culture Collection (Manassas, VA) and grown as described (18),
were treated with colcemid for 18 h prior to electroporation using
a Bio-Rad gene pulser unit set at 500 microfarads and 250 V to
transfect 10 µg of plasmid DNA into approximately 106
cells. The cells were grown on glass coverslips that had been treated
with 0.1 mg/ml poly-L-lysine to reduce cell loss during subsequent selective permeabilization steps. At 24 h
post-transfection, the cells were washed twice with PBS.
For complete permeabilization, cells were fixed in methanol for 6 min
at 4 °C and blocked with 1% BSA in PBS. Proteins were detected by
incubation with 5.2 µg/ml M2 anti-FLAG antibody and 1.4 µg/ml
rabbit anti-
To selectively permeabilize the plasma membrane, cells were incubated
at 4 °C for 45 min in sucrose buffer (1% BSA, 0.3 M sucrose, 0.1 M KCl, 2.5 mM MgCl2, 1 mM EDTA, 10 mM HEPES, pH 7.4) containing 0, 1, 1.5, or 2 µg/ml digitonin and with the same concentrations of M2 FLAG
and Analysis of Truncated Glycosylated Proteins--
For expression
of various NPC1 constructs, COS-7 cells were grown in 10-cm diameter
dishes and transfected using 4 µg of DNA, 20 µl of LipofectAMINE,
and 30 µl of Plus reagent, according to the manufacturer's
recommendations. The transiently expressed proteins were analyzed by
immunoprecipitation and SDS-polyacrylamide gel electrophoresis (PAGE),
essentially as described previously (27). Briefly, at 36 h
post-transfection, the cells were metabolically labeled with
[35S]methionine for 16 h. Cells were harvested using
PBS, 2 mM EDTA and centrifuged at 800 × g
for 10 min. The cell pellet was resuspended in buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 2 mM
EDTA, 10 µg/ml leupeptin, 5 µg/ml pepstatin A, 25 µg/ml
N-acetyl-leucinal-leucinal-norleucinal, 1 µM/ml phenylmethylsulfonyl fluoride, and 0.1 mM Pefabloc), disrupted by sonication using a Branson
sonifier (Smith-Klein, Philadelphia, PA), and centrifuged at
22,000 × g for 10 min. The resultant cell membrane
pellet was resuspended in 0.5 ml of 0.5 M NaCl, 50 mM Tris, 2 mM EDTA, 1% Igepal, supplemented
with the above proteinase inhibitors, using 10 passages through a
22-gauge needle, prior to rocking for 15 min and centrifugation at
22,000 × g for 10 min. The solubilized membranes were
incubated for 1 h with 2.8 µg/ml of anti-FLAG M2, followed by a
45-min incubation with protein G-agarose beads (Roche Molecular
Biochemicals). The immunoprecipitates were washed with buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Igepal, 1 mM EDTA, 0.25% gelatin, 0.2% NaN3),
resuspended in SDS-gel loading buffer, heated for 10 min at 65 °C,
and subjected to SDS-PAGE analysis (28). N-Glycosidase F
digestions were carried out as described previously (27).
Predicted Membrane Topology of NPC1--
The predicted NPC1
protein sequence was analyzed to identify potential transmembrane (TM)
or hydrophilic domains. Using the TMpred algorithm (29) at EMBnet-CH,
the TM domains and topology of the hydrophilic loops of NPC1 were
evaluated. The NPC1 protein is predicted to have 15 TM regions (Fig.
1A). The assignment of predicted TM regions 4-8 is based on
their relative hydrophobicity and their high degree of similarity to
the sterol-sensing domains of SCAP and HMG-R (30, 31) (Fig.
1A, SSD). Based on these observations and computer analyses,
the predicted model for NPC1 topology is shown in Fig. 1B
and consists of 15 TM domains, 5 hydrophilic loops ranging in size from
~50 amino acids (loop B) to ~265 amino acids (loop A), and a
27-amino acid cytoplasmic tail containing a potential
endosomal/lysosomal dileucine targeting motif. In addition, the model
predicts that the amino terminus of NPC1 (loop A) remains in the lumen
upon entering the endoplasmic reticulum (ER).
Characterization of Full-length Epitope-tagged NPC1
Proteins--
To validate the locations of the major hydrophilic loops
A to E in the predicted model (Fig. 1B), a series of
epitope-tagged, full-length NPC1 proteins was constructed and analyzed.
These epitope-tagged NPC1 proteins were expressed in human NPC1
fibroblasts to determine whether the addition of the tags interfered
with their correct processing, transport, and/or function. Transfected cells were costained with filipin and the anti-FLAG antibody to determine whether cells expressing the FLAG-tagged NPC1 were corrected for the NPC1 phenotype, thus producing filipin-negative cells. 50-100
NPC1-positive cells were scored for correction. For all constructs
~60-90% of positive cells appeared to be corrected, with the
exception of the loop C construct for which about 22% of cells were
corrected. Representative fields for each construct are shown in Fig.
3; human NPC1 cells positive for NPC1
expression (left panels; arrows) show a dramatic
decrease in filipin staining (right panels;
arrows) compared with neighboring cells, which are negative
for NPC1 expression. Only one construct, which contained the tag in
loop C (Fig. 3C), did not appear to complement the NPC1
phenotype. Upon careful inspection it is clear that NPC1 containing the
loop C tag does not colocalize with the stored cholesterol and thus is
unable to mobilize cholesterol, similar to results obtained by
mutagenesis of the NPC1 tail (32). These results indicate that the
FLAG-tagged NPC1 proteins are functional and, with the exception of the
loop C construct, were correctly targeted to the endosomal/lysosomal
system, the site of cholesterol accumulation in NPC1 cells. Thus, these
epitope-tagged NPC1 proteins remained functionally active and could be
used to determine the membrane topology of NPC1.
Epitope Mapping by Selective Permeabilization and
Immunofluorescence--
The full-length FLAG-tagged NPC1 constructs
were transiently expressed in COS-7 cells following electroporation.
Because COS-7 cells are easy to transfect and maintain, the majority of
experiments were carried out using these cells. Initial studies showed
no differences in the processing, distribution, and functional
integrity of the expressed proteins, indicating that NPC1 is
topologically identical whether expressed by human or simian cells
(data not shown). To determine the positions of the hydrophilic
domains, selective membrane permeabilization and immunofluorescence
microscopy were used. At 24 h post-transfection, the cells were
subjected to immunofluorescence analysis under one of three conditions: no permeabilization (absence of detergent); selective permeabilization of the plasma membrane using low concentrations of digitonin; or full
permeabilization by methanol fixation. All NPC1 proteins were detected
with the M2 FLAG antibody and a fluorescein-conjugated secondary
antibody. As a control for permeabilization, the intralysosomal protein
Fig. 4 shows the results of
immunofluorescence detection of proteins from the FLAG-tagged
constructs. Representative cells are shown for each condition. In fully
permeabilized cells, a bright, vesicular staining pattern for the
intraorganelle marker
To further confirm the location of loop E, two different approaches
were used. First, an additional construct containing the FLAG tag at a
different position within loop E was expressed and analyzed as above.
The expressed protein did not mislocalize to the plasma membrane. As
shown in Fig. 4F, failure to detect the FLAG-tagged protein
in digitonin-treated cells confirmed the previous conclusion that loop
E is luminal. Second, an antibody directed against loop E was used to
detect its epitope in permeabilized cells that expressed wt NPC1, as
above, lending further support to the luminal location of loop E (data
not shown).
Notably, the above results indicate that elimination of TM3 from our
topological model reverses the orientation of the SSD. In this
orientation, the amino terminus of the SSD is luminal, in complete
agreement with the topological models for the HMG-R and SCAP SSDs,
further supporting the notion that the SSD plays an important role in
cholesterol homeostasis in these proteins. Also, in the absence of TM3,
the position of the NPC1 tail shifts from the cytoplasm to the lumen.
Because the carboxyl-terminal dileucine (LLNF) motif normally functions
in the cytoplasm to target proteins to the endosomal/lysosomal system
(33), the precise number of TM domains and, therefore, the correct
orientation of the tail needed to be established. Thus, a construct in
which the tail was removed and replaced by a FLAG tag was used. As
anticipated, the epitope tag was undetected in nonpermeabilized cells
but was accessible following treatment with digitonin, indicating a
cytosolic location for the tail (Fig. 4G). This is
consistent with other proteins that contain the dileucine lysosomal
targeting motif at their carboxyl terminus. However, for the model to
correctly reflect the location of the tail, one of the proposed TM
domains after TM13, most probably TM14, which has a lower hydropathy
score than TM15 (Fig. 1A), would have to be eliminated as a
potential transmembrane domain.
Expression and N-Glycosylation Analysis of Truncated NPC1
Proteins--
To further confirm the NPC1 topology suggested by the
above results, a number of truncated NPC1 constructs, whose sequences terminated after TM1, 2, 3, 4, 6, and 13 (Fig. 2), were expressed and
analyzed as above. Based on the predicted model, constructs terminating
after TM1, 4, and 6 were expected to have a FLAG tag on the cytosolic
side of the membrane. pLaTM1 protein was undetected in nonpermeabilized
cells but showed distinct plasma membrane staining in digitonin-treated
cells, suggesting that its FLAG tag was cytosolic (Fig.
5A). pLbTM2 and pLcTM3 were
also detected at the plasma membrane and because nonpermeabilized cells
were stained, this indicated a luminal position for both, as expected in the absence of TM3 (Fig. 5, B and C).
Interestingly, the pLdTM4 protein did not localize to the plasma
membrane but showed a predominantly vesicular appearance, suggesting
the presence of a targeting signal that either facilitated retrieval
from the plasma membrane or caused intracellular sequestration (Fig.
5D). Because the tag was detected in digitonin-treated
cells, its cytosolic location was confirmed, further supporting the
absence of TM3. pLdTM6 gave similar results, although its location was
less vesicular and displayed a Golgi and/or ER pattern of distribution
(Fig. 5E). pLeTM13 protein had a distribution similar to
that of TM6 but was not detected in digitonin-treated cells, indicating
a luminal tag position (Fig. 5F). These results confirmed
previous observations that TM3 is not present and that loops A, C, D,
and E are luminal.
Because the results obtained using the truncated NPC1 proteins were in
complete agreement with the results obtained using the full-length
FLAG-tagged NPC1 proteins, we extended our studies to investigate the
level of glycosylation, if any, of each truncated protein. Loops that
are glycosylated would have to enter the ER lumen, lending further
support to their luminal location. The truncated proteins were
expressed, immunoprecipitated, and then deglycosylated using
N-glycosidase F, prior to SDS-PAGE analysis (Fig.
6). Comparison of the estimated molecular
weights of the deglycosylated and untreated proteins allowed the extent
of N-linked glycosylation to be determined (Table
I). For pLaTM1 protein, there was a clear
band shift after N-glycosidase F treatment (Fig. 6A), indicating that the amino terminus (loop A) was heavily
glycosylated and therefore located in the ER lumen. Because the average
molecular mass of one high mannose-type oligosaccharide is about
2 kDa, the shift of approximately 11 kDa probably represents
glycosylation at all five potential sites. The protein expressed from
pLbTM2 also showed an approximate shift of 11 kDa, and because there was no apparent increase in the extent of deglycosylation in comparison to the shift seen for pLaTM1, it was presumed that the glycosylation site in loop B was not utilized and that this loop was cytoplasmic, as
predicted. The results for pLcTM3 expression are also in agreement with
the proposed model. A band shift of about 16 kDa indicated that loop C
was glycosylated, and it was therefore located in the lumen (Fig.
6A).
The results from the larger truncated proteins were less conclusive
than those for loops A to C. The pLdTM6 construct was substituted for
the pLdTM4 construct because of inefficient immunoprecipitation of the
pLdTM4 protein (Fig. 6B), even though immunofluorescent staining indicated that this protein was efficiently expressed in COS-7
cells. Following immunoprecipitation of the pLdTM6 protein, SDS-PAGE
analysis revealed multiple bands, thus complicating estimations of the
band shift (Fig. 6B). A similar problem was encountered with
the pLeTM13 and full-length NPC1 proteins (not shown). However, following deglycosylation, both the pLeTM6 and pLeTM13 proteins showed
a qualitatively greater shift than pLcTM3, indicating that loops D and
E were glycosylated and thus located in the lumen (Fig. 6B
and Table I). The additional bands may represent protein aggregation,
or they may reflect additional forms of post-translational modification. Interestingly, there is a 5- to 8-kDa discrepancy between
the predicted and observed molecular masses for the deglycosylated pLaTM1, pLbTM2, and pLcTM3 proteins (Table I). This may reflect additional post-translational modification of loop A, which contains six consensus sites for myristoylation. Further studies are necessary to determine whether NPC1 is subject to other types of
post-translational processing.
Together, these data independently verify the positions of hydrophilic
domains A-C and confirm the absence of TM3. Removal of TM14 to
correctly orient the tail results in the topological model of NPC1
shown in Fig. 7. This model consists of
13 transmembrane domains, 3 large luminal and 4 small hydrophilic
loops, 6 small cytoplasmic loops, and a cytoplasmic tail. Elimination
of the proposed TM3 (Fig. 1) results in a reversal of the location of domains D and E, thus placing the putative SSD in the same orientation as those of the HMG-R and SCAP proteins. Because the carboxyl terminus
tail is cytosolic, an additional membrane domain has been removed
between TM13 and 15 in our modified model.
The data obtained in these studies are consistent with a
topological model in which NPC1 has 13 TM domains, a luminal amino terminus and a cytosolic carboxyl-terminal tail. The results from the
glycosylation analyses of truncated NPC1 proteins and the results
obtained from investigating the antibody accessibility of FLAG-tagged
NPC1 proteins in selectively permeabilized cells are also in agreement
with this model. The prediction for TM3 has been determined to be
incorrect. These studies have shown that loop D is luminal, not
cytosolic as predicted, thus indicating that loops C and D are fused
and that the proposed TM3 does not span the membrane. In addition, loop
E, which is predicted to be cytosolic, is in fact luminal, suggesting
that the orientation of protein domain from loop D to at least loop E
is reversed. Thus, the orientations of loops D-E as well as
transmembrane domains 4, 6, and 13, and presumably all intervening
sequences, are reversed with respect to the predicted model (Fig.
1B). The loss of TM3 from the topological model is not
surprising in view of the hydrophobicity profile in which TM3 has the
lowest score compared with the other predicted membrane-spanning
regions of NPC1 (Fig. 1A). Finally, immunolocalization of
the NPC1 tail in cells with selectively permeabilized plasma membranes
has confirmed its cytoplasmic location, which is expected, because the
tail contains a dileucine targeting motif (33, 34) that normally
functions in the cytosol to target proteins to an endosomal/lysosomal compartment.
We therefore propose a new model for the topology of NPC1 (Fig. 7),
which is in agreement with our extensive experimental data. In this
model, the original TM3 does not span the membrane and the proposed
loops C and D form one luminal domain. In addition, because our amended
model reverses the orientation of the middle portion of the protein but
maintains the cytosolic tail, the 14th predicted TM domain, which is
very close to TM15, is presumed to be absent. Therefore, our data for
NPC1 is consistent with a membrane glycoprotein that contains 13 transmembrane domains oriented as in Fig. 7, with a luminal amino
terminus and cytosolic carboxyl-terminal tail. The three largest
hydrophilic domains are luminal, and the majority of the remaining
portions of the NPC1 protein is embedded within the membranes. Of
interest, all three luminal hydrophilic domains are approximately the
same length (~250 amino acids), although the importance of this
observation is currently unclear.
The results from our topological analyses have added to current
speculation regarding the possible functional role of the domain from
amino acids 615 to 797 (6, 35). This region of NPC1 is homologous to
the SSD of HMG-R and SCAP, two cholesterol-regulated proteins (22, 31).
In HMG-R, the putative SSD is required for regulation by enzyme
degradation, which occurs in the presence of excess cholesterol (22,
36). In SCAP, a D443N mutation in the SSD causes sterol insensitivity
(20). The topologies of HMG-R and SCAP are known (30, 31), and the
current studies have shown that the topological orientation of the NPC1
SSD is identical to those of HMG-R and SCAP, supporting the notion that the SSD in NPC1 is functionally important. A recent study has confirmed
the functional importance of the NPC1 SSD, using site-directed mutagenesis (32). However, as in the case of HMG-R and SCAP, the exact
function of the SSD is not clear. For example, it is not known whether
this domain directly interacts with sterols, such as cholesterol, or
whether it interacts with another sterol-regulated protein.
Alternatively, the SSD may interact with lipids other than cholesterol.
Further studies are necessary to determine the function of this domain.
Even more extensive homology is seen between the membrane domains of
NPC1 and the morphogen receptor, PTC, which not only share a similar
putative SSD but have additional homologous TM regions. PTC has been
implicated in embryogenesis, carcinoma formation, and cholesterol
homeostasis (37), a role that is further strengthened by observations
that its ligand, sonic hedgehog (24, 25), is active when
covalently bound to cholesterol (38). In light of the fact that the
SSDs of HMG-R, SCAP, and NPC1 are in the same topological orientation,
it is tempting to speculate that the SSD in PTC is oriented similarly,
and this information would facilitate analysis of this receptor. Thus,
knowledge of the topology of NPC1 may also aid in functional studies of
the PTC protein.
In conclusion, we have provided extensive experimental evidence to
support a model for the topology of the NPC1 membrane glycoprotein, indicating that its putative SSD is functionally significant, based on
its identical orientation to homologous sequences. Knowledge of the
topology of NPC1 and the functional data obtained in these studies will
aid the complete elucidation of NPC1 function.
*
These studies were supported in part by a grant from the Ara
Parseghian Medical Research Foundation and a grant from the March of
Dimes Foundation.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.
Published, JBC Papers in Press, May 19, 2000, DOI 10.1074/jbc.M002184200
The abbreviations used are:
NPC, Niemann-Pick
type C;
HMG-R, 3-hydroxy-3-methylglutaryl-coenzyme A reductase;
SCAP, sterol regulatory element binding protein cleavage-activating protein;
ER, endoplasmic reticulum;
SSD, sterol-sensing domain;
TGN, trans-Golgi network;
PTC, Patched;
TM, transmembrane;
wt, wild-type;
PAGE, polyacrylamide gel electrophoresis;
Topological Analysis of Niemann-Pick C1 Protein Reveals That the
Membrane Orientation of the Putative Sterol-sensing Domain Is Identical
to Those of 3-Hydroxy-3-methylglutaryl-CoA Reductase and Sterol
Regulatory Element Binding Protein Cleavage-activating
Protein*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (37K):
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Fig. 1.
Hydropathy plot and predicted topology
of NPC1. A, hydropathy profile of NPC1 showing the 15 predicted TM domains numbered sequentially. The box above TM
domains 4-8 indicates the location of the putative SSD. B,
a pictorial representation of the predicted topology of NPC1 is shown
with cylinders denoting TM domains, including the putative
SSD, in boldface. Heavy lines depict the large
hydrophilic portions (loops A-E) above and below the
cylinders to indicate their luminal or cytosolic location,
respectively. The letter F within the ovals
denotes the locations of the FLAG tags.
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Fig. 2.
FLAG-tagged truncated NPC1 constructs used in
these studies. The positions of FLAG peptide tags introduced into
truncated NPC1 proteins are shown. Their predicted topology is
indicated by cylindrical transmembrane domains and hydrophilic loops
(heavy lines), which are either luminal or cytosolic in
location (above or below the TM domains, respectively).
-galactosidase A (-Gal A) polyclonal antibody (27) in
blocking solution for 45 min, followed by a wash with PBS, and then
incubation with 2 µg/ml anti-mouse IgG-FITC and 2 µg/ml anti-rabbit
IgG-rhodamine-X secondary antibodies in blocking solution. After
further washing, the cells were mounted in Fluoromount G and
visualized using a Nikon Eclipse fluorescence microscope equipped with
a charge-coupled device (CCD) camera (Nikon, Melville, NY).
-Gal A antibodies used for complete permeabilization. The cells
were then fixed with methanol and incubated in blocking solution for 30 min before addition of secondary antibodies and visualization as above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (33K):
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Fig. 3.
Correction of human NPC1 cells by transient
expression of epitope-tagged NPC1 proteins. Human NPC1 fibroblasts
were transiently transfected with constructs containing full-length
NPC1 cDNAs epitope-tagged in loops A-E and cytoplasmic tail
(A-F, respectively). NPC1-positive cells were identified by
immunofluorescence microscopy using an anti-FLAG antibody (NPC1
panels; arrows). Cholesterol storage was visualized by
filipin staining (Filipin panels). Arrows
indicate corrected cells, positive for NPC1 expression.
-Gal A was detected using a specific polyclonal antibody and a
rhodamine-conjugated secondary antibody. Pilot studies indicated that
concentrations of digitonin below 3 µg/ml specifically permeabilized the plasma membrane but not intracellular membranes, as demonstrated by
the inability of the -Gal A antibody to detect its target protein
within lysosomes (not shown). Under these conditions, cytosolic protein
epitopes were detected while luminal ones, including the -Gal A
control protein, were negative. Nonpermeabilized cells were used as a
negative control while transfection efficiency and locations of
expressed proteins were analyzed in fully permeabilized cells.
-Gal A was observed, indicating complete
antibody access. However, in digitonin-permeabilized cells there was no
-Gal A staining, confirming that permeabilization was selective for
the plasma membrane. The six full-length NPC1 proteins, containing FLAG
tags in domains A-E and the cytoplasmic tail, were analyzed (Fig. 4). Detection of the loop A-tagged NPC1 protein in the fully but not the
partially permeabilized cells indicated that loop A was luminal. Similarly, detection of the loop B-tagged protein in
digitonin-permeabilized cells, accompanied by a negative -Gal A
signal, indicated that this epitope, and thus loop B, was cytosolic
(Fig. 4B). In contrast, the FLAG tags in loops C and D were
both undetectable in digitonin-treated cells, suggesting their luminal
location (Fig. 4, C and D). These results
indicated that the orientation of loops D and E was reversed with
respect to our predicted model, implying that the predicted TM3 domain
does not span the membrane. This suggests that loops C and D comprise a
single luminal loop and loop E is shifted from a cytoplasmic position
to the luminal one. The results for loop E confirmed its luminal
location, assuming elimination of TM3. However, its distribution was
surprising. This protein was detected in large quantities at the plasma
membrane of nonpermeabilized cells (Fig. 4E). Interestingly,
the membrane staining appeared speckled and uneven, perhaps indicating
that some form of aggregation occurred at the cell surface. However,
this phenomenon does not apparently interfere with NPC1 function,
because the protein successfully reversed the cholesterol storage
observed in filipin-stained human fibroblasts (Fig. 3E). The
mechanism by which a FLAG tag in this luminal loop causes
mislocalization of NPC1 to the plasma membrane is currently being
investigated.
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Fig. 4.
Immunolocalization of FLAG epitopes in NPC1
proteins of selectively permeabilized cells. The FLAG-tagged,
full-length NPC1 proteins were transiently expressed in COS-7 cells.
Proteins were detected by immunofluorescence analysis in
nonpermeabilized (Non), completely permeabilized
(Complete), and selectively permeabilized
(Digitonin) cells to determine the accessibility of the FLAG
antibody to the FLAG tags. NPC1 and the control protein were
colocalized using the FLAG antibody and -Gal A antibody,
respectively. Proteins tagged at loops A, C, and D (panels
A, C, and D, respectively) were undetected
in selectively permeabilized cells, indicating a luminal location,
whereas the loop B and tail protein was detected (panels B
and G, respectively), showing that they were cytosolic. For
loop E two different tagged NPC1 proteins were expressed (panels
E and F), and both showed a luminal location. The first
was observed at the plasma membrane surface of nonpermeabilized cells,
whereas the second protein was undetectable in permeabilized
cells.
View larger version (36K):
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Fig. 5.
Localization of FLAG epitopes in truncated
NPC1 proteins in semipermeabilized cells. The FLAG-tagged
truncated NPC1 proteins were transiently expressed in COS-7 cells and
detected by immunofluorescence, as described in Fig. 4. In
A-F, expression of proteins from the pLaTM1, pLbTM2,
pLcTM3, pLdTM4, pLdTM6, and pLeTM13 constructs (see Fig. 2),
respectively, in nonpermeabilized (Non), completely
permeabilized (Complete), and selectively permeabilized
(Digitonin) cells is shown. The pLaTM1, pLdTM4, and pLdTM6
proteins were detected in selectively permeabilized cells indicating
their cytosolic location. Proteins from pLbTM2 and pLcTM3 were detected
in nonpermeabilized cells at the plasma membrane, and that of pLeTM13
was undetected in selectively permeabilized cells, confirming that
their FLAG-tagged epitopes were luminal.
View larger version (76K):
[in a new window]
Fig. 6.
Determination of glycosylated domains by
N-glycosidase F treatment and SDS-PAGE analysis of
truncated NPC1 proteins. The truncated NPC1 proteins shown in Fig.
1A were transiently expressed in COS-7 cells and
metabolically radiolabeled using [35S]methionine. The
NPC1 proteins were immunoprecipitated with the M2 FLAG antibody and
were subsequently treated with N-glycosidase F (+ lanes) and then subjected to SDS-PAGE analysis, followed by
autoradiography. Proteins were separated through either 10%
(A) or 6% (B) acrylamide gels. B,
arrows indicate the approximate positions of the NPC1
protein and solid horizontal lines show the positions of the
molecular mass marker bands indicated on the left. All
untreated NPC1 proteins appeared as diffuse bands due to heterogeneity
in the extent of their glycosylation.
Summary of results from the glycosylation analyses of the truncated
NPC1 proteins
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Fig. 7.
Topological model for NPC1. A
topological model based on experimental data obtained in these studies
is shown. Roman numerals indicate transmembrane domains; the
SSD spans the TMIII-TMVII region; a key is shown at the
bottom of the figure. Hydrophilic loops are labeled sequentially from
A (amino-terminal) to N (carboxyl-terminal).
Large loops A, C, and I span the amino acid sequences for residues
25-264, 370-621, and 854-1098, respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
FOOTNOTES
To whom correspondence should be addressed: Dept. of Human
Genetics, P. O. Box 1498, Mount Sinai School of Medicine, Fifth Ave.
at 100th St., New York, NY 10029. Tel.: 212-659-6720; Fax: 212-348-3605; E-mail: Ioanny01@doc.mssm.edu.
ABBREVIATIONS
-Gal A, -galactosidase A;
FITC, fluorescein isothiocyanate;
PBS, phosphate-buffered saline;
BSA, bovine serum albumin.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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