From the Institut de Génétique et de Biologie
Moléculaire et Cellulaire, UPR 6520 CNRS/U184
INSERM/Université Louis Pasteur, BP 163, 67404 Illkirch, C.U. de
Strasbourg, France, § Laboratoire de Physiologie
Générale, URA 1446 CNRS, Université Louis
Pasteur, 67084 Strasbourg Cedex, France, ¶ Department of
Biochemistry, Sciences II, University of Geneva, 30 quai E. Ansermet,
1211 Geneva 4, Switzerland, Service d'Anatomie Pathologique
Générale, Centre Hospitalier Universitaire de Hautepierre,
67098 Strasbourg, France, and ** U189 INSERM/Fondation
Gillet-Mérieux, Faculté de Médecine et Centre
Hospitalier Lyon-Sud, 69921 Oullins Cedex, France
Received for publication, July 14, 2000, and in revised form, October 26, 2000
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ABSTRACT |
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MLN64 is a transmembrane protein that
shares homology with the cholesterol binding domain (START domain) of
the steroidogenic acute regulatory protein. The steroidogenic
acute regulatory protein is located in the inner membrane of
mitochondria, where it facilitates cholesterol import into the
mitochondria. Crystallographic analysis showed that the START domain of
MLN64 is a cholesterol-binding domain. The present work was
undertaken to determine which step of the intracellular cholesterol
pathway MLN64 participates in. Using immunocytofluorescence, MLN64
colocalizes with LBPA, a lipid found specifically in late endosomes.
Electron microscopy indicates that MLN64 is restricted to the limiting
membrane of late endosomes. Microinjection or endocytosis of specific
antibodies shows that the START domain of MLN64 is cytoplasmic.
Deletion and mutagenesis experiments demonstrate that the
amino-terminal part of MLN64 is responsible for its addressing.
Although this domain does not contain conventional dileucine- or
tyrosine-based targeting signals, we show that a dileucine motif
(Leu66-Leu67) and a tyrosine residue
(Tyr89) are critical for the targeting or the proper
folding of the molecule. Finally, MLN64 colocalizes with cholesterol
and Niemann Pick C1 protein in late endosomes. However, complementation
assays show that MLN64 is not involved in the
Niemann Pick C2 disease which, results in cholesterol lysosomal
accumulation. Together, our results show that MLN64 plays a role at the
surface of the late endosomes, where it might shuttle cholesterol from
the limiting membrane to cytoplasmic acceptor(s).
Cholesterol, the essential sterol found in vertebrates, has
several functions, which include modulating the fluidity and
permeability of membranes, serving as precursor for steroid hormones
and bile acid synthesis, and covalently modifying proteins. Animal
cells obtain cholesterol by de novo synthesis in the
endoplasmic reticulum or receptor-mediated uptake of plasma
lipoproteins (1). Most of cells acquire cholesterol from low density
lipoprotein (LDL).1 LDL is
endocytosed and transported to early endosomes and then to late
endosomes/lysosomes for degradation. Free cholesterol generated from
LDL in late endosomes/lysosomes is then redistributed in the cell.
Although the regulation of cholesterol content in cells has been
extensively studied, little is known about the mechanisms of its
intracellular transport. Only a few molecules involved in this pathway
have been identified, including steroidogenic acute regulatory protein
(StAR), whose expression is tissue-specific, or the recently identified
Niemann Pick C1 protein (2, 3).
MLN64 cDNA was identified from a breast cancer-derived metastatic
lymph node cDNA library by differential hybridization using malignant (metastatic lymph node) versus nonmalignant
(breast fibroadenoma and normal lymph node) tissues. Chromosomal
mapping showed that the MLN64 gene is located in the q12-q21
region of the long arm of chromosome 17 (4). This region is altered in
20-30% of breast cancers, the most common modification being the
amplification of the proto-oncogene c-erbB-2 (5-7). The
proto-oncogene c-erbB-2 is a marker of poor prognosis and
tumor aggressiveness in breast cancers; its overexpression in tumors
has been correlated to hormone therapy failure (8). In breast cancers,
an invariable coamplification and consequent overexpression of
MLN64 and c-erbB-2 in 22.5% of the cases tested
(98 cases) was observed, suggesting that overexpression of
MLN64 could be of clinical relevance for breast cancer
development and/or progression (9).
MLN64 cDNA encodes for a protein of 445 residues containing four
potential transmembrane regions at its amino-terminal part. In
addition, MLN64 shares a conserved COOH-terminal region with StAR called the StAR homology domain (SHD) (10). Recently, a larger domain, including the SHD, has been defined as the StAR-related lipid transfer (START) domain (20). A wide variety of proteins involved
in different cell processes possess a START domain, such as the
phosphatidylcholine transfer protein, the signal-transducing protein
p122-RhoGAP, or a putative acyl-CoA thioesterase. Interestingly, it has
been shown that mutations in the StAR gene, which lead to COOH-terminal
truncated proteins in which the SHD domain is deleted, are responsible
for congenital adrenal hyperplasia, a disease characterized by severely
impaired steroidogenesis (12-14). The functional relationship between
MLN64 and StAR was previously investigated. It was shown that, like
StAR, MLN64 can enhance steroidogenesis in an in vitro
assay. Removal of the SHD domain resulted in the complete loss of
steroidogenic activity, while removal of the NH2-terminal
region of MLN64 increased this activity (15).
StAR is a mitochondrial protein that regulates the acute production of
steroids in the adrenal glands and gonads in response to corticotropin
and luteinizing hormone, respectively. StAR regulates the rate-limiting
step of steroidogenesis, which is the transfer of cholesterol from the
outer to the inner mitochondrial membrane, where it is converted into
pregnenolone (16). Investigations into the mechanism of action of StAR
have shown that StAR is a sterol transfer protein that acts directly on
the mitochondria (17, 18). In addition, StAR has been shown to be a
sterol transfer protein in vitro (19). Recently, the
three-dimensional structure of the START domain of MLN64 was solved,
and its ability to bind cholesterol at an equimolar ratio was reported
(20).
MLN64 is likely to be involved in cholesterol transport, and
cholesterol is the precursor of all steroid hormones. Since
c-erbB-2 and MLN64 are coamplified and
overexpressed in breast cancer, a role for MLN64 in facilitating
intratumoral biosynthesis of steroid hormones can be postulated, which
may relate to the hormonal resistance of part of the
c-erbB-2-expressing tumors.
This study was aimed at identifying the exact subcellular localization
of MLN64 to define where MLN64 is likely to play a key role during
cholesterol trafficking.
Cell Culture--
The MCF7 human breast cancer and the SK-OV-3
human ovarian cancer cell lines were provided by the American Type
Culture Collection (ATCC, Manassas, VA) and routinely maintained in our
laboratory and cultured as recommended. The Chinese hamster ovary (CHO)
cell line was a kind gift of Dr. L. Liscum (Tufts University, Boston, MA).
The NPC2 fibroblast strain was obtained from case 16 in Vanier et
al. (21). Human lipoprotein-deficient serum and human LDL were
prepared in the laboratory as described previously (21).
Plasmids--
To produce stable cell lines expressing MLN64, a
1.5-kilobase BamHI fragment corresponding to the open
reading frame of the human MLN64 cDNA (GenBankTM
accession number X80198) was cloned into the BamHI site of the pCMVneo vector (22), thus generating the pCMVneo-MLN64 plasmid. To
map the domain responsible for the sorting of MLN64,
NH2-terminal deletion mutants of pSG5 MLN64 (10) were
constructed. pSG5 MLN64-(30-445) was obtained in two steps. An
intermediate plasmid was constructed by creating two in-frame
NheI sites at amino acid positions 2 and 28 by site-directed
mutagenesis using the QuikChange Site-Directed Mutagenesis kit
(Stratagene, La Jolla, CA) and the synthetic oligonucleotides 1 (5'-GGGGCCCACC AGGATGGCTA GCCTGCCCAG GGAGCTGA) and 2 (5'-GGGCTCCTCA CTGTCCGCTA GCCAGAGCCT CTCCTCGC). Sequences corresponding to amino acids
2-29 were then excised by a NheI digest, and the remaining plasmid was recircularized to produce pSG5 MLN64-(30-445). pSG5 MLN64-(47-445) and pSG5 MLN64-(54-445) were obtained in a similar way. For pSG5 MLN64-(47-445), the two in-frame NheI sites
at amino acid positions 2 and 45 were obtained by site-directed
mutagenesis using the synthetic oligonucleotides 1 and 3 (5'-GCCTGAGAAG
CGAAGGGCTA GCTCTGATGT CCGCCGCAC). For pSG5 MLN64-(54-445), the two
in-frame NheI sites at amino acid positions 2 and 52 were
obtained by site-directed mutagenesis using the synthetic
oligonucleotides 1 and 4 (5'-CTCTGATGTC CGCCGCGCTA GCTGTCTCTT
CGTCACCT). pSG5 MLN64-(1-218) was obtained by insertion of an in-frame
stop codon at amino acid position 219 by site-directed mutagenesis
using the following oligonucleotide: 5'-GCAGGGTCTG ACTAGTAATC AGATGAAG.
pSG5 MLN64-(1-171) was obtained by insertion of an in-frame stop codon
at amino acid position 172 by site-directed mutagenesis using the
following oligonucleotide: 5'-GGTTCCTTGA CTTTTAAATC CTACCCCAGG. pSG5
MLN64-(1-145) was obtained by insertion of an in-frame stop codon at
amino acid position 146 by site-directed mutagenesis using the
following oligonucleotide: 5'-CTCTGAGCTG CTTTAAAAAG GGGCATTTG.
pSG5 MLN64 LL1 was constructed by mutating
Leu61-Leu62 to
Ala61-Ser62 by site-directed mutagenesis using
the following synthetic oligonucleotide: 5'-ACTTCGTCACC TTCGACGCTA
GCTTCATCTC CCTGCTC. Similarly, pSG5 MLN64 LL2 was constructed by
mutating Leu66-Leu67 to
Ala66-Ser67 (5'-CCTGCTCTTC ATCTCCGCTA
GCTGGATCAT CGAACTG), pSG5 MLN64 LL3 by mutating
Leu109-Leu110 to
Ala109-Ser110 (5'-CTTCCGCTTC TCTGGAGCTA
GCCTAGGCTA TGCCGTGC), pSG5 MLN64 LL4 by mutating
Leu144-Leu145 to
Ala144-Ser145 (5'-GGTCATCCTC TCTGAGGCTA
GCAGCAAAGG GGCATTTG), pSG5 MLN64 LL5 (and Y3) by mutating
Tyr152-Leu153 to
Ala152-Ser153 (5'-CAAAGGGGCA TTTGGCGCTA
GCCTCCCCAT CGTCTCTT), pSG5 MLN64 Y1 by mutating Tyr89 to
Val89 (5'-GCAGGAGATC ATCCAGGTTA ACTTTAAAAC TTCC), pSG5
MLN64 Y2 by mutating Gly112-Tyr113 to
Ala112-Ser113 (5'-CTCTGGACTG CTCCTAGCTA
GCGCCGTGCT GCAGCTCC).
The pCR3.1 NPC1 expression plasmid was a kind gift of Dr. E. Ikonen
(National Public Health Institute, Helsinki, Finland).
Stable Cell Lines--
MCF7 cells were transfected by calcium
phosphate coprecipitation with either the pCMVneo-MLN64 expression
vector or the pCMVneo vector, both linearized by HindIII.
The medium was changed after 20 h, and on the following day, the
selection was begun in the same medium supplemented with 400 µg/liter
G418 (Life Technologies, Inc.). After 2 weeks of selection, resistant
clones were subcloned, and expression of MLN64 was assessed by Western
blot as described previously (10).
Antibodies--
The rabbit polyclonal 605 (pAbMLN64-Ct) and
mouse monoclonal 2BE2F4 (mAbMLN64-Ct) antibodies were raised against
the synthetic peptide HSAKPPTHKYVRGENG corresponding to residues
369-384 of human MLN64 as described previously (10, 23). In addition, a rabbit polyclonal antibody 1611 (pAbMLN64-Nt) was raised against the
peptide MSKLPRELTRDLERSLPAV corresponding to residues 1-19 of human
MLN64. The 6C4 monoclonal anti-LBPA antibody was described previously
(24). The M1G8 monoclonal anti-cathepsin D antibody was a generous gift
from Dr. M. Garcia and Prof. H. Rochefort (INSERM U148, Montpellier,
France). The monoclonal anti-CD63 antibody was purchased from Chemicon
(Temecula, CA). The rabbit polyclonal anti-NPC1 antibody was a kind
gift of Dr. E. Ikonen (National Public Health Institute, Helsinki,
Finland). Cy3-conjugated affinity-purified donkey anti-mouse IgG- and
Cy2-conjugated affinity-purified goat anti-rabbit IgG were purchased
from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).
Biotin-conjugated donkey anti-mouse antibody was purchased from Vector
Laboratories (Burlingame, CA).
Immunocytofluorescence--
SK-OV-3 and MCF7 cells were grown to
70% confluence on glass coverslips. After washing with
phosphate-buffered saline (PBS), cells were fixed 10 min at room
temperature in 4% paraformaldehyde in PBS and permeabilized for 10 min
with 0.1% Triton X-100 in PBS. After blocking in 1% bovine serum
albumin in PBS, cells were incubated at room temperature with the
primary antibodies, either pAbMLN64-Ct or mAbMLN64-Ct together with
anti-cathepsin D M1G8, anti-LBPA 6C4, anti-CD63, or anti-NPC1. Cells
were washed three times in PBS and incubated 1 h with Cy3- and
Cy2-conjugated secondary antibodies (1:400). Cells were washed three
times in PBS, and in some cases, nuclei were counterstained with
Hoechst-33258 dye. Slides were mounted in Aqua Poly/Mount (Polysciences
Inc., Warrington, PA). Observations were made with a confocal
microscope (Leica TCS4D; Heidelberg, Germany) or with a fluorescence
microscope (Leica DMLB 30T).
Immunoelectron Microscopy--
MCF7 cells were grown on glass
coverslips to 70% confluence. After washing with PBS, cells were fixed
45 min at room temperature in 2.5% glutaraldehyde in PBS followed by a
15-min incubation in 1% sodium borohydrure in PBS. Cells were then
permeabilized for 20 min with 0.1% saponin in PBS. After blocking for
30 min in 5% normal donkey serum in PBS, cells were incubated
overnight at 4 °C with mAbMLN64-Ct antibody (1:1000 dilution). After
three washes with PBS, cells were incubated 1.5 h with
biotin-conjugated donkey anti-mouse antibody. Cells were then processed
for immunoperoxidase labeling using the Vectastain Elite ABC standard
kit (Vector Laboratories, Burlingame, CA) according to the
manufacturer's instructions, which was followed by incubation with
0.0125% diaminobenzidine and 0.005% H2O2 in
0.05 M Tris buffer, pH 7.6. Cells were postfixed with 2.5%
glutaraldehyde in PBS and then with 2% OsO4 for 30 min. They were then dehydrated with graded concentrations of ethanol and
conventionally embedded in epoxy resin. Glass coverslips were dissolved
in hydrofluoric acid, and ultrathin sections were observed under
a transmission electron microscope (Hitachi 7500, Japan).
Endocytosis and Microinjection of Antibodies--
MCF7 cells, on
glass coverslips, were washed three times with Dulbecco's modified
Eagle's medium without serum.
For endocytosis of antibodies, cells were incubated for 1 h with
either anti-cathepsin D M1G8, mAbMLN64-Ct, or pAbMLN64-Nt antibodies. Antibodies were used at the same concentration as for immunofluorescence.
Antibodies were microinjected into the cell cytoplasm together with
lysine fixable dextran fluorescein 40000 MW (Molecular Probes, Inc.,
Eugene, OR) as described previously (25). Microinjections were followed
by an incubation of the cells for 1 h. Antibodies were used 10 times more concentrated than for immunofluorescence.
Detection of either internalized or microinjected antibodies was
performed by incubating fixed cells with the secondary antibody as
described above (see "Immunocytofluorescence").
Transfection of NPC2 Fibroblasts and Filipin
Staining--
Niemann-Pick C2 fibroblasts were grown on glass
coverslips to 70% confluence and were transfected with the expression
vector pSG5 MLN64 with FuGENE6 transfection reagent (Roche Molecular Biochemicals). After transfection, cells were cultured in medium supplemented with 5% lipoprotein-deficient serum for 24 h and then changed to medium supplemented with 5% lipoprotein-deficient serum and 50 µg/ml human LDL for an additional 24 h of culture. Cells were then processed for immunofluorescence to identify
transfected cells with mAbMLN64-Ct anti-MLN64 as described above (see
"Immunocytofluorescence") except that permeabilization with Triton
X-100 was omitted. Staining of free cholesterol was performed after
fixation using 50 µg/ml filipin (Sigma) for 30 min.
MLN64 Is a Late Endosomal Protein--
To assess the subcellular
localization of the MLN64 protein, we performed indirect
immunofluorescence experiments using two cancer-derived cell lines. A
human ovarian cancer cell line, SK-OV-3, that overexpresses MLN64 at
both the RNA and protein levels and a human breast cancer cell line,
MCF7, that does not express detectable levels of MLN64 mRNA were
chosen (4, 10). A punctuate cytoplasmic staining was observed using an
anti-MLN64 antibody in the SK-OV-3 cells (Fig.
1A, a), whereas no
protein expression could be detected in MCF7 cells (Fig. 1A,
c). MCF7 cells overexpressing MLN64 (MCF7/MLN64) showed a
similar punctuate staining as observed for SK-OV-3 cells (Fig.
1A, b). To identify the subcellular structures
where the MLN64 protein resides, MCF7/MLN64 cells were colabeled with
antibodies directed against known organelle-resident proteins or
lipids. Double staining experiments using MLN64 and cathepsin D or
LampI, two markers of both endosomes and lysosomes (26, 24), showed a
large overlap of both signals, the relative quantity of both proteins
being variable from one vesicle to another (Fig. 1B, a-c, and data not shown). Double labeling experiments with
MLN64 and CD63, a marker for all types of endosomes (27), showed also a
large overlap of both signals, with the relative quantity of both
proteins again being variable from one vesicle to another (Fig.
1B, d-f). Colocalization of MLN64 and LBPA, a
lipid restricted to late endosomes (24), showed that both signals
completely overlapped (Fig. 1B, g-i). Thus,
among the endosome/lysosome vesicles, MLN64 appeared to specifically
reside in the late endosomes. In addition, we noted that the
immunocytofluorescence staining of MLN64 appeared as a ring on most of
the endosomes (Fig. 1B, g-i). When the cells are
costained with anti-LBPA, a lipid exclusively located in late endosomes
(24), MLN64 staining surrounded LBPA-stained spheres (Fig.
1B, i).
The Transmembrane Helices-containing Domain Is Responsible for the
Addressing of MLN64 to Late Endosomes--
To identify endosomal
addressing signals, we analyzed the MLN64 protein sequence for known
motifs. MLN64 possesses four putative transmembrane domains (10). As a
control of the functionality of these transmembrane domains, we decided
to analyze potential N-glycosylation sites, which have been
shown to direct luminal proteins to endosomes/lysosomes (28). MLN64
contains two potential sites of N-glycosylation at positions
219 and 311; we performed site-directed mutagenesis of these amino
acids (Asn219
Three NH2-terminal deletions were constructed by
site-directed mutagenesis in the eukaryotic expression vector pSG5
MLN64 (Fig. 2A). Subcellular
localization was assayed by transient transfection in MCF7 cells
followed by immunofluorescence using pAbMLN64-Ct. The localization of
mutated proteins was compared with a marker of late endosomes (data not
shown). Deletions of the NH2-terminal part to position 29 or 46 (pSG5 MLN64-(30-445) and pSG5 MLN64-(47-445)) showed a
localization (Fig. 2B, b) similar to the
wild-type protein (Fig. 2B, a). Further
NH2-terminal deletion (pSG5 MLN64-(54-445)) involving the
transmembrane domains of the protein led to a mislocalization of the
protein, which then exhibited a reticular pattern characteristic of the
endoplasmic reticulum (Fig. 2B, c). Similarly,
three COOH-terminal deletions were constructed (Fig. 2A),
and subcellular localization was assayed using pAbMLN64-Nt. The
localization of mutated proteins was compared with a marker of late
endosomes (data not shown). When the COOH-terminal part of the protein
is deleted from amino acids 219 or 172 to 445 (pSG5 MLN64-(1-218),
pSG5 MLN64-(1-171)), the protein remained in endosomes (Fig.
2B, d and e). Further deletion (pSG5
MLN64-(1-145)) including the transmembrane helices-containing domain
led to a modified localization of the protein, which then resided in
the Golgi apparatus (Fig. 2B, f).
NH2- and COOH-terminal deletions showed that the minimal
domain for sorting to the endosome was included within amino acids
47-171 of the MLN64 protein.
Mutation of Dileucine 66-67 or of Tyrosine 89 Impairs the
Localization of MLN64--
Further deletions of the protein would
include the transmembrane helices-containing domain and could therefore
modify the overall structure of MLN64 and its anchorage to the
membrane. Thus, we decided to perform point mutations within the
sequence located from residue 47 to 172 to identify amino acids
involved in the sorting. Most of the membrane proteins located in the
endosome/lysosome exhibit dileucine or tyrosine type addressing signals
(30). We therefore decided to systematically mutate tyrosine and
dileucine motifs within the minimal region necessary to target the
protein to endosomes. Five dileucine motifs (LL) are present in this
region of the protein (Fig.
3A, LL1-LL5). Mutations of
four of them (LL1 and LL3-LL5) did not change the localization of the
protein (Fig. 3B, a; data not shown) when
compared with a marker of late endosomes (data not shown). In contrast,
mutation of LL2 located at positions 66 and 67 led to a mislocalization
of the protein to the endoplasmic reticulum (Fig. 3B,
b). Three different tyrosines (Y1-Y3) are present between
amino acids 47 and 172. Y2 is included in the previously studied
GYXXZ motif (Table I). Mutations of Y2 or Y3 did not change
the subcellular localization of MLN64 (Fig. 3B,
c; data not shown) when compared with a marker of late
endosomes (data not shown). On the contrary, the mutation of Y1
(residue 89) modified the subcellular localization of the protein,
which then resided in the endoplasmic reticulum (Fig. 3B,
d). Thus, two motifs, the dileucine motif 66-67 and the
tyrosine 89 are important for proper addressing of MLN64 to late
endosomes.
MLN64 Is Located on the Limiting Membrane of Late
Endosomes--
The immunocytofluorescence staining of MLN64 (Fig.
1B, g-i) suggested that MLN64 localizes to the
limiting membrane of endosomes. To confirm this localization of MLN64,
we performed immunoelectron microscopy on MCF7/MLN64 cells using the
mAbMLN64-Ct antibody. The immunolabeling was restricted to round
vesicles with an endosome shape (31) that were about 700 nm in diameter
(Fig. 4A). These labeled
vesicles were scattered through the cytoplasm, with frequent perinuclear accumulation (Fig. 4A). The labeling of MLN64
was restricted to the cytoplasmic surface of the vesicles. No internal membranous structures of the endosomes were labeled. Moreover, the
labeling on the limiting membrane of endosomes appeared as patches
(Fig. 4B), which showed an uneven localization of the protein within the membrane. Additional punctuate labeling, which was
not associated with clear vesicular structures, appeared in the
cytoplasm of cells (Fig. 4B). The labeling of MLN64 is
mainly observed on the cytoplasmic face of the limiting membrane of
endosomes. No immunoreactivity was detected in the parental MCF7 cells,
indicating that the staining was specific for MLN64 (data not
shown).
The START Domain and the NH2 Terminus of MLN64 Are
Cytoplasmic--
Immunoelectron microscopy strongly suggested that the
START domain of MLN64 is facing the cytoplasm and not the lumen of
endosomes. To confirm this hypothesis, we first performed endocytosis
of mAbMLN64-Ct antibody or anti-cathepsin D by MCF7/MLN64 living cells.
Cathepsin D is a protease located in the lumen of endosomes/lysosomes (32). Antibodies directed against cathepsin D can be taken up by living
cells permitting the visualization of the protein in endosomes/lysosomes (26). Indeed, a punctuate staining typical of
cathepsin D was observed (Fig.
5A, a). On the
opposite, no staining could be detected for MLN64 (Fig.
5A, b). The complementary experiment consisting
of microinjection, into the cytoplasm of MCF7/MLN64 living cells, of
anti-cathepsin D or mAbMLN64-Ct antibodies was performed. Cells
microinjected with anti-cathepsin D exhibited no staining (Fig.
5B, a-c). On the opposite, cytoplasmic
microinjection of mAbMLN64-Ct led to a punctuate cytoplasmic signal
(Fig. 5B, d-f). Since the antibody used is
directed against the COOH terminus of MLN64, these findings indicate
that the START domain of MLN64 is cytoplasmic (Fig. 5C).
MLN64 possesses four putative transmembrane domains, which implies that
if the COOH terminus of the protein is cytoplasmic then the
NH2 terminus is also in the cytoplasm. We performed the
same experiment using the pAbMLN64-Nt antibody directed against the
NH2 terminus of the protein. No staining was obtained when
the pAbMLN64-Nt antibody was taken up by endocytosis by MCF7/MLN64
living cells (Fig. 5A, c), while cytoplasmic
microinjection of pAbMLN64-Nt antibody led to a punctate cytoplasmic
signal (Fig. 5B, g-i). No staining was present
when mAbMLN64-Ct antibodies were neutralized with their cognate antigen
prior to microinjection in MCF7/MLN64 cells or when mAbMLN64-Ct or
pAbMLN64-Nt antibodies were microinjected into MCF7 cells (data not
shown). Taken together, these results show the NH2 terminus
and the COOH terminus of MLN64 are cytoplasmic (Fig.
5C).
MLN64 Colocalizes with Cholesterol and NPC1 but Does Not Correct
Late Endosomal Accumulation of Cholesterol in NPC2 Patient
Fibroblasts--
Niemann-Pick C disease is characterized by a late
endosomal accumulation of LDL-derived cholesterol (2, 33, 34). Two complementation groups were identified, and NPC1, the protein defective
in the first complementation group of NPC disease, is located in the
endosomes/lysosomes (35, 36). Since MLN64 is located in the late
endosomes, we postulated that MLN64 might be a candidate
gene for the defect of the second complementation group of Niemann-Pick
C disease (NPC2). We first performed colocalization experiments in CHO
cells between MLN64 and cholesterol using filipin, a fluorescent
molecule that specifically binds free cholesterol (33). The majority of
MLN64-positive vesicles were also positive for cholesterol (Fig.
6A, a-c). We also
examined the effect of drugs that block the transport of cholesterol
out of lysosomes on the localization of MLN64 and cholesterol. We
treated CHO cells with U18666A, a hydrophobic amine that induces the
accumulation of cholesterol in lysosomes (1). In these cells, MLN64 was present on the surface of cholesterol-loaded vesicles (data not shown).
We next performed colocalization experiments between MLN64 and NPC1.
MLN64 and NPC1 colocalized in CHO cells, the relative quantity of both
proteins being variable from one vesicle to another (Fig.
6A, d-f). Since MLN64 and NPC1 were located in
the same subset of endosomes, we checked if the expression of MLN64 in NPC2 fibroblasts could complement the phenotype of accumulation of
cholesterol in late endosomes. NPC2 fibroblasts were transiently transfected with MLN64, fed with LDL, processed for immunofluorescence, and stained with filipin. MLN64 was present at the surface of cholesterol-loaded late endosomes in NPC2 cells (Fig. 6B,
a-c). None of the 50 cells transfected with the MLN64
expression vector exhibited a corrected late endosomal accumulation of
cholesterol (Fig. 6B, a-c). In addition, the
cDNA encoding MLN64 was sequenced from NPC2 fibroblasts, and no
mutations were found (data not shown). Therefore, alteration of
MLN64 is not responsible for NPC2 disease.
MLN64 and StAR belong to a novel protein family involved in
cholesterol transport. They share a specific domain, the START domain.
This 200-210-amino acid domain is present in a wide range of proteins
involved in diverse cell functions (11), including potential
cholesterol transfer. Among the START domain-containing proteins, MLN64
and StAR are the most closely related in sequence (11). Mutations or
truncations within the START domain of StAR cause congenital adrenal
hyperplasia, an autosomal recessive disease characterized by markedly
impaired gonadal and adrenal steroid hormone synthesis. This finding
suggests that this domain is functionally relevant. In addition, the
START domain of MLN64 and StAR was shown to bind cholesterol at an
equimolar ratio (20). The crystal structure of the START domain of
MLN64 was solved, revealing that the protein encompasses a hydrophobic
tunnel large enough to bind a single cholesterol molecule (20).
In animal cells, cholesterol can be obtained either by de
novo synthesis or receptor-mediated lipoprotein uptake.
Cholesterol can be used as a structural component of membranes, be
converted into cholesteryl esters, be effluxed to extracellular
acceptors, or be metabolized into steroid hormones or bile acids
depending on the cell type (1, 2). Mechanisms of cholesterol transport between intracellular compartments remain unclear. Only a few proteins
involved in these processes have been identified. StAR has been shown
to be implicated in the transport of cholesterol into the mitochondria
(37). In contrast to StAR, MLN64 was not present in the mitochondria
but in distinct undefined vesicular structures (10). Besides human
cancers that show abnormal overexpression, MLN64 is
expressed at basal levels in all cells and tissues, and its promoter
harbors the classical feature of a housekeeping gene promoter.2 Therefore, MLN64
fulfills the criteria of a protein involved in an essential cellular
function such as cholesterol transport. Here, we have studied where
MLN64 is likely to play a role in intracellular cholesterol movement.
MLN64 protein was found to colocalize largely with cathepsin D, LampI,
CD63, and LBPA, different markers of endosomes/lysosomes. More
precisely, the staining of MLN64 completely overlapped with the
staining of LBPA, a late endosomal lipid, demonstrating that MLN64 is
localized to late endosomes. Addressing protein to endosomes/lysosomes can be achieved by several means (30, 38), and redundant pathways have
been suggested in eukaryotic cells (39, 40). We show in this study that
putative N-glycosylation of MLN64 is not the sorting
determinant of the protein as is common with luminal proteins of
endosomes/lysosomes (28), which strongly suggests that the potential
transmembrane domains of the protein are functional. MLN64 contains
several potential conventional endosome/lysosome targeting signals
typical of membranous proteins; using serial deletion constructs, we
defined a minimal addressing domain. This domain, spanning amino acids
47-171, which comprises the four putative transmembrane domains, is
necessary for correct sorting to late endosomes. Among the classical
endosomal/lysosomal determinants that contain tyrosine- or
dileucine-based motifs (30), five dileucines (LL1-LL5) and three
tyrosines (Y1-Y3) are present in the minimal targeting domain of
MLN64. Alteration by mutagenesis of the dileucine motif LL2 (amino
acids 66 and 67) or of the tyrosine Y1 (amino acid 89) resulted in the
modification of the subcellular localization. The mutated protein
exhibited a reticular pattern specific to the endoplasmic reticulum.
Although mutations of LL2 and Y1 modify the subcellular localization of
MLN64, these two sites do not fulfill the criteria of targeting motifs.
Typical dileucine- and tyrosine-based signals are located in the
cytoplasmic region of proteins; when these signals are mutated, the
protein is routed to the plasma membrane. Within the MLN64 protein
structure, LL2 is located in the first putative transmembrane helix and
Y1 in a luminal loop; when mutated, the protein is localized in the ER,
suggesting a misfolding of the protein. Thus, we can conclude that
within the endosomal addressing minimal region of MLN64, no
conventional targeting signals could be identified. However, the
dileucine motif LL2 and the tyrosine residue Y1 are critical for the
proper folding of this domain and/or for the targeting of the protein
to late endosomes.
By immunoelectron microscopy, MLN64 labeling shows a patchy pattern on
the limiting membrane of endosomes. This uneven distribution reveals a
compartmentalization within the same continuous membrane of endosomes,
as is the case for Rab proteins (41).
Interestingly, late endosomes have recently been shown to be an
important organelle for the trafficking of cholesterol derived from
LDL. Indeed, multivesicular internal membranes of late endosomes, which
contain high amounts of LBPA, regulate cholesterol transport (34).
Immunoelectron microscopy images obtained using an antibody against
MLN64 COOH terminus, which contains the START domain, suggest that this
part of the protein might be cytoplasmic. We have further investigated
the localization of the MLN64-START domain by microinjection and
endocytosis experiments using the mAbMLN64-Ct antibody. Antibodies
incorporated by endocytosis allowed the detection of intraendosomal
proteins such as cathepsin D (26). By contrast, antibodies directly
microinjected into the cytoplasm of cells recognized antigens present
in the cytoplasm that are not sequestered in subcellular vesicles. A
specific signal was obtained when the antibody raised against the COOH
terminus was directly injected into the cytoplasm, whereas no signal
could be detected when the same antibody was endocytosed by cells.
Therefore, the functional START domain of MLN64 is cytoplasmic. MLN64
possesses four putative transmembrane domains, which implies that both
the COOH and the NH2 termini of the protein are
cytoplasmic. By endocytosis and microinjection of the NH2
terminus-specific antibody, we showed that both the NH2 and
the COOH termini of MLN64 are cytoplasmic.
In the autosomal recessive Niemann-Pick type C disease, a
neurodegenerative endosomal/lysosomal storage disorder, LDL-derived cholesterol accumulates in the late endosomes (33, 34). In over 95% of
the cases of NPC disease, the recently identified gene NPC1
is mutated. There is evidence for the existence of a second
complementation group, which concerns a few families of NPC patients
(21, 42). The gene inactivated in this complementation group, named the
NPC2 gene, remains unknown. Therefore, MLN64 appeared as a potential candidate gene for this disease. We have shown
that MLN64 and cholesterol as well as NPC1 colocalized in CHO cells,
showing that the two proteins might act in the same compartments.
However, transient transfection of an expression vector encoding for
MLN64 could not restore a normal phenotype to fibroblasts from NPC2
patients. Transfected fibroblasts still accumulated LDL-derived
cholesterol in the late endosomes. Therefore, inactivation of MLN64 is
not responsible for the NPC2 disorder.
Taken together, these observations suggest that MLN64 acts on late
endosome cholesterol traffic. The MLN64 amino-terminal transmembrane-containing domain is anchored in the limiting membrane of
late endosomes, and its COOH-terminal START domain is cytoplasmic. The
START domain of MLN64 can interact with the membrane of late endosome,
where it could deplete cholesterol by shuttling it to a cytoplasmic
acceptor site.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
MLN64 is a late endosomal protein.
A, SK-OV-3 (a), MCF7 (c), and stably
transfected MCF7/MLN64 cell lines (b) were fixed,
permeabilized, and then labeled with pAbMLN64-Ct antibody
(green). Nuclei were counterstained with
Hoechst-33258 dye (blue). SK-OV-3 and MCF7/MLN64 exhibited
similar punctuate MLN64 staining, whereas MCF7 was not stained.
B, MCF7/MLN64 cells were fixed, permeabilized, and then
incubated with pAbMLN64-Ct antibody (B, a,
d, and g) and anti-cathepsin D (B,
b), anti-CD63 (B, e), or
anti-LBPA (B, h). Overlays of B
(a) and B (b), of B
(d) and B (e), and of B
(g) and B (h) are shown in B
(c), B (f), and B
(i), respectively. The yellow staining
indicates colocalization. MLN64 colocalized with cathepsin D
(B, c) or CD63 (B, f),
although the relative quantity of both proteins from one vesicle to
another one was variable (arrows, stronger signal for
cathepsin D or CD63; arrowheads, stronger signal for MLN64).
MLN64 colocalized completely with LBPA. Scale
bar, 10 µm except for insets, showing 2 times
higher magnification in B, g, h, and
i.
Ala219 and Asn311
Ser311) (Table I). The
localization of mutated proteins was compared with a marker of late
endosomes (data not shown). Neither single mutants nor the double
mutant have a modified subcellular localization. These data indicate
that putative N-glycosylation of MLN64 are not involved in
the sorting of MLN64, which strongly suggests that the transmembrane
domains of the protein are functional. The contribution of another
sorting signal typical of endosomal transmembrane protein was therefore
tested. The GYXXZ motif (where Z represents an
aliphatic or aromatic residue) is responsible for the sorting of the
Lamp proteins to lysosome and endosome membranes (29). MLN64 contains
one such motif at positions 112-116. Site-directed mutagenesis of this
motif did not alter the endosomal localization of the protein (Table
I). Therefore, this motif is not involved in the sorting of MLN64.
Since common signals were not responsible for the addressing of MLN64,
we decided to map the MLN64 endosomal sorting domain using recurrent
deletions in the NH2 and COOH termini of the protein.
Sequences and positions of the potential N-glycosylation sites and
GYXXZ type sorting motif of MLN64
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Fig. 2.
The transmembrane helices-containing domain
is responsible for the targeting of MLN64 to late endosomes.
A, scheme of the deletion constructs of MLN64 and
subcellular localization of the resulting proteins. The
numbers correspond to the first and the last amino acid of
the MLN64 wild type and mutant constructs. The four potential
transmembrane domains are represented by dark
boxes. ER, endoplasmic reticulum. B,
immunofluorescence analysis of the subcellular localization of
MLN64-(1-445) (a), MLN64-(47-445) (b),
MLN64-(54-445) (c), MLN64-(1-218) (d),
MLN64-(1-171) (e), and MLN64-(1-145) (f)
revealed by immunocytofluorescence with pAbMLN64-Ct (a-c)
or with pAbMLN64-Nt (d-f) antibodies. Scale
bar, 10 µm.
View larger version (56K):
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Fig. 3.
Mutations of dileucine 66-67 or of tyrosine
89 impairs the localization of MLN64. A, positions of
the five dileucine motifs (LL1-LL5) and of the three tyrosines (Y1-Y3)
that were mutated within the MLN64 transmembrane helices-containing
domain. The domain responsible for the targeting of MLN64 (amino acids
47-171) is represented by the gray box.
Transmembrane helices are represented by the four
dark boxes. The numbers indicate the
amino acid residues. B, immunofluorescence analysis of the
subcellular localization of four of the mutants revealed with
pAbMLN64-Ct antibody. MLN64 LL1 (B, a) and MLN64
Y2 (B, c) had a similar subcellular localization
as the wild type MLN64 (Fig. 2B, a). On the
opposite MLN64 LL2 (B, b) and MLN64 Y1
(B, d) mutants showed an endoplasmic reticulum
staining. Scale bar, 15 µm.
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Fig. 4.
MLN64 is located on the limiting membrane of
late endosomes. MCF7/MLN64 cells were labeled with the mAbMLN64-Ct
antibody. The labeling was detected by electron microscopy.
A, perinuclear concentration of immunolabeled endosomes
(arrowheads). The labeling was restricted to the cytoplasmic
face of endosomes. The Golgi apparatus (G), nucleus
(n), and mitochondria (m) were unlabeled.
B, higher magnification of the area boxed in
A showed the patchy distribution (arrowheads) of
immunolabeling on the cytoplasmic surface of the limiting membrane of
endosomes. The arrows indicate small punctuate staining
within the cytoplasm. Scale bar, 2 µm
(A) and 500 nm (B).
View larger version (59K):
[in a new window]
Fig. 5.
The START domain and the NH2
terminus of MLN64 are cytoplasmic. A, MCF7/MLN64 cells
were incubated in a medium containing either anti-cathepsin D
monoclonal antibody M1G8 (a), mAbMLN64-Ct antibody
(b), or pAbMLN64-Nt antibody (c). Cells were then
fixed, permeabilized, and incubated with Cy3-conjugated secondary
antibody (red). Cells incubated with anti-cathepsin D
antibody exhibited a punctuate staining (a), whereas cells
incubated with mAbMLN64-Ct (b) or pAbMLN64-Nt (c)
were not stained. Scale bar, 15 µm.
B, MCF7/MLN64 cells were microinjected with either M1G8
(a-c), mAbMLN64-Ct (d-f), or pAbMLN64-Nt
(g-i) conjointly with dextran fluorescein. Cells were then
fixed, permeabilized, and incubated with Cy3-conjugated secondary
antibody (red; a, d, and
g). Microinjected cells were visualized with dextran
fluorescein (green; b, e, and
h). Nuclei were counterstained with Hoechst-33258 dye
(blue; b, e, and h).
Overlays of a and b, d and
e, and g and h are shown in
c, f, and i, respectively. By
microinjection of the antibodies in the cells' cytoplasm, a punctate
staining could be observed with mAbMLN64-Ct (d) and
pAbMLN64-Nt (g) but not with anti-cathepsin D antibody
(a). Scale bar, 15 µm. C,
model of topology for MLN64 on the limiting membrane of the late
endosome.
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Fig. 6.
MLN64 colocalizes with cholesterol and NPC1
but does not correct late endosomal accumulation of cholesterol in NPC2
patient fibroblasts. A, CHO cells were transfected
either with pSG5 MLN64 alone (a-c) or with pSG5 MLN64 and
pCR3.1 NPC1 (d-f). Cells were then processed for
immunofluorescence either with mAbMLN64-Ct antibody (a)
together with filipin staining (b) or after classical Triton
X-100 permeabilization with mAbMLN64-Ct antibody (d) and
anti-NPC1 antibody (e). Overlays of a and
b and of d and e are shown in
c and f, respectively. A large majority of MLN64
positive vesicles were filled with cholesterol (c). MLN64
colocalized with NPC1, although the relative quantity of both proteins
from one vesicle to another one was variable (f).
Scale bar, 10 µm. B, NPC2
fibroblasts were transfected with the expression vector pSG5 MLN64,
incubated with LDL, and processed for immunofluorescence with the
pAbMLN64-Ct antibody (a; red) and filipin
staining (b; blue). Overlay of a and
b is shown in c. MLN64 expression (c;
left cell versus right
cell) could not correct cholesterol accumulation in late
endosomes (c). Scale bar, 10 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank S. Chan for critical reading of the manuscript; E. Ikonen for providing the NPC1 plasmid and the anti-NPC1 antibody; H. Rochefort and M. Garcia for providing the anti-cathepsin D antibodies; Dr. L. Liscum for providing the CHO cell line; G. Duval, P. Eberling, M. J. Klein, I. Stoll, J. L. Vonesch, and N. Messaddeq for technical assistance; and D. Stocco, E. Lalli, B. Murphy, G. Millat, A. Boulay, A. L. Gall, S. Ribieras, J. L. Linares, and C. H. Régnier for helpful discussions.
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FOOTNOTES |
---|
* This work was supported by funds from the Institut National de la Santé et de la Recherche Médicale, Center National de la Recherche Scientifique, Hôpital Universitaire de Strasbourg, Bristol-Myers Squibb Pharmaceutical Research Institute, Association pour la Recherche sur le Cancer, and the Fondation de France. The Ligue Nationale Française contre le Cancer and the Comités du Haut-Rhin et du Bas-Rhin also supported this work (équipe labelisée).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.
Recipient of an Allocation du Ministère de l'Education
Nationale, de la Recherche et de la Technologie.
To whom correspondence should be addressed: IGBMC, 1 rue
Laurent Fries, BP163, 67404 Illkirch Cedex, France. Tel:
33-3-88-65-34-23; Fax: 33-3-88-65-32-01; E-mail:
cat@igbmc.u-strasbg.fr.
Published, JBC Papers in Press, October 26, 2000, DOI 10.1074/jbc.M006279200
2 F. Alpy, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: LDL, low density lipoprotein; StAR, steroidogenic acute regulatory protein; SHD, StAR homology domain; START, StAR-related lipid transfer; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; NPC, Niemann-Pick C.
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