From the Medical Research Council Toxicology Unit, University of Leicester, Leicester LE1 9HN, United Kingdom
Received for publication, October 21, 2002, and in revised form, December 26, 2002
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ABSTRACT |
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Neuropathy target esterase (NTE), the human
homologue of a protein required for brain development in
Drosophila, has a predicted amino-terminal transmembrane
helix (TM), a putative regulatory (R) domain, and a hydrophobic
catalytic (C) domain. Here we describe the expression, in COS cells, of
green fluorescent protein-tagged constructs of NTE and mutant proteins
lacking the TM or the R- or C-domains. Esterase assays and Western
blots of particulate and soluble fractions indicated that neither the
TM nor R-domain is essential for NTE catalytic activity but that this
activity requires membrane association to which the TM, R-, and
C-domains all contribute. Experiments involving proteinase treatment
revealed that most of the NTE molecule is exposed on the cytoplasmic
face of membranes. In cells expressing a moderate level of NTE and all
cells expressing Neuropathy target esterase
(NTE)1 is the human homologue
of a protein required for brain development in Drosophila
(1, 2). mRNA encoding NTE is expressed in embryonic mouse
neurons, suggesting the possibility of a similar function in mammals
(3). NTE was identified originally in adult vertebrate neural tissue as
a protein reactive with the organophosphates (OP), which cause a
syndrome of axonal degeneration (4, 5). Elucidation of the part played by NTE in OP-induced neuropathy and neural development is of obvious neurobiological interest. However, NTE is present not only in neurons
but also in a variety of non-neural tissues including intestine,
placenta, and lymphocytes (5, 6). Furthermore, the existence of a
putative homologue in Saccharomyces cerevisiae (7) suggests
that NTE may be involved in a fundamental process common to cells from
yeast to neurons.
NTE is conveniently detected in vitro by its ability to
catalyze OP-sensitive hydrolysis of an artificial substrate, phenyl valerate (8). Using this assay, NTE has been shown to be firmly membrane-associated. Differential centrifugation of brain homogenates resulted in an enrichment of NTE in microsomal fractions containing elements of endoplasmic reticulum (ER), Golgi, and plasma membrane; attempts to further resolve NTE by density gradient centrifugation of
microsomes were unsuccessful (9). In neural sections, immunoreactive NTE staining fills neuronal cell bodies, excluding the nucleus, and
extends into the proximal axon; in addition, the rate of accumulation of NTE at a peripheral nerve ligature indicates that it is conveyed along axons by vesicular fast transport (10). These observations are
consistent with an ER/Golgi location for NTE.
In keeping with the membrane-bound character of NTE, hydropathy
analysis of its primary sequence predicts a transmembrane helix (TM)
near the amino terminus (residues 10-32). Further examination of the
sequence of the 1327 residues of NTE indicates two functional domains:
1) an amino-terminal putative regulatory domain of ~700 residues that
includes areas with similarity to cyclic AMP-binding proteins; and 2) a
carboxyl-terminal catalytic/esterase domain containing the active site
serine residue (Ser-966), which reacts with OPs (7). Various
carboxyl-terminal constructs of NTE have been expressed in
Escherichia coli to define the minimum polypeptide with
OP-sensitive phenyl valerate esterase activity. A hydrophobic recombinant protein called NEST (NTE amino acids 727-1216) had this
property and, although lacking the amino-terminal TM of NTE, associated
firmly with phospholipid membranes and required this association for
its esterase activity (11, 12). In the present study, we expressed
various green fluorescent protein (GFP)-tagged constructs of NTE in COS
cells to relate the protein's molecular features to its enzymatic
activity and intracellular distribution in eukaryotic cells.
Cells, Antibodies, and Other Products--
COS-7 cells were
obtained from the European Collection of Animal Cell Culture (ECACC
number 87021302). Antibodies to GFP and to calnexin were purchased from
Zymed Laboratories Inc. and StressGen, respectively.
TRITC and peroxidase conjugates of anti-rabbit IgG were from Sigma, and
colloidal gold (10 nm)-conjugated anti-mouse IgG was from British
Biocell International Ltd. [35S]methionine (43.5 TBq/mmol) was from PerkinElmer Life Sciences.
DNA Cloning and Mutagenesis--
For generation of a full-length
NTE carrying the GFP tag at the C terminus, two primers were designed
to bring the human NTE cDNA in-frame into the pEGFP-N1 vector
(Clontech) between SalI and
BamHI sites (forward primer, 5'-AGATCGGTCGACCAGCTGGAATC-3'; reverse primer, 5'-TGTCGAGGATCCCAGGCATCTGT-3'). A 4-kb PCR product was
generated from the human NTE clone D16 (7) using Pfu DNA polymerase (Stratagene) and cloned into pEGFP-N1 to produce
pNTE-GFP (Fig. 1).
To generate a construct deleting the first 42 amino acid residues,
which include the TM at the N terminus, we designed a forward primer
(5'-TGCCAAGAATTCCAGCCATGGATGGCCCCC-3')
to bring in a Kozak consensus, an EcoRI site, and a
translation start codon. This primer was paired with a reversed primer
(5'-TGTCGAGGATCCCAGGCATCTGTGGCTGAG-3') with a
BamHI site to remove the original stop codon to amplify a
4-kb PCR fragment from NTE clone D16. The PCR fragment was cloned into
the EcoRI/BamHI sites of vector pEGFP-N1.
To construct an expression vector for the catalytic domain of human NTE
lacking the first 680 residues of the full-length protein, a forward
primer (5'-ATAGCCAAGCTTCCCGAGGCCGCCATGGGT-3') was designed to make use
of an internal HindIII site of human NTE cDNA and bring
in a Kozak consensus for optimal transcription. This forward primer,
with a reverse primer (5'-TGTCGAGGATCCCAGGCATCTGTGGCTGAG-3'), was used
in a PCR reaction to amplify a 2-kb DNA fragment, which was cloned into
the HindIII and BamHI sites of the vector
pEGFP-N1 to make pNEST-GFP (Fig. 1).
To generate a construct of NTE-GFP (designated
To generate a construct lacking the catalytic domain (
Mutation of the active site serine (Ser-966) to alanine by
site-directed mutagenesis was achieved by using
QuikChangeTM site-directed mutagenesis kit (Stratagene) and
the primers 5'-GTGGGCGGCACGGCCATTGGCTCTT-3' and
5'-AAGAGCCAATGGCCGTGCCGCCCAC-3'. The mutation was verified by DNA
sequencing and enzymatic activity assay.
Cell Culture and Fluorescent Microscopy--
COS cells were
cultured in Dulbecco's modified Eagle's medium with Glutamax-I
(Invitrogen), 10% fetal bovine serum, and 2% of both
penicillin and streptomycin at 37 °C, 5% CO2.
Transfection was carried out using PolyFect Transfection Reagent
(Qiagen) according to the manufacturer's protocol. For
fluorescent microscopic study, cells were plated at 4 × 104/well in an 8-well Lab-Tek II chamber slide (Nalge Nunc
International) and transfected with various constructs after 24 h
of culture. 24-48 h after transfection, cells were fixed with 2%
paraformaldehyde in phosphate-buffered saline for 10 min at room
temperature and permeabilized by methanol/acetone (50:50) at Subcellular Fractionation, Esterase Assay, and Western
Blotting--
COS cells were plated at 0.8 × 106 in
10-cm dishes and cultured for 24 h before transfection.
Forty-eight hours after transfection, cells were harvested by
trypsinization. The cell pellet was resuspended in TE buffer (50 mM Tris-HCl, 1 mM EDTA, pH 8.0), homogenized with 10 passages through a 25-gauge hypodermic needle, and centrifuged at 100 × g at 4°C for 2 min. The
supernatant fraction was further centrifuged at 100,000 × g at 4°C for 45 min in an OptimaTM
TLX ultracentrifuge using a TLA120 rotor (Beckman). After removing the
soluble cytosolic fraction, the particulate fraction was washed once by
resuspension and centrifugation and finally resuspended in the original
volume of TE buffer. Protein concentration was measured using a Bio-Rad
protein assay kit (Bio-Rad Laboratories). NTE (phenyl valerate)
esterase assay was carried out as described previously (11). Soluble
and particulate fractions were run on SDS-PAGE (4-20% gradient gels),
blotted, and probed with anti-GFP antiserum (1:1000) followed by
peroxidase-labeled anti-rabbit IgG (1:1000) with final detection by
enhanced chemiluminescence using Pierce reagents essentially as
described previously (11). Relative levels of immunoreactive GFP in
soluble and particulate fractions were determined by densitometry of
the Western blots.
Proteinase K Digestion of Sealed Membrane Vesicles Isolated from
COS Cells Expressing NTE-GFP--
Cells transfected with NTE-GFP for
48 h were harvested and homogenized in hypotonic TE buffer, and
the post-nuclear supernatant was subjected to 100,000 × g ultracentrifugation as above. The membrane pellet was
resuspended in the same volume of TE buffer containing 0.25 M sucrose and re-homogenized and used as sealed membrane
vesicles. For proteinase K digestion, an aliquot of the membrane
vesicles was incubated on ice for 30 min with proteinase K (4 µg/ml)
with or without 1% Triton X-100. The digestion was terminated with 4 mM phenylmethanesulfonyl fluoride (PMSF) and a 30-min
incubation on ice to allow complete inhibition of proteinase K before
SDS-PAGE and Western blotting with anti-GFP. As a control, the same
blots were probed with an antibody (StressGen, catalog number SPA-865;
1:2500) directed to the N terminus of calnexin, an ER protein; this
procedure has been reported to detect a 70-kDa intralumenal fragment of
calnexin (13).
In Vitro Transcription/Translation and Protection from
Proteinase K Digestion by Pancreatic Microsomes--
cDNA
encoding NTE was subjected to coupled transcription/translation
in vitro at 30 °C for 75 min using
[35S]methionine and a STP3 kit from Novagen, according to
the manufacturer's instructions, in the absence or presence of canine
pancreatic microsomes (Promega; 1 µl). Subsequently, proteinase K
(0.2 µg) was added, and mixtures were incubated for 30 min at
4 °C. Reactions were stopped with 2 mM PMSF. After
incubation for a further 20 min, samples were run on SDS-PAGE and
blotted onto a nitrocellulose membrane, which was then subject to
autoradiography. As a control, parallel reactions were run using
cDNA encoding MADM, a mammalian protein cloned in this laboratory
(14), which (unlike NTE) has an amino-terminal signal peptide and a
carboxyl-terminal TM.
Relative Fluorescence Analysis, Cell Sorting, and Electron
Microscopy--
Transfected COS cells were harvested by
trypsinization and resuspended in culture medium containing 10% fetal
bovine serum and incubated at 37 °C for 30 min to allow recovery
from trypsinization. In initial experiments, relative expression levels
of fluorescent protein were determined using a BD Biosciences FACS
Vantage system. Prior to electron microscopy, cells were sorted into
pure GFP-expressing populations and were then spun down in a swing-out
rotor at 3000 × g for 30 min at 4 °C. The pellets
were processed for routine electron microscopy and for post-embedding
immunogold labeling as described previously (15). Disrupted cell
fractions were also used for immunogold labeling as described by
Johnston et al. (16).
Amino-terminal TM Facilitates Membrane Association, but Neither TM
nor the R-domain Is Essential for a Catalytically Active Conformation
of NTE in COS Cells--
Constructs of NTE tagged at the carboxyl
terminus with GFP, including the full-length protein, deletion mutants
with either the amino-terminal TM segment (
Phenyl valerate esterase assays on soluble and particulate fractions
from the cells transfected with the various constructs indicated that
all the activity was confined to the latter fraction (data not shown);
this may reflect the fact that association with phospholipids is
required for NTE esterase activity (11, 12). Esterase activities in
particulate fractions from NTE-GFP- and
Mean fluorescence levels in NEST-GFP-transfected COS cell populations
were about half of those in NTE-GFP transfected cells, but esterase
activity in particulate fractions from NEST-GFP-expressing cells was
only ~5% of those from cells expressing the full-length protein
(Table I). However, Western blotting of soluble and particulate fractions indicated that, whereas ~90% of immunoreactive GFP in NTE-GFP-transfected cells was confined to the latter fraction, only
~30% of GFP in NEST-GFP-transfected cells was particulate (Fig.
2). Thus normalized for the amount of
NEST present, the esterase activity in particulate fractions of
NEST-transfected COS cells is actually about one-third of that in
NTE-transfected particulates. Similarly, ~60% of immunoreactive GFP
was present in the particulate fractions of cells transfected with the
When expressed in E. coli, NEST is able to fold to a
catalytically active conformation in association with the bacterial
membrane (11). The present results suggest that recombinant constructs of NTE lacking the N-terminal TM can also adopt a catalytically active
conformation once they become associated with membranes in eukaryotic
cells; thus, the major function of the TM is to facilitate more
efficient association with membrane. Interestingly, hydropathy analysis
predicts at least one TM near the amino terminus of all the eukaryotic
NTE homologues but none in YCHK, a 34-kDa E. coli protein
with homology to a ~200 residue region surrounding the active site
of NTE (7).
Most of the NTE Molecule Is Exposed on the Cytoplasmic Surface of
Intracellular Membranes--
Because NTE lacks a signal sequence, it
is likely to associate with intracellular membranes with the great
majority of the molecule exposed to the cytoplasm. To confirm this
possibility, we showed that proteinase K treatment (either in the
absence or presence of Triton X-100) of sealed membrane vesicles from
NTE-GFP-transfected COS cells reduced the size of the
GFP-immunoreactive polypeptide from ~180 kDa (NTE-GFP) to ~30 kDa
(similar to GFP itself), indicating that the membranes did not protect
NTE from proteolysis (Fig. 3A). By contrast, a 70-kDa
fragment of calnexin (i.e. its intralumenal domain; see
Ref.13) was clearly protected until the membrane vesicles were
disrupted by treatment with Triton X-100 (Fig. 3A). In a second approach to the question of the topology of NTE with
intracellular membranes, we subjected NTE cDNA to in
vitro transcription and translation in the absence or presence of
pancreatic microsomes. The resulting polypeptide was not protected by
microsomes from degradation by added proteinase K, suggesting a
predominantly cytoplasmic disposition (Fig. 3B). By
contrast, in a parallel control experiment, the polypeptide formed by
transcription/translation of MADM (a mammalian membrane protein with an
amino-terminal signal sequence TM; Ref. 13), which is imported into the
lumen of the microsomes, was protected from proteinase K digestion
(Fig. 3B).
NTE Has an ER-like Distribution, and Its Overexpression Disrupts
the ER via a Non-enzymatic Property of the Cytoplasmic
C-domain--
In COS cells expressing either NEST-GFP or
An ER-like localization was observed for fluorescence in essentially
all cells expressing
The ultrastructure of COS cells with intense juxtanuclear expression of
NTE-GFP (Fig. 5b) was compared
with that of GFP-vector control cells (Fig. 5a) to
investigate reasons for the abnormal distribution of calnexin. Cells
expressing high levels of NTE-GFP exhibited fine tubular structures
(20-40 nm in diameter) contiguous with the ER, which was often
distended to result in vesicles containing fine flocculent material
(Fig. 5b). In many of these cells the tubular structures
were aggregated in the cytoplasm to form irregular clusters up to 7 µm in diameter. Immunogold labeling of resin sections demonstrated
the localization of NTE-GFP to these membrane clusters (Fig.
5c). Greater resolution was obtained by immunogold labeling
of disrupted cell fractions (16), which clearly revealed the presence
of NTE-GFP on the cytoplasmic face of these membrane clusters, whereas
adjacent Golgi, mitochondrial, lysosomal, and nuclear membranes were
not labeled (Fig. 5d). Electron microscopy of cells
expressing
Recently, we demonstrated that purified recombinant NEST catalyzes
hydrolysis of membrane lipids in vitro (17). We wondered whether the abnormal membrane structures observed in COS cells overexpressing NTE and
The abnormal ER morphology induced by overexpression of NTE is
reminiscent of that described in COS cells overexpressing the inositol
1,4,5-triphosphate receptor (18), malfolded cytochrome P450 (19), and
microsomal aldehyde dehydrogenase (20). All of these proteins are
anchored in the ER membrane by at least one TM and have large
cytoplasmic domains. It has been suggested that the ER membranes
aggregate by the head-to-head association of the cytoplasmic domains of
these proteins (20). Our present experiments showing that microsomal
membranes do not protect NTE from proteinase K digestion (Fig. 3)
indicate that the protein is probably anchored in the ER membrane via
its amino-terminal TM with residues 33-1327 exposed to the cytoplasm.
This topology is also consistent with the pattern of immunogold
labeling of disrupted cell fractions (Fig. 5d). When NTE is
overexpressed, intermolecular association of the cytoplasmic
hydrophobic C-domains could give rise to ER aggregation by the general
mechanism noted above. The fact that overexpression of C-NTE, fluorescence was distributed in an endoplasmic reticulum (ER)-like pattern. Cells expressing high levels
of NTE showed aberrant distribution of ER marker proteins and
accumulation of NTE on the cytoplasmic surface of ER-derived tubuloreticular aggregates. Deformation of the ER was also seen in
cells expressing
R-NTE or enzymatically inactive S966A-NTE but not
TM-NTE. The data suggest that NTE is anchored in the ER via its TM,
that its R- and C-domains also interact with the cytoplasmic face of
the ER, and that overexpression of NTE causes ER aggregation via
intermolecular association of its C-domains.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
R-NTE; Fig. 1) which
retained the first 157 amino acids (including the predicted TM) but
lacked residues 158-673 (i.e. the putative regulatory domain), we designed two primers (forward,
5'-AAGCTTGCTAGCGAATTCCCCCACGC-3'; reverse,
5'-GCGGCAAAGCTTCAGGAAGAGTGGCTTCT-3') to amplify a short fragment of DNA
from human NTE clone D16 containing the 5' untranslated region, the
translation start codon, and the TM. This PCR product was cut with
NheI and HindIII and ligated into the long arm of pNTE-GFP pre-cut with NheI and HindIII and
recovered from an agarose gel.
C-NTE-GFP;
Fig. 1) the appropriate region of human D16 NTE cDNA was amplified
by PCR with the same forward primer used to make
R-NTE (above) and a
new reverse primer (5'-TTGATGGGATCCAAGGTGCCCTCGGGAA-3'). The PCR
product was cut with NheI and BamHI and cloned
into the vector backbone of the construct pNTE-GFP to replace the
full-length NTE-encoding sequence.
20 °C
for 10 min. After three washes with phosphate-buffered saline, the
cells were blocked with 3% bovine serum albumin in phosphate-buffered
saline for 2 h at room temperature and probed with anti-calnexin
(1:400) followed by TRITC-labeled anti-rabbit IgG (1:400) with three
washes between and after antibody reaction. Slides were mounted with Vectorshield ® mounting medium (Vector Laboratories).
Fluorescent images were acquired by confocal scanning using an
argon-krypton laser and a Leica TCS-4D confocal imaging system.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
TM), the putative
regulatory (
R) domain or the catalytic (
C) domain removed, and a
truncated polypeptide of similar length to bacterially expressed NEST
(11) were made as shown in Fig. 1. FACS
analysis revealed that, in the transfected cells, the relative mean
levels of fluorescent protein expression were quite similar; mean
values for cells transfected with NTE-GFP were about 2-fold higher than
those with NEST-GFP, whereas cells transfected with the
TM- and
R- constructs showed intermediate values (Table
I).
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Fig. 1.
NTE-GFP constructs used for transient
transfection experiments. Full-length NTE (1327 amino acids) was
tagged with GFP at the carboxyl terminus. The predicted TM at residues
10-32 is represented with a vertical line, and the position of the
active site, serine 966 (S966) is indicated. The putative
regulatory domain (R) and the catalytic domain (C) are shown as
striped and dotted areas, respectively.
Also shown are the constructs TM-GFP-(
1-42),
NEST-GFP-(
1-680),
R-NTE-GFP-(
158-673), and
C-NTE-GFP-(
681-1327).
Relative mean fluorescence levels and esterase activities of various
NTE-GFP proteins in particulate fractions of transfected cells
R-NTE-GFP-transfected cells
were roughly proportional to the relative mean fluorescence intensity
of the cells (Table I). The fact that
R-NTE has essentially
identical catalytic activity as the full-length protein indicates that
the R-domain is neither required for, nor does it substantially
inhibit, NTE phenyl valerate esterase activity. Sequence homology
within the R-domain with proteins that bind cyclic AMP (7) suggests
that this nucleotide might modulate NTE catalytic activity. However, we
have found no effect of cyclic AMP on phenyl valerate esterase activity
in particulate fractions from NTE-transfected COS cells (data not shown).
TM construct (Fig. 2) and, thus, when data in Table I are normalized on this basis, the esterase activity in particulate fractions from
TM-NTE-GFP-expressing cells is about half of that in
NTE-GFP-transfected cells.
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Fig. 2.
Soluble/particulate distribution of
GFP-tagged NTE constructs in transfected COS cells. COS cells were
transfected with nothing (Nil), GFP vector, NEST-GFP;
TM-GFP, or NTE-GFP. After 48 h the cells were harvested and
homogenized, and the soluble (S) and particulate
(P) fractions were subjected to Western blotting with an
antiserum to GFP as described under "Experimental Procedures."
Migration of molecular mass standard proteins is indicated to the
left of the figure.
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Fig. 3.
Microsomal membranes do not protect NTE from
proteinase K digestion. A, sealed membrane vesicles
were isolated from COS cells transfected with NTE-GFP and treated with
proteinase K (PK) as detailed under "Experimental
Procedures." As a control, the ER protein calnexin was probed with an
antibody to its intralumenal N terminus. The C terminus of calnexin is
cytoplasmic and therefore cleaved by proteinase K, reducing its
molecular mass from 90 to 70 kDa (see Ref. 13). Calnexin is
completely degraded by proteinase K in the present of Triton X-100
(TX). By contrast, the membrane vesicles did not protect
NTE-GFP from proteinase K digestion. B, cDNAs encoding
either NTE or MADM (a lumenal protein used here as control) were
transcribed and translated in the presence of pancreatic microsomes
(MS) and then exposed to proteinase K (PK) and
analyzed by SDS-PAGE and autoradiography as described under
"Experimental Procedures." The presence of microsomal membrane
partially protects lumenal MADM, but not NTE, from degradation by
proteinase K.
TM-NTE-GFP, fluorescence was distributed in a pattern distinct from
that in cells expressing GFP itself and was partially cytoplasmic and
partially coincided with that of the ER marker calnexin (Fig.
4). This pattern is consistent with the
soluble/particulate distribution of these recombinant proteins detected
by Western blotting and supports the view that both the C- and
R-domains of NTE contribute to its association with the cytoplasmic
face of the ER membrane.
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Fig. 4.
Distribution of various GFP-tagged NTE
protein constructs and calnexin in transfected COS cells.
Cells were transfected with GFP vector or various NTE-GFP constructs as
indicated on each panel image for 48 h and then fixed,
permeabilized, immunostained for the ER-marker calnexin
(red), and visualized by confocal microscopy as described
under "Experimental Procedures." Note the marked differences in
calnexin distribution in cells expressing R-NTE-GFP or high levels
of NTE-GFP compared with the relatively normal calnexin staining in
cells expressing other deletion constructs of NTE.
C-NTE-GFP but in only a minority of cells
expressing full-length NTE-GFP (Fig. 4). In a majority of COS cells
expressing NTE-GFP, intense fluorescence was observed in the
juxtanuclear area, and those expressing
R-NTE-GFP commonly showed
even more intense and bizarre patterns of fluorescence (Fig. 4).
Calnexin appeared to colocalize with juxtanuclear areas of intense
expression of NTE-GFP or
R-NTE-GFP in a pattern that was clearly
abnormal (Fig. 4). Although some cells expressing
TM-NTE-GFP also
showed relatively intense juxtanuclear fluorescence, calnexin
distribution in these cells was relatively normal (Fig. 4). Similar
morphological observations, (and those relating to esterase activity)
were also made with transfected human HeLa and N2a mouse neuroblastoma
cells using protein disulfide isomerase rather than calnexin as an ER
marker (data not shown). Furthermore, the intense juxtanuclear
fluorescence was not readily dispersed by treating the cells with
brefeldin A (data not shown), an indication that NTE-GFP was not
associated with the Golgi apparatus (16).
R-NTE-GFP revealed even more markedly abnormal membrane
structures than those observed with the full-length protein. These
tubuloreticular membrane structures were up to 3 µm in diameter and
contiguous with the ER (Fig. 5, e and f).
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Fig. 5.
Ultrastructural membrane abnormalities in
cells overexpressing NTE-GFP. Transmission electron micrographs of
COS7 cells. a, a cell transfected with vector alone showing
the presence of dense, osmiophilic droplets of the transfection fluid
within the cytoplasm (arrowheads); bar, 2.5 µm.
b, a cell overexpressing full-length NTE-GFP showing
fine tubular structures (20-40 nm diameter) adjacent to the nucleus
(*); bar, 500 nm. c, a cell overexpressing
full-length NTE-GFP showing immunogold localization of GFP in a cluster
of tubules adjacent to the nucleus (*); bar, 500 nm.
d, disrupted cell overexpressing full-length NTE-GFP
showing localization of GFP on the cytoplasmic face of membrane
clusters; bar, 250 nm. e, a cell
overexpressing R-NTE-GFP showing two complex tubuloreticular
membrane structures (curved arrows) and a normal
Golgi apparatus (straight arrow); bar,
2.5 µm. f, detail of region outlined by the
box in panel e showing continuity
between the tubuloreticular structures and the ER; bar, 500 nm.
R-NTE, but not in those with
C-NTE, might reflect hydrolysis of membrane lipid. However, the same aberrant membrane clusters were observed in cells expressing the enzymatically inactive S966A mutant forms of both NTE-GFP and
R-NTE-GFP (data not
shown). Thus, disruption of the ER and formation of the tubuloreticular structures appear to reflect a non-enzymatic property of the catalytic domain of overexpressed NTE.
TM-NTE-GFP
does not cause gross redistribution of calnexin indicates that this ER
aggregation requires NTE to be anchored via its TM. The leading role of
the C-domain in the aggregation is emphasized by the relatively normal morphology of cells expressing
C-NTE. To some degree, the R-domain may hinder intermolecular association of C-domains, and this may reflect the exacerbated ER abnormality in cells expressing
R-NTE.
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ACKNOWLEDGEMENTS |
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We thank Kul Sikand and Roger Snowden for assistance with confocal microscopy and FACS, respectively, Tim Smith and Judy McWilliam for help with sample preparation for electron microscopy, and Shawn Bratton and Marion MacFarlane for constructive criticism.
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FOOTNOTES |
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* This work was supported by the Medical Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Fax: 44-116- 2525616;
E-mail: yl8@le.ac.uk.
Published, JBC Papers in Press, January 3, 2003, DOI 10.1074/jbc.M210743200
1 The abbreviations used are: NTE, neuropathy target esterase; C-domain, catalytic domain; ER, endoplasmic reticulum; FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein; MADM, mammalian disintegrin metalloprotease; MS, microsomes; NEST, NTE esterase domain; OP, organophosphate; PMSF: phenylmethanesulfonyl fluoride; R-domain, regulatory domain; TM, transmembrane helix; TRITC, tetramethylrhodamine isothiocyanate.
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