From the Department of Biochemistry and
§ Electron Microscopy, University of Connecticut Health
Center, Farmington, Connecticut 06030-3305 and the
¶ Department of Pediatrics, Children's Hospital, Harvard Medical
School, Boston, Massachusetts 02115
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ABSTRACT |
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Previous studies identified two intrinsic
endoplasmic reticulum (ER) proteins, 11 A number of proteins are resident and catalytically active in the
lumen of the endoplasmic reticulum
(ER).1 The best characterized
motif targeting this compartment is for soluble proteins bearing
C-terminal sequence KDEL (1, 2). The molecular signals responsible for
the insertion and retention of membrane proteins in the ER with a
lumenal orientation of their functional domains are poorly understood.
Most ER proteins are targeted for membrane insertion by a hydrophobic
signal peptide at the N terminus (3). During translation, the signal
peptide is recognized by a cytosolic RNA/protein complex termed the
signal recognition particle (SRP) (4). Upon binding a signal peptide, the SRP arrests the protein synthesis until it has facilitated transfer
of the nascent polypeptide-ribosome complex to the translocation channel (5). The signals controlling folding and eventual topology of
ER lumenal proteins after the nascent chain is transferred to the
translocation channel are unknown. One early critical event in the
multistep folding process is whether or not the signal peptide is
removed. If the signal peptide is removed, then the newly formed N
terminus is generally located in the lumen of the ER. In cases where
the signal peptide is not removed, the resulting N-terminal hydrophobic
segment may function as a membrane anchor, directing the transmembrane
insertion of the nascent polypeptide. Therefore, membrane proteins with
uncleaved signal peptide may have either cytosolic or lumenal
orientation. Mutagenesis experiments on a number of membrane proteins
have revealed that the charge distribution, the length of the membrane
segment, as well as the charge of the segments following the membrane
segment are some of the factors that determine the specific topology of
such proteins (6). Detailed analysis of these factors have been
complicated by use of multispanning model proteins with a lack of
agreement on the topology. Moreover, additional ambiguities are
encountered in trying to overexpress polytopic eukaryotic membrane
proteins in prokaryotic hosts. Previously, we identified the membrane
binding anchor of cytochrome b5 (7), cytochrome b5 reductase
(8), cytochromes P-450 (9-11), epoxide hydrolase (12), In the present study, we investigated whether the N-terminal membrane
binding segment of 11 Materials--
Chemical products were purchased from Sigma
unless otherwise noted. COS-7 cells were from American Type Culture
Collection (ATCC). Cell reagents, restriction, and modification enzymes
were from Life Technologies, Inc. The GFPS65T mutant protein in
pEGFP-N1 vector and an anti-GFP mouse monoclonal antibody were from
CLONTECH. Anti-stearyl CoA-desaturase polyclonal
antibody was raised in a rabbit by injecting keyhole limpet
hemocyanin-conjugated 20-residue synthetic peptide corresponding to
residues 338-358 of stearyl CoA-desaturase antigen (20). Avian
anti-b5 and anti-reductase were raised against the polar
moieties of rat cytochrome b5 and cytochrome b5
reductase (19). Anti-calreticulin rabbit polyclonal antibody was from
Affinity Bioreagents. Alkaline phosphatase-conjugated anti-mouse and
anti-rabbit IgG were from Sigma. Biotin-coupled goat anti-mouse IgG was
from Molecular Probes. Iodocarbocyanine (Cy3)-streptavidin and
Cy3-conjugated goat anti-rabbit IgG were from Jackson Immunoresearch
Laboratories. Proteinase K and 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) were from Roche Molecular Biochemicals. Gold-labeled secondary antibody was from Amersham Pharmacia Biotech.
Construction of Plasmids--
All expression vectors were driven
by the cytomegalovirus promoter-enhancer contained in the pEGFP-N1
vector. The N tag-GFP chimeras were constructed by inserting the coding
region of GFP at the C terminus of the first 23 and 34 amino acids
corresponding to lumen targeting signal (LTS) of 11-
The resulting PCR products were purified and digested with
EcoRI-BclI and BclI-NotI
for N-tags and GFP, respectively and inserted into
EcoRI-NotI-digested pEGFP-N1 vector in one-step
ligation. All the N-tag-GFP chimeras and their mutated or deleted
variants were constructed using the experimental procedure described above.
The expression vector pEN1.rab E3 N-b5 red.(31-300) encodes a fusion
protein consisting of rabbit esterase 3 LTS, followed by three novel
amino acids (PPV) encoded by the restriction site for AgeI,
by amino acids 31-300 of bovine liver NADH-cytochrome b5
reductase, and FLAG epitope TAG (LARIKRTGDGSHKSS). This fusion protein
was expressed in pEN1 vector obtained by removing the coding sequence
of GFP from pEGFP-N1. The rab E3 N-b5 red.(31-300) was constructed as
follows. First, the sequence corresponding to amino acids 31-300 of
reductase was amplified by PCR using as a template the 940-bp DNA
fragment coding for entire bovine liver microsomal reductase
inserted in pGEM3zf(+) (21) with a pair of primers,
5'-GATCCACCGGTCCCGGCCATCACGCTCGAGAAT-3' matching with the amino acids
31-37 of beef reductase preceded by AgeI site, and
5'-AGAGTCGCGGCCGCTTTAGCTACTCTTGTGGCTCCCATCTCCAGTTCTCTTAATCCTGGCTAAGAAGGCGAAGCAGCGTTCTTT-3' matching with amino acids 294-300. This oligonucleotide included the desaturase epitope, stop codon, and NotI site. The PCR
fragment was purified, digested with AgeI-NotI
and inserted along with the EcoRI-AgeI fragment
corresponding to LTS of rabbit esterase3 into
EcoRI-NotI-digested pEN1 vector. The integrity of
the constructs were confirmed by DNA sequencing.
Cell Culture and Transient Transfection--
COS-7 cells were
cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented
with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin,
and 100 mg/ml streptomycin. The cells were seeded at approximately 30%
confluence onto 35-mm dishes at 37 °C under 5% CO2.
After 24 h, when the cells became 60-70% confluent, they were
transfected using the LipofectAMINE reagent according to the
manufacturer's instructions. The cell staining was observed 48-72 h
after transfection under Zeiss LSM 410 confocal microscope.
Immunoblot Analysis--
Particulate components and the soluble
fraction of the cells were separated as described previously (22).
Insoluble material was removed by centrifugation, samples resolved by
SDS-PAGE, and Western blot analysis was performed using anti-mouse
monoclonal antibody against GFP. Alkaline phosphatase-conjugated
antibodies were used as secondary antibodies for protein visualization.
Isolation and Protease Digestion of Microsomes from COS-7
Cells--
Microsomal membranes were prepared as described by Ref. 23.
The pellet fraction was enriched in cytochrome b5 by adding 10 µl of 0.5 mM pure detergent free cytochrome
b5 (24). The mixture was resuspended in a solution
containing 250 mM sucrose, 10 mM
potassium-Hepes, pH 7.2, 50 mM potassium chloride, 2 mM magnesium chloride, and 2 mM calcium
chloride to a final volume of 0.2 ml. Protease digestions were
performed at 4 °C for 30 min with increasing concentrations of
proteinase K in presence or absence of 0.5% Triton X-100. AEBSF (4 mM) was used to terminate the protease digestions. After 15 min of incubation on ice, the membranes were solubilized with SDS gel
loading buffer, and the proteins subjected to SDS-PAGE and
immunoblotted as described above.
Selective Permeabilization and Immunofluorescence
Analysis--
Permeabilization of cells using digitonin, streptolysin
O, and saponin was performed 48 h after transfection, as described previously (25). Immunocytofluorescent staining was conducted by
incubation of the cells with a 1:100 dilution of anti-GFP mouse monoclonal antibody in the permeabilization buffers containing 0.1%
BSA. The cells were then rinsed three times and then incubated with a
1:100 dilution of biotin-coupled goat anti-mouse immunoglobulin. After
the wash, cells were incubated with streptavidin
coupled-iodocarbocyanine (Cy3) diluted 1:100 in the permabilization
buffers containing 3% BSA. The cells were rinsed several times before
mounting in Mowiol 4.88 and then viewed under Zeiss LSM 410 confocal microscope.
Electron Microscopic Immunogold Labeling--
Transfected COS-7
cells with rab E3 N-GFP were fixed with 3% paraformaldehyde plus
0.15% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH
7.4. The cells were allowed to fix for about 1 h at room
temperature, the fixative was removed, and the cells stored at 4 °C
in 1% paraformaldehyde in 0.1 M cacodylate, pH 7.4. The cells were removed from the flask by scraping and then pelleted in
0.2% BSA in PBS. The cells were resuspended in 0.1% BSA/PBS, transferred to a tube containing 3% low-gelling agarose and pelleted again. The agarose was chilled on ice, and the tip of the tube was cut
off. The embedded cell pellet was rinsed in 0.1 M
cacodylate buffer, pH 7.4, dehydrated in methanol, infiltrated, and
embedded at
Immunogold labeling was done essentially as described elsewhere (26).
Nonspecific binding was blocked with 1% BSA plus 1% instant milk in
PBS. The sections were labeled overnight at 4 °C with anti-GFP mouse
monoclonal antibody diluted 1:50 in 1% BSA and 5% normal goat serum
in PBS. After rinsing with PBS, the bound immunoglobulins were
visualized by incubating with 10-nm diameter gold-labeled secondary
antibody diluted 1:15 in PBS for 1 h at room temperature. After
thorough rinsing with PBS and distilled water, the sections were
stained with uranyl acetate and lead citrate and observed in Philips
CM10 TEM.
The N Termini of 11 The N Termini Targets ER Localization--
To confirm that the
fluorescence distribution along the reticular network observed in Fig.
2 is characteristic of ER localization, we compared the fluorescence
pattern of cells expressing rab E3N-GFP (Construct e) with a known ER
marker, calreticulin. The fluorescence pattern of Construct e (Fig.
2F) is almost identical to the calreticulin staining (Fig.
2G). Indeed, the combined fluorescence revealed a yellow
staining indicative of the colocalization of the rab E3N-GFP-associated
green fluorescence and the calreticulin-associated red
fluorescence as evidenced by Fig. 2H.
Electron Microscopic Immunogold Labeling--
To confirm these
results at the ultrastructural level, ultrathin cryosections were
analyzed in double-labeling experiments with the immunogold technique,
using anti-GFP antibodies to identify the ER. Electron microscopic
examination of thin sections of rab E3 N-GFP (Construct e) transfected
cells labeled with anti-GFP antibodies revealed the presence of gold
particles over the nuclear envelope and the ER (Fig.
3, A and B).
Clusters of heavily labeled ER membranes were found in the perinuclear
region. The gold particles were clearly associated with periphery of
the ER cisternae. Although the labeling was most frequently seen in
association with ribosome-studded membranes, cisternae of apparently
smooth ER were also labeled (Fig. 3C). The labeling obtained
with the polyclonal and monoclonal anti-GFP preparations was
approximately equivalent. Nonspecific background labeling was
relatively low; very few particles were found over the nuclear
chromatin, mitochondria, or other structures.
Transmembrane Topology of Rab E3N-GFP, Construct e--
To
determine the orientation of Construct e in the membrane, we performed
immunofluorescent staining of selectively permeabilized COS-7 cells by
saponin, digitonin, and streptolysin O (SLO). As previously reported,
digitonin (20 µg/ml) and SLO (200 units/ml) selectively permeabilized
plasma membrane leaving internal membranes including ER intact, whereas
saponin permeabilized all the membranes (29). For control experiments,
in cells expressing untagged GFP treated with digitonin (Fig.
4A), or saponin (Fig.
4C), the antibodies stained the nucleus as well as the
cytoplasm as expected. Cells treated with SLO (Fig. 4B)
showed a similar staining except for the nuclear membrane, which was
not permeabilized by SLO. Cells transfected with cDNA for rab
E3N-GFP (Construct e) and permeabilized with either digitonin (Fig.
4D) or SLO (Fig. 4E) failed to stain with the
antibodies to GFP even though these cells expressed the GFP construct
as indicated by the green fluorescent emission. However, after
permeabilization with saponin (Fig. 4F), all the cells
expressing GFP chimeras became reactive to the GFP antibody. The fact
that the GFP fluorescence pattern uniformly overlapped with the GFP
antibody staining pattern only when all the cell membranes were
completely permeabilized with saponin indicates that the GFP tagged
with E3 LTS is oriented toward the lumenal side of ER (Fig.
4F).
Evidence for Lumenal Localization by Proteinase K Assay--
To
confirm the lumenal topography of rab E3N-GFP (Construct e), membranes
from cells expressing Construct e were subjected to proteinase K
digestion in the presence and absence of detergent. As seen in Fig.
4G, in the intact membranes Construct e was not susceptible
to proteinase K digestion. Proteolysis of Construct e could be
demonstrated when detergent was added to intact membranes prior to
incubation with the protease. Taken together, these results are
consistent with the lumenal orientation of the expressed chimera. The
proteolysis of cytochrome b5 was used as a positive control in this proteolysis protection assay. Cytochrome b5 is an
integral membrane protein oriented toward the cytosolic side of ER, and is readily released from the membrane upon proteolysis (19, 24).
Cytochrome b5 added to a membrane preparation is
spontaneously incorporated into the membrane. As shown in Fig.
4G, proteolysis of membrane fractions in the presence or
absence of detergent results in the conversion of cytochrome
b5 to a species with faster mobility upon SDS-PAGE. The
latter species lacks the C-terminal 40 residue membrane anchor, but
retains its catalytic activity (7). Cleavage of cytochrome
b5 was observed in intact microsomes of cells containing
Construct e at protease concentration where no cleavage of the
expressed protein occurred (Fig. 4G).
Mutagenesis of the Lumenal Targeting Sequence--
Sequences of
11
When the single Gly residue in region II was replaced by Glu, and the
resulting chimera (Construct b) expressed in cells, such mutation led
to the expression of the protein with an apparent cytosolic and nuclear
localization (Fig. 6A).
Deletion of six residues in region II as in the Construct d also led to
a cytosolic and nuclear localization of the protein (Fig.
6B).
Reorientation of NADH Cytochrome b5 Reductase Conferred by the
LTS--
To test the utility of LTS as an in vivo
trafficking signal for proteins other than GFP, we made a fusion
protein between the polar segment of microsomal NADH cytochrome
b5 reductase and the N terminus of rab E3. The catalytically
active polar segment of the reductase consists of some 270 residues,
and lacks the hydrophobic 28-residue N-myristoylated
membrane-anchoring domain at the N terminus present in the intact
protein (8). The native reductase has a cytosolic orientation. To
assess whether the LTS can redirect the reductase derivative to the
lumen of ER, we fused the LTS of rabbit E3 to the N terminus of the
polar segment of the reductase. In addition, we added to the C terminus
of the reductase the highly antigenic C-terminal sequence of stearyl CoA desaturase as an epitope tag. We termed the chimera rab E3-N-b5 red
(31-300) or Construct g. The orientation of Construct g in the ER
membrane was examined by proteinase K digestion. As shown in Fig.
7, digestion of intact microsomes
(lane 1-3 and 5) revealed a
protease-resistant fragment approximately of 35-40 kDa in mass, indicating that the construct was intralumenal. When microsomal membranes were disrupted with 0.5% Triton X-100, the protein became sensitive to proteinase K digestion and was completely degraded (lane 4). The proteinase K protection assay
results confirm our previous results of the GFP chimera orientation and
show that soluble as well as membranous proteins with a cytoplasmic
orientation wearing 11 11 Fig. 1B displays the mutations and deletions of the chimeras
constructed by fusion of N-terminal domain of 11 The subcellular localization and orientation of the constructed GFP
chimeras is summarized in Fig. 8
(A and B). The most important finding
of this study is that fusion of the N-terminal sequences from both
11-hydroxysteroid
dehydrogenase, isozyme 1 (11
-HSD) and the 50-kDa esterase (E3),
sharing some amino acid sequence motifs in their N-terminal
transmembrane (TM) domains. Both are type II membrane proteins with the
C terminus projecting into the lumen of the ER. This finding implied
that the N-terminal TM domains of 11
-HSD and E3 may constitute a
lumenal targeting signal (LTS). To investigate this hypothesis we
created chimeric fusions using the putative targeting sequences and the
reporter gene, Aequorea victoria green fluorescent protein.
Transfected COS cells expressing LTS-green fluorescent protein chimeras
were examined by fluorescent microscopy and electron microscopic
immunogold labeling. The orientation of expressed chimeras was
established by immunocytofluorescent staining of selectively
permeabilized COS cells. In addition, protease protection assays of
membranes in the presence and absence of detergents was used to confirm lumenal or the cytosolic orientation of the constructed chimeras. To
investigate the general applicability of the proposed LTS, we fused the
N terminus of E3 to the N terminus of the NADH-cytochrome b5 reductase lacking the myristoyl group and N-terminal
30-residue membrane anchor. The orientation of the cytochrome
b5 reductase was reversed, from cytosolic to lumenal
projection of the active domain. These observations establish that an
amino acid sequence consisting of short basic or neutral residues at
the N terminus, followed by a specific array of hydrophobic residues
terminating with acidic residues, is sufficient for lumenal targeting
of single-pass proteins that are structurally and functionally unrelated.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
9 stearyl coenzyme A desaturase (13), and three forms of flavin-containing monooxygenases (14). All of these ER proteins appeared to be oriented
toward the cytosolic side of the ER membrane by single or multispanning
membrane segments. In further studies to define the proteins found or
oriented in the lumenal compartment of the ER, we identified two
isoforms of 60-kDa carboxylesterases (15) and the lumenal NADP
glucose-6-phosphate dehydrogenase (16). The esterases and the
glucose-6-phosphate dehydrogenase were devoid of any transmembrane
segments. The lumenal esterases carried a C-terminal segment HIEL- and
HTEL-related KDEL, the ER lumen retention motif (1). The primary
structure of the glucose-6-phosphate dehydrogenase failed to display
sequence segments or a motif that would be responsible for its lumenal
orientation or retrieval. We then extended our studies to
oligosaccharyltransferase complex (OT) (18). The OT from mammalian
tissues was found to be a trimolecular complex consisting of
ribophorins I and II and a 50-kDa protein. The predicted membrane
orientation of OT indicated that both ribophorins and the 50-kDa
proteins have large N-terminal lumenal domains and a short C-terminal
cytoplasmic domain (18). Studies on the orientation of the 11
-HSD
(17) and the 50-kDa esterase/N-deacetylase (E3) (19) revealed that both
proteins shared a short N-terminal cytoplasmic domain and a large
C-terminal lumenal domain.
-HSD and E3 may act as a targeting sequence for
the lumenal orientation of proteins in the ER. Here we show that a
transmembrane segment consisting of basic residues at the N terminus
followed by an array of specific 17 hydrophobic residues terminating
with acidic residues constitute a lumenal targeting signal for a set of
single-membrane-spanning proteins that are otherwise structurally and
functionally unrelated.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
HSD and esterase
3, respectively and variants thereof, as follows (see Fig. 1). First,
cDNA encoding residues 1-34 of human esterase 3 preceded by Kozak
sequence and EcoRI site at 5' end and extended by
BclI site at 3' was synthesized using two long overlapping
oligonucleotides
(5'-CTTCGAATTCCCACCATGGGAAGAAAATCGCTGTACCTTCTGATTGTGGGGATCCTCAATTT-3' as sense long oligo and
5'-TGGATTGATCATTCTCCATGGCTCCTCAACGTTATCTGGGAGAGGCGTATAAATATAATG-3' as
antisense long oligo) and two short oligonucleotides
(5'-CTTCGAATTCCCACCATG-3' as sense short oligo and
5'-TGGATTGATCATTCTCCA-3' as antisense short oligo) and the chain
polymerase reaction (PCR). Second, the coding sequence of GFP was
amplified from pEGFP-N1 by PCR using a 5'-oligonucleotide, which
contains a BclI restriction site
(5'-ATGGTGATCAAGGGCGAGGAGCTG-3') and 3'-oligonucleotide containing NotI site (5'-CCTCTACAAATGTGGTATGGC-3'). This was done
in order to remove the Kozak sequence preceding the first ATG codon of GFP in pEGFP-N1 vector so that the initiation codon introduced in the
N-tag sequences will be used by N-tag-GFP chimeras.
20 °C in Lowicryl K4M resin. After polymerization with
ultraviolet light (365 nm), thin sections of the embedded cells were
cut with a diamond knife and collected on Formvar-coated, 200-mesh
nickel grids.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-HSD and Esterase 3 (E3) Determines Their
Subcellular Localization--
The proposed LTS of rat, rabbit, and
human 11
-HSD aligned with sequences of E3 of rabbit and human are
shown in Fig. 1A. The two
protein families share a short positively charged N termini (region I),
followed by an array of some 17 hydrophobic residues containing an
aromatic cluster Ala-Tyr-Tyr-X-Tyr or Gly-Tyr-Tyr-Tyr cluster (region II), terminating with di-glutamyl residues (region III). Sequence similarity beyond the N termini could not be found in
these two protein families (19). To ascertain whether N terminus of
11
-HSD is a determinant of its subcellular localization in mammalian
cells, constructs were generated encoding N terminus of 11
-HSD fused
to GFP (11
-HSD N-GFP) (Fig. 1B). The A. victoria GFP is widely recognized as a powerful tool in cell
biology serving as a reporter for monitoring localization and dynamics
of intracellular proteins and organelles (27). When rab 11
-HSD N-GFP
(Construct a) was transiently expressed in COS-7 cells and examined by
fluorescent microscopy, a staining pattern characteristic of ER
localization was seen (Fig.
2A). Expression of native GFP
showed green fluorescent signals in both the cytoplasm and nucleus
(Fig. 2B) consistent with the previous studies (28). To
explore whether the N-terminal domain of E3 also imparts GFP a membrane
localization, GFP constructs hum and rab E3N-GFP (Constructs c and e,
respectively) were generated and expressed in COS-7 cells. Cells
expressing Constructs c and e showed fluorescence pattern of ER
localization, which was similar to that seen with Construct a (Fig. 2,
A, C, and D). Analysis of the membrane
fractions of cells expressing the Construct e by SDS-PAGE, followed by
immunoblotting with GFP antibody showed the localization of GFP
construct in the insoluble fraction. Upon SDS-PAGE, a decreased
mobility of rab E3 N-GFP as compared with untagged GFP was observed,
implying that the signal sequence like N terminus of the construct was
not removed by the signal peptidase (Fig. 2E). As
anticipated, untagged GFP expressed in cells was found only in the
soluble fraction.
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Fig. 1.
N-terminal sequences of native proteins and
GFP constructs. A, N-terminal sequences of rat, rabbit,
and human 11 -HSD are aligned with those of rabbit and human E3. The
positions of N-terminal amino acid residues are numbered
below the sequence. Conserved amino acids are enclosed in
boxes. Dashes were inserted to maintain alignment
of conserved residues. The LTS is denoted by regions I, II and III as
indicated above. The Protein Identification Resource accession numbers
for the rat, rabbit, and human 11
-HSDs are A34430, A55573, and
A41173, respectively. Rabbit and human E3 accession numbers are A58922
and A53856. B, amino acid sequences of LTS of native
proteins and the derivatives thereof with altered LTS fused to GFPs,
which were prepared for the experiments described here. Amino acid
residues or segments that were altered are indicated by
letters in boldface or by dashed
line.
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Fig. 2.
Expression of
rab11 -HSD N-GFP, hum E3 N-GFP, rab E3 N-GFP,
and native GFP in COS-7 cells. Evidence for the colocalization
with the ER marker calreticulin. Representative fluorescence images of
COS 7 cells transfected with GFP and the indicated fusion proteins are
shown. A, Rab 11
-HSD N-GFP (Construct a). B,
GFP control. C, Hum E3 N-GFP (Construct c). D,
Rab E3 N-GFP (Construct e). The cells were cultured for 48 h after
transfection and observed under Zeiss LSM 410 confocal microscope
(magnification, ×40). E, immunoblot analysis of rab E3
N-GFP (Construct e) and native GFP in cellular subfractions. Cells
transfected with cDNA for GFP and rab E3 N-GFP were harvested
48 h after transfection, disrupted, and subjected to
centrifugation. The whole cell extract (W), soluble fraction
(S), and pellet fraction (P) (50 µg of protein)
were subjected to SDS-PAGE and immunoblotted with 5 µg/ml of an
anti-GFP mouse monoclonal antibody. F, G, and
H, colocalization of rab E3 N-GFP (green) with
the ER marker calreticulin (red) in COS-7 transfected cells
permeabilized with 0.2% saponin. F, fluorescence image of
COS-7 cells expressing rab E3 N-GFP (green). G,
the same cell as in F was costained by incubation with a
rabbit polyclonal antibody against the ER-resident protein calreticulin
followed by incubation with Cy3-conjugated goat anti-rabbit IgG. The
cells were then observed under confocal microscope to visualize the
staining pattern of the fluorescent dye Cy3 (red).
H, merged images of F and G reveal
considerable colocalization of brightest signal for rab E3 N-GFP and
calreticulin in yellow (magnification ×40).
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Fig. 3.
Electron microscopic immunogold labeling of
rab E3 N-GFP transfected cells with anti-GFP antibodies.
A and B, illustrate clusters of ER in the
perinuclear region. In A, the asterisks (*)
indicate dilated regions of ER with membrane-associated gold particles.
In B, the arrows indicate regions where labeling
of the ER is particularly prominent. In both panels, the
arrowheads indicate gold particles associated with the
nuclear envelope. Nucleus is denoted by n. In C,
several smooth-surfaced membranes are labeled (arrows).
Scale bar = 0.25 µm.
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Fig. 4.
Immunocytofluorescent staining of selectively
permeabilized COS-7 cells and the protease sensitivity of the expressed
chimeras. COS-7 cells transfected with cDNA coding for GFP
(A-C), and rab E3 N-GFP (D-F) were
permeabilized either with digitonin (20 µg/ml), streptolysin O (200 units/ml), or saponin (0.2%) before incubation with anti-GFP mouse
monoclonal antibody. Bound antibody was visualized by incubation with
biotin-coupled goat anti-mouse and then with Cy3-streptavidin as
described under "Experimental Procedures" (magnification, ×40).
G, protease sensitivity of rab E3 N-GFP and cytochrome
b5 in membranes. The microsomal membranes of COS-7 cells
transfected with cDNA coding for rab E3 N-GFP fusion protein and
enriched in cytochrome b5 were incubated with the indicated
concentration of proteinase K for 30 min on ice in the absence
(lanes 1-3) or presence (lane
4 and 5) of 0.5% Triton X-100. The mobility of
rab E3 N-GFP and cytochrome b5 was analyzed by SDS-PAGE and
immunoblot with 5 µg/ml of an anti-GFP mouse monoclonal antibody or
with 1:1,000 dilution of an anti-cytochrome b5 chicken polyclonal
antibody.
-HSD from different mammalian species contain two Lys residues in
region I. The region I of human E3 contains an Arg-Lys sequence. In the
rabbit E3 sequence, the Arg residue is replaced by Val, implying that
in region I a single lysyl residue rather than two basic residues do
not alter the orientation of E3 in the membrane. Replacement of the
single Lys residue by Ile as in rab E3N(K4/I)-GFP (Construct f) led to
ER localization of the resulting chimera as evidenced by
immunofluorescence (Fig. 5A).
The fluorescence image of cells expressing Construct f is similar to
that seen for rab E3N-GFP (Construct e). The membrane localization of
Construct f was confirmed by subcellular fractionation and immunoblot
analysis. As seen in Fig. 5B, the expressed protein was
present in pellet and absent in the soluble fraction. To establish the
orientation of the expressed protein, cells were selectively permeabilized with saponin, digitonin, SLO, and analyzed by
immunofluorescence microscopy (Figs 5, C-E). Again, the
staining pattern of such permeabilized cells was identical to that
obtained with Construct e (Fig. 4, D-F). This result
indicates that positively charged residues are not an essential
topogenic signal for the lumenal orientation.
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Fig. 5.
Expression of rab E3 N(K4/I)-GFP in COS-7
cells to analyze the importance of positively charged residue of region
I in the targeting to the ER. A, subcellular
localization of rab E3 N(K4/I)-GFP (Construct f) determined by
fluorescence microscopy (magnification, ×40). The (K4/I)
symbol indicates the position of positively charged residue within E3
region I in which the single lysine was mutated to an isoleucine
residue. B, immunoblot analysis of COS-7 cells expressing
Construct f and fractionated as described in Fig. 2. The fluorescence
image and the immunoblot show the same intracellular localization as
the constructs E3 N-GFP in Fig. 2 (C and D).
Selective permeabilization with digitonin (C), SLO
(D), and saponin (E) of cells expressing
Construct f was done as described in Fig. 4.
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Fig. 6.
Effect of mutations in the hydrophobic region
of 11 -HSD and E3 LTS on the localization of
GFP chimera proteins. A, fluorescence microscopy of
COS-7 cells transfected with rab 11
-HSD N(G10/E)-GFP (Construct b)
in which the glycine residue in position 10 was mutated to glutamic
acid residue. B, fluorescence image of COS-7 cells
transfected with hum E3 N(
16-21)-GFP (Construct d) in which the
hydrophobic segment was shortened by deletion of the six residues
AYYIYT. A strong labeling pattern of nucleus, ER, and cytoplasm is seen
for the mutant chimera proteins as compared with the ER labeling
pattern seen for the wild chimera proteins in Fig. 2 (C and
D) (magnification, ×40). C, immunoblot analysis
of cells expressing GFP, Construct b, and Construct a.
or E3 LTS are oriented toward the lumenal
side of ER.
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Fig. 7.
Evidence for the lumenal orientation of rab
E3 N-b5 red.(31-300) in microsomal membranes of transfected
cells. Microsomes containing the chimeric protein (Construct g)
were incubated with the indicated concentrations of proteinase K in the
presence (lane 4) or absence (lanes
1-3 and 5) of 0.5% Triton X-100. Samples of the
proteolysis reaction mixture were subjected to SDS-PAGE analysis,
followed by immunoblot with 1:2,000 dilution of anti-FLAG polyclonal
antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-HSD and E3 are two unrelated ER lumenal proteins with type
II orientation (17, 19). Analysis of their covalent structures implied
that the LTS for 11
-HSD and E3 is encoded in the N-terminal segment.
The N-terminal amino acid sequence of 34 residues of this group of
proteins from several mammalian species is shown in Fig. 1A.
For discussion purposes, the LTS sequence is divided into regions I,
II, and III. In addition to the positive charge at the N-terminal
residue, features of region I that are shared by several species
include a single lysyl residue. A stretch of aliphatic and aromatic
hydrophobic residues (region II) follows region I, and continues into a
segment of negatively charged residues (region III). A striking feature
of region I is the variability of the first three residues and the
conservation of a single lysyl residue. Region II is a hydrophobic
segment containing a cluster of tyrosyl residues. In region III, there
is a conserved Asn-X-Glu-Glu segment. The remaining sequence
of the C-terminal ectodomain shared no obvious homology between the
dehydrogenases and the esterases. Despite that the bulk of the
polypeptide chain is translocated across the membrane, the N-terminal
leader peptide like sequence in this group of proteins escapes signal
peptidase cleavage and serves as the membrane anchor.
-HSD or E3 to the N
terminus of GFP. These constructs were expressed in COS 7 cells, and
analysis of their cellular targeting was accomplished by combination of
fluorescence microscopy and electron microscopic immunogold labeling.
The topology of expressed proteins was established by
immunocytofluorescent staining of selectivity permeabilized COS cells.
Protease protection assay of membranes in the presence and absence of
detergents was also used to distinguish the membrane sidedness of the
GFP chimeras.
-HSD and E3 proteins to GFP resulted in the lumenal localization
of the chimera in the ER membrane. This finding implied that the
information (LTS) for the lumenal orientation of 11
-HSD and E3 is
encoded in the N-terminal segment of some 20 to 25 residues.
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Fig. 8.
Targeting proteins to the lumen of ER with
LTS. A, summary of subcellular localization of all the
constructs expressed. The constructs are abbreviated a-g as
shown in Fig. 1B. The abbreviations for the subcellular
localizations are N, ER, C, S, L, and Cyt,
nucleus, endoplasmic reticulum, cytosolic, secreted, lumen, and
cytoplasmic side, respectively, proposed membrane topology of the
LTS-GFP constructs. N and C represent the N and C
termini of the native and chimera proteins. The chimeras are displayed
in the upper part, and the native proteins in the lower part of the
model. My at the N terminus of native cytochrome
b5 reductase denotes a myristoyl residue.
As displayed in the amino acid sequences of the native proteins from different species, the presence of two neutral or two basic residues in region I of LTS are not essential for the lumenal targeting. This is based on the assumption that the proteins from different animal species share an identical orientation. The proposed essential di-arginine sequences (30) at the cytoplasmic N terminus of type II membrane proteins are not indispensable for the ER localization for the group of proteins described here. Deletion of the single lysyl residue in region I (Construct f) led to ER targeting of the GFP chimera with a lumenal ER orientation (Fig. 5). Therefore, the absence of basic residues in region I does not alter the lumenal targeting of the protein. Clearly, the positively charged residues are not required for the ER localization, but the extreme negative charge of region III would conform to the N > C charge role for the lumenal orientation.
In order to further characterize the essential residues of the LTS, several chimeras of LTS fused to GFP were constructed and their subcellular localization analyzed. The Construct b bearing a single negative charge in the center of region II led to the expression of protein with fluorescence pattern similar to the control GFP (Fig. 6A). Immunoblot of the subcellular fractions of cells expressing Construct b indicated its electrophoretic mobility upon SDS-PAGE identical to native GFP, implying that a truncation of the N terminus of the chimera had occurred (Fig. 6C), but the protein failed to enter the secretory pathway as anticipated. This implied that the signal peptidase was not responsible for the cleavage of the N-terminal signal. What may have compensated for the translocation deficiency of Construct b? It is most likely that the introduction of a glutamyl residue in region II of the LTS resulted in a conformational change interpreted by the cytosolic quality control system as a misfolded protein. Proteolytic elimination of malfolded proteins, including uncomplexed subunits of protein assemblies, is a common defense mechanism of the cell. Mutant forms of cystic fibrosis transmembrane conductance regulator expressed in mammalian cells, or carboxypeptidase Y mutants that fail to translocate across the membrane and accumulate on the surface of ER are readily degraded by the cytoplasmic proteosome pathway (31, 32). One reason we observed the GFP tag in cells expressed with Construct b could be the resistance of the GFP molecule toward proteolysis. Expression of the N terminus of Construct b fused to a polypeptide that is more sensitive to proteolysis than the GFP molecule most likely would result in the degradation of the entire construct.
A striking feature of region II of the parent proteins is the presence
of a cluster of tyrosyl residues (AYYXY or GYYY). Repeats of
tyrosyl residues (AYPYYA) are found in the transmembrane domain of the
48-kDa subunit of microsomal OT. OT has a short C terminus oriented
toward the cytosol, and a single transmembrane domain with the bulk of
the protein molecule positioned in the lumen of ER (18). The T-cell
receptor delta chain displays a GYYYYV sequence in its membranous
segment (33). That a tyrosine-containing motif mediates ER retention of
CD3- chain has also been reported (34). To determine the topogenic
importance of the tyrosyl residue cluster GFP chimera, Construct d
lacking the tyrosyl residues in region II was generated. Construct d
upon its expression in COS cells displayed fluorescence in the nucleus
and cytosol (Fig. 6B). This fluorescence pattern of intracellular
structures was similar to that observed by the untagged GFP. These
findings suggest that a cluster of tyrosyl residues is essential for
the correct folding and the lumenal targeting. Further analysis will be
needed to establish whether replacement of these residues with
phenylalanines or glycines provides an appropriate context for the
function of the conserved tyrosyl residues. Deletion of five to six
residues in region III of the 11
-HSD-GFP constructs does not affect
the lumenal orientation of the constructs (Fig. 2A).
Presently, the 11-HSD and the E3 protein are the only type II,
static lumenal ER proteins with a single N-terminal transmembrane segment that have been characterized. The asialoglycoprotein receptor (35) and the influenza virus neuramidinase (36) are type II plasma
membrane proteins. The glycoprotein, paramyxovirus
hemagglutinin-neuroaminidase (HN) is a type II membrane protein
localized to the Golgi cisternae (37). The predicted amino acid
sequence of the simian virus HN protein includes a 17-residue
cytoplasmic tail, a 19-residue membranous segment, and a large
523-residue C-terminal ectodomain (38). Newly synthesized HN
oligomerizes into tetramers before transport from ER to Golgi, and
alterations of the C-terminal ectodomain can prevent ER to Golgi
transport (39). The large family of glycosidases and
glycosyltransferases, although type II transmembrane proteins are all
located in the Golgi cisternae. Their common features include an
N-terminal cytoplasmic tail, a transmembrane domain, and a large
catalytic domain oriented toward the lumenal side of the membrane (40).
Two mutually complementary models have been proposed to explain the
mechanism of Golgi retention of the glycosyltransferases mediated by
their transmembrane domains. One model postulates the retention through
oligomerization, which prevents proteins from entering transport
vesicles (41, 42). The other model suggests that their retention
depends on the length of a membrane-spanning domain, which can be
accommodated by the specific lipid composition of Golgi complex
membrane (43, 44). It has been pointed out that neither the
oligomerization nor the membrane lipid composition alone can explain
the sorting of Golgi proteins.
Having established that the N-terminal domain of 11-HSD or E3 can
mediate lumenal localization of the following downstream GFP molecule,
we sought to test the general applicability of this finding to
targeting of ER proteins that display a cytosolic orientation in their
native state. The flavoprotein NADH cytochrome b5 reductase has an amphiphilic structure in which the hydrophilic, catalytic domain
of some 270 residues is linked to a membrane-anchoring, hydrophobic
domain that serves to orient the catalytic site of the reductase at the
membrane-aqueous interface to permit a rapid electron transfer to
cytochrome b5 (8). The hydrophobic domain at the N terminus
of the reductase consists of some 28 residues as well as
N-myristoylation of the N-terminal glycine residue (45)
(Fig. 8B). Fusion of the LTS of rabbit E3 to the N terminus of the polar segment of the reductase (Construct g) resulted in the
lumenal targeting of the fusion protein (Figs. 7 and
8B).
Whether the topogenic signal described here is position-independent as
regards to lumenal targeting of proteins, in addition to identifying
the structural features that prevent these constructs transiting to the
Golgi, are the topics for further studies. In conclusion, a static
lumenal targeting signal of proteins reported here should add to the
repertoire of techniques for studies on the structure, organization,
and processes of the lumen of ER.
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ACKNOWLEDGEMENT |
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We thank Dr. C. S. Ramaro (University of Alabama at Birmingham) for constructing and expressing Constructs a and b.
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FOOTNOTES |
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* This work was supported by Grant R01 GM-26351 from the National Institutes of Health (to J. O.).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: Dept. of
Biochemistry, School of Medicine of the University of Connecticut
Health Center, Farmington, CT 06030-3305. Tel.: 860-679-2211; E-mail: ozols{at}sun.uchc.edu.
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ABBREVIATIONS |
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The abbreviations used are:
ER, endoplasmic reticulum;
11-HSD, 11
-hydroxysteroid dehydrogenase;
E3, liver microsomal 50-kDa esterase/N-deacetylase;
GFP, green fluorescent protein;
LTS, lumenal targeting signal;
PAGE, polyacrylamide gel electrophoresis;
PBS, phosphate-buffered saline;
SLO, streptolysin O;
OT, oligosaccharyltransferase;
AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride;
PCR, polymerase chain
reaction;
BSA, bovine serum albumin.
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REFERENCES |
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