From the Department of Microbiology and Immunology, Emory
University School of Medicine, Atlanta, Georgia 30322
Transmembrane glycoproteins with type 1 topology
can be retrieved to the endoplasmic reticulum (ER) by a retrieval
signal containing a di-lysine (KK) motif near the C terminus. To
investigate the structural requirements for ER retrieval, we have
constructed mutants of the simian immunodeficiency virus (SIV) envelope
(Env) protein with cytoplasmic tails of different lengths and
containing a KK motif at the
3 and
4 positions. Such proteins were
found to be retained intracellularly when the signal was located 18 amino acids or more away from the membrane spanning domain. The retrieval signal was found to be functional even when placed at the
distal end of the wild-type SIV Env protein with 164 amino acids in the
cytoplasmic tail, as shown by the lack of proteolytic processing and
lack of cell surface expression of the mutant proteins. However,
proteins with a cytoplasmic tail length of 13 amino acids or less
having the di-lysine motif at the
3 and
4 positions were not
retrieved to the ER since they were found to be processed and
transported to the cell surface. The surface-expressed proteins were
found to be functional in inducing cell fusion, whereas the proteins
retained intracellularly were defective in fusion activity. We also
found that the KK motif introduced near an amphipathic helical region
in the cytoplasmic tail was not functional. These results demonstrate
that the ability of the KK motif to cause protein retrieval and
retention in the endoplasmic reticulum depends on the length and
structure of the cytoplasmic domain. The ER retrieval of the mutant
proteins was found to correlate with increased intracellular binding to
COP proteins.
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INTRODUCTION |
For type 1 integral membrane proteins, a KKXX or
XKXX motif (K residues at the
3 or
3 and
4
positions) functions as an efficient
ER1 retrieval and retention
signal (1, 2). The E3/19k protein of adenovirus was found to contain
the sequence DEKKMP at the carboxyl terminus, which determined its ER
localization (3) and caused a block in cell surface expression when
transferred to chimeric proteins (1). Subsequent studies have
demonstrated a requirement for lysine residues at the
3 and
4
positions from the C terminus for ER retrieval (4-8), which appears to
be mediated by a 7-subunit receptor called the coatomer complex
(9-12).
The cytoplasmic tail length for type 1 transmembrane glycoproteins
ranges from a few to several hundred amino acids. It has not been
established whether there is a minimum or maximum length of
intracytoplasmic amino acids required for the efficient ER retrieval of
proteins containing the KK motif. To investigate the possible role of
the position of such a signal within the cytoplasmic domain, we have
used the envelope glycoprotein of simian immunodeficiency virus (SIV).
This envelope glycoprotein was chosen because of the following
properties: (a) it is a type 1 transmembrane protein with an
unusually long cytoplasmic tail of 164 amino acids; (b)
mutants with various truncations in the cytoplasmic tail have been
shown to be efficiently transported to the cell surface (13, 14) which
enabled us to investigate the effect of placing a KK motif at different
positions in the cytoplasmic tail; (c) the cytoplasmic tail
includes two amphipathic helical regions which enabled us to determine
the effect of placing the KK motif near such a helical region; and
(d) the effect of the KK motif on surface expression can be
evaluated by functional analysis of membrane fusion activity. The SIV
Env protein is synthesized as a precursor (gp160); during its transport
to the cell surface, about 10-15% of gp160 is cleaved by a cellular
protease resulting in the generation of surface (SU) and transmembrane
(TM) subunits (13, 15). In the present study, we have constructed a
series of mutant proteins with lysine residues at
3 and
4 positions in the cytoplasmic domain and analyzed their intracellular processing, cellular localization, and ability to induce membrane fusion.
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EXPERIMENTAL PROCEDURES |
Cells, Virus, and Reagents--
HeLa T4 (16) cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum. HUT 78 cells were maintained in RPMI 1640 supplemented with 10%
fetal calf serum. The recombinant vaccinia virus vTF7-3, which
expresses bacteriophage T7 RNA polymerase in infected cells (17), was obtained from the National Institutes of Health AIDS Research and
Reference Reagent Program. The rhesus antiserum to SIV was kindly
provided by Dr. P. Marx (Aaron Diamond AIDS Research Center, New York,
NY). The cDNA encoding
COP was kindly provided by Dr. T. Kreis
(University of Geneva, Geneva, Switzerland), and the antiserum to
COP was purchased from Sigma.
Cloning of SIV Envelope Mutant Genes Carrying ER Retention
Signals--
Polymerase chain reaction was employed to construct
SIV239 env mutants. Briefly, oligonucleotide primers which
incorporated coding sequence for lysines at
3 and
4 positions
followed by a stop codon were synthesized. The 5
-primer AAT ACG ACT
CAC TAT AGG GCG AA was used with a panel of 3
-primers to obtain the
desired mutants. Primers 3
-AGA GAA GAA TTC TTA ATA CCC CTT CTT TAA CTT AGC TAG CAT TTG T (SIVenv6RS), 3
-CTG GAA GAA TTC TTA TGG GGA TTT TTT
CAC TGG CCT ATA CCC CTG (SIVenv13RS), 3
-TTG GAT GAA TTC CTA CTG GAA
TTT TTT GGG TGG GGA AGA GAA CAC (SIVenv18RS), 3
-ACC GCC GAA TTC TTA
GTC TCT TTT TTT GCC TTC TCT GGT TGG CAG T (SIVenv37RS), 3
-TAG GTA GAA
TTC TTA AGT CCT TTT TTT TTC TCG AAT CCT CTG TAG (SIVenv103 RS), and
3
-TAT TTC GAA TTC TCA CAA GAG TTT TTT CTC AAG CCC TTG TCT AAT
(SIVenv164RS) were used. In addition to having the altered coding
sequence for lysines and stop codon, the 3
-primers also incorporated
an EcoRI restriction site. Polymerase chain reaction was
performed using pBSKS+ vector (Stratagene) containing the
SIVmac239 env gene as the template. The sense and antisense
primers were mixed with the template and appropriate buffer. Vent
polymerase (New England Biolabs) was added, and polymerase chain
reaction was performed for 30 cycles. After amplification, the products
were purified, cut with EcoRI and XbaI, and
cloned into the pBSKS+ vector that was cut with appropriate
enzymes. The introduced mutations were confirmed by sequencing the
plasmid DNA using Sequenase Version 2 (U. S. Biochemical Corp.)
following the protocol recommended by the manufacturer.
Transfection, Radioimmunoprecipitation, and Protein
Analysis--
Transfection and protein analyses were done as described
previously (5). Briefly, HeLa T4 cells (5 × 105) were
infected with vaccinia virus vTF7-3 (multiplicity of infection 10),
and DNA (5 µg) and Lipofectin (10 µg) were added to the cells. At 7 h post-transfection, the cells were starved in medium lacking methionine and cysteine and then labeled with 100 µCi of
[35S]methionine and -cysteine (Amersham Life Sciences,
Inc.) for 30 min. For better detection of the TM proteins, the cells
were labeled with 100 µCi of [3H]leucine after
preincubation in medium lacking leucine. At the end of the labeling,
the label was removed, and Dulbecco's modified Eagle's medium with
fetal calf serum was added and chased for different times. Cells were
lysed in radioimmune precipitation buffer and clarified, and proteins
were immunoprecipitated with SIV-specific antiserum from an infected
rhesus monkey. The immunoprecipitated proteins were extensively washed
and analyzed by SDS-PAGE and autoradiography. For separation of the
precursor and gp120, and for detection of the TM proteins, aliquots
were analyzed on both 7.0 and 10.0% SDS-PAGE. The media collected at
different chase times were immunoprecipitated and analyzed
similarly.
Immunofluorescence--
At 7 h post-transfection, the expression
and cellular localization of proteins were analyzed after fixing cells
for 10 min with paraformaldehyde (3.6%), permeabilizing for 5 min with
Nonidet P-40, incubating with primary antibodies specific to the Env
protein of SIV, and then using secondary mouse anti-monkey antibodies conjugated with fluorescein isothiocyanate. The coverslips were washed
with phosphate-buffered saline, mounted on glass slides, and viewed in
a Nikon fluorescence microscope.
Cell Fusion Assay--
HeLa T4 cells were infected with vTF7-3
and transfected with plasmids as described above. At 8 h
post-transfection, the cells were detached from the dishes using
versene and cocultured with an equal number of CD4+ HUT 78 cells. The cells were photographed using a modulation contrast imaging
system (18) attached to a Nikon Diaphot microscope.
Assay for Intracellular Interaction of
COP and SIV Env
Proteins--
HeLa T4 cells transfected with plasmid DNA were labeled
with [35S]methionine and -cysteine for 3 h, washed
with phosphate-buffered saline, permeabilized with saponin (19), and
incubated with
COP antibody (Sigma) for 30 min at 4 °C. The
supernatant was collected, and it was determined that the SIV Env
proteins did not leak out due to permeabilization. The cells were then
washed in phosphate-buffered saline to remove the
COP antibody and lysed in CHAPS buffer (20). The lysate of the transfected cells was
divided into two equal portions; one portion was reacted with SIV
antiserum to analyze the cell-associated Env proteins, and protein A
was added to the other portion to detect the
COP-Env protein
complexes. The proteins were analyzed using SDS-PAGE and a
PhosphorImager (Molecular Dynamics).
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RESULTS |
SIVmac239 Env Protein Mutants with KK Residues at
3 and
4
Positions Are Efficiently Retrieved to the ER--
We have used the
SIVmac239 Env protein to investigate the function of the
KKXX retrieval signal in proteins with cytoplasmic domains
of various lengths. Fig. 1 depicts the
mutations introduced in the cytoplasmic tail, which all contained two
lysine residues as the
3 and
4 amino acids from the C-terminal end.
Using the recombinant vaccinia virus-based transient T7 expression
system (19), cells transfected with plasmid DNA expressing wt or mutant SIV Env proteins were labeled with [35S]methionine and
-cysteine for 30 min and chased for up to 6 h. With the wt
SIVmac239 Env protein, the precursor protein of 160 kDa was synthesized
during the 30-min pulse (Fig. 2,
lane 1). During 3- and 6-h chases, a fraction of the
precursor protein was cleaved into the SU component gp120 and the TM
component gp41 (Figs. 2A, lanes 2 and
3, and 2B, lane 1). These results are
consistent with earlier observations (13, 21, 22).

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Fig. 1.
Schematic representation of the cytoplasmic
domain of the SIVmac239 envelope protein and mutants with the retrieval
signal motif. The last four amino acids of each protein are
indicated, with lysines at the 3 and 4 positions. The
numbers correspond to the number of amino acids
in the cytoplasmic tail. The mutants are designated as SIV
env followed by the number of amino acids in the cytoplasmic tail
starting from the membrane-spanning domain and RS (retrieval
signal).
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Fig. 2.
Intracellular expression of SIV Env and
retrieval signal mutants. HeLa T4 cells were infected with vTF7-3
and transfected with plasmid DNA encoding the wt envelope,
SIVenv18RS, SIVenv37RS, or SIVenv164RS. A, 7 h
post-transfection, the cells were labeled with
[35S]methionine and -cysteine for 30 min and chased for 3 or 6 h in the presence of unlabeled methionine and cysteine.
Samples were immune-precipitated, analyzed using SDS-PAGE, and
visualized using autoradiography. Lane 1 shows the wt SIV
Env proteins labeled during the 30-min pulse; lane 2, 3-h
chase; lane 3, 6-h chase; lane 4, proteins
expressed by plasmid SIVenv18RS; lanes 5 and 6,
proteins during the 3- and 6-h chase; lane 7, proteins
expressed by plasmid SIVenv37RS; lanes 8 and 9,
proteins during the 3- and 6-h chase; lane 10, proteins
expressed by plasmid SIVenv164RS; and lanes 11 and
12, proteins during the 3- and 6-h chase. Pre denotes the precursor protein; gp120 and TM
denote the surface and transmembrane subunits. B, the lack
of proteolytic processing of the mutant proteins was further confirmed
by labeling the transfected cells with [3H]leucine and
analyzing the TM proteins (asterisks) by immunoprecipitation and 10% SDS-PAGE. Lane 1, wt TM subunit; lanes
2-4, TM in cells expressing plasmids SIVenv18RS,
SIVenv37RS, and SIVenv164RS, respectively; lanes 5-7,
TM proteins in cells expressing plasmids SIVenv6RS, SIVenv13RS, and
SIVenv103RS, respectively. The numbers on the right side denote the sizes of molecular weight
markers.
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To investigate the structural features necessary for a functional ER
retrieval signal, we placed KK residues at the
3 and
4 positions of
SIV Env proteins having cytoplasmic tail lengths of 18, 37, or 164 amino acids (Fig. 1). Immunoprecipitation of the protein encoded by
construct SIVenv18RS showed the presence of a precursor protein (Fig.
2A, lane 4) which was not proteolytically cleaved
during the chase periods, as evidenced by the lack of SU or TM subunits
(Figs. 2A, lanes 5 and 6, and
2B, lane 2). With the mutant protein encoded by
construct SIVenv37RS, which has 37 amino acids in the cytoplasmic tail
and lysines at
3 and
4 positions, similar results were obtained
(Figs. 2A, lanes 7-9 and 2B,
lane 3). To exclude the possibility that the lack of
cleavage of proteins encoded by the constructs SIVenv18RS and
SIVenv37RS was due to the deletions in the cytoplasmic tail, we also
analyzed truncated proteins with cytoplasmic tails of 18 and 37 amino
acids but which lacked the di-lysine motif. These proteins were
efficiently cleaved and transported to the cell surface, as evidenced
by cell surface immunofluorescence staining and secretion of gp120 into the media (data not shown). This demonstrates that the lack of proteolytic processing was a consequence of di-lysine-mediated ER
retrieval and retention.
To determine whether the signal is functional when placed in a protein
with 164 amino acids in the cytoplasmic tail, we mutated the coding
sequence of full length SIV Env to introduce di-lysines at
3 and
4
positions. When this construct (SIVenv164RS) was analyzed for protein
expression, a precursor protein similar in size to wt SIV Env was
synthesized during the 30-min pulse. With chases up to 6 h, this
protein did not undergo proteolytic processing (Figs. 2A,
lanes 10-12 and 2B, lane 4),
indicating its retrieval and retention in the ER.
To further investigate the cellular localization of these proteins,
their site of expression was analyzed by indirect immunofluorescence. In permeabilized cells, the wild-type SIV Env protein exhibited a
reticular staining pattern that extended throughout the cells (Fig.
3, panel A). As expected, the
cells also showed surface expression of the envelope protein
(panel E). The intracellular staining pattern of the mutant
proteins resembled that of the wild-type protein (Fig. 3, panels
B-D). However, in contrast to the wild-type Env protein, no cell
surface staining was detected for any of the three mutants
(panels F-H), confirming that those proteins are
efficiently retrieved and retained in the ER. Thus a di-lysine motif
functions as a retrieval signal in proteins with a cytoplasmic tail 164 amino acids in length.

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Fig. 3.
Cellular localization of SIV Env and
retrieval signal mutant proteins. 7 h after transfection, the
cells were fixed with 3.6% paraformaldehyde or fixed and permeabilized
with 1% Nonidet P-40 and immunolabeled as described under
"Experimental Procedures." Panels A-D show the
intracellular fluorescent staining patterns, and panels E-H
show surface fluorescence. Panels A and E,
wild-type SIV Env; panels B and F, cells
transfected with plasmid SIVenv18RS; panels C and
G, cells transfected with plasmid SIVenv37RS; panels
D and H, cells transfected with plasmid
SIVenv164RS.
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SIV Envelope Proteins with a Di-lysine Motif near the Membrane
Spanning Domain Are Transported to the Cell Surface--
To determine
if ER retrieval signals placed closer to the membrane spanning domain
are functional, we analyzed the mutant proteins encoded by constructs
SIVenv6RS and 13RS and having 6 or 13 amino acids in the cytoplasmic
tail and lysines at
3 and
4 positions from the C terminus. The
proteins encoded by both constructs were found as a precursor during
the 30-min pulse, and chases for 3 and 6 h resulted in cleavage
into SU and TM components (Fig. 4). These
data indicate that the mutant proteins are not retained in the ER but
undergo proteolytic processing in a post-ER-Golgi compartment (15,
23).

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Fig. 4.
Expression of SIV Env proteins having a
retrieval signal close to the transmembrane domain. Upper
and lower panels represent the proteins resolved using 7 and
10% SDS-PAGE, respectively. Lane 1 shows the proteins
labeled during the 30-min pulse, and lanes 2 and
3 show the proteins during a 3- and 6-h chase, respectively, from cells transfected with plasmid SIVenv6RS. Lane 4 shows
the proteins labeled during the 30-min pulse, and lanes 5 and 6 show the proteins during a 3- and 6-h chase,
respectively, from cells transfected with plasmid SIVenv13RS.
Pre denotes the precursor protein; gp120 and
TM denote the surface and transmembrane components. The
numbers on the right side denote the sizes of
molecular weight markers.
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For both the mutant proteins, the intracellular immunofluorescence
pattern resembled that of the wild-type protein (Fig.
5, A and B).
Although the mutants contained the di-lysine motif, cell surface
expression was observed on unpermeabilized cells (panels D
and E). Thus, these results support the conclusion that a
cytoplasmic domain of a minimum length is needed for efficient retrieval and retention in the ER by a di-lysine containing signal.

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Fig. 5.
Cellular localization of SIV Env proteins
having a retrieval signal proximal to the membrane spanning
domain. HeLa T4 cells were transfected, fixed, and stained as in
Fig. 3. Panels A-C show the intracellular fluorescent
staining patterns, and panels D-F show surface
fluorescence. A and D, cells transfected with
plasmid SIVenv6RS; B and E, cells transfected
with plasmid SIVenv13RS; and C and F,
untransfected HeLa T4 cells.
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A Di-lysine Motif near an Amphipathic Helical Region of the
Cytoplasmic Tail of SIV Env Does Not Function as a Retrieval
Signal--
The cytoplasmic domain of HIV is postulated to form
amphipathic helical structures between amino acids 770-794 and
824-856 (24), which may associate with the inner surface of the plasma membrane (25). Both of these regions are conserved in HIV and SIV
genomes (26). To determine whether a retrieval signal positioned near
such an amphipathic region could be functional, we analyzed the
construct SIVenv103RS, which encoded a protein truncated after 103 amino acids in the cytoplasmic domain. During a 30-min pulse, a
precursor protein of approximately 155 kDa was synthesized in transfected cells (Fig. 6A).
After a 3- or 6-h chase, the precursor was cleaved into SU and a TM
subunit of approximately 36 Kda, indicating that the retrieval signal
was not functional (Figs. 6A, lanes 1-3 and
2B, lane 7).

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Fig. 6.
A retrieval signal near the amphipathic
helical region of SIV Env is not functional. HeLa T4 cells were
infected with vTF7-3 and transfected with plasmid SIVenv103RS.
Labeling and immunoprecipitation were done as described for Fig. 2, and
the proteins were resolved on SDS-PAGE and visualized by
autoradiography. A, intracellular expression of protein
encoded by plasmid SIVenv103RS. Upper and lower
panels represent the proteins resolved using 7 and 10% SDS-PAGE,
respectively, and the numbers on the right side indicate the sizes of molecular weight markers. Lane 1,
30-min pulse; lane 2, 3-h chase; lane 3, 6-h
chase. B, media were collected at the end of 3- and 6-h
chase periods and immunoprecipitated and analyzed by SDS-PAGE.
Lane 1, 3-h chase; lane 2, 6-h chase. Pre denotes the precursor protein; gp120 and
TM denote the surface and transmembrane components.
C, immunofluorescence of cells transfected with plasmid
SIVenv103RS. Cells were processed as in Fig. 3. Panel a,
intracellular fluorescence; panel b, cell surface
fluorescence.
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The lack of recognition of the retrieval signal was confirmed by
detection of gp120 in the extracellular medium from cells transfected
with plasmid SIVenv103RS (Fig. 6B, lanes 1 and
2). The protein exhibited a reticular immunofluorescence
pattern within the cell (Fig. 6C, panel a) and
was readily detected on the cell surface (panel b). These
results indicate that the di-lysine motif did not function as an
effective ER retrieval signal when positioned near an amphipathic
helical region.
Membrane Fusion Properties of SIV env Mutants--
In cells
transfected with expression vectors or SIV-infected cells, membrane
fusion occurs when the cell surface-expressed Env protein interacts
with the CD4 receptor on neighboring cells (13, 27). Hence, we
determined if the mutants with the di-lysine motif in different length
cytoplasmic tails were functional in inducing the formation of
syncytia. Coculture of HUT 78 cells with HeLa T4 cells expressing the
wt SIV Env protein revealed syncytium formation (Fig.
7A). In contrast, the mutant
proteins encoded by plasmids SIVenv18RS, SIVenv37RS, and SIVenv164RS
failed to induce membrane fusion (panels B-D). These
results are consistent with the observed defect in proteolytic
processing and cell surface transport. In contrast, the mutant proteins
expressed from plasmids SIVenv6RS and SIVenv13RS with the KK motif
close to the membrane spanning domain were able to cause extensive
membrane fusion (panels E and F), and there was
no apparent difference between fusion activity of these mutants and
truncated proteins with tail length of 6 and 13 amino acids which
lacked the retrieval signal. These results are consistent with earlier
data indicating increased fusogenic properties of cytoplasmic tail
truncation mutants (13). The mutant protein encoded by construct
SIVenv103RS, which has the retrieval signal near the amphipathic
region, also induced membrane fusion (panel G). Thus, the
results with the syncytium assay are consistent with the other results
showing cell surface expression versus ER retrieval of the
Env mutants.

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Fig. 7.
Membrane fusion activity of SIV mutants.
HeLa T4 cells were infected and transfected as described for Fig. 2. 8 h post transfection, cells were detached and co-cultured with
CD4+ HUT 78 cells. Panel A shows syncytia in
cells transfected with the plasmid encoding wt SIV Env; panels
B-D show the lack of syncytia in cells transfected with plasmids
SIVenv18RS, SIVenv37RS and SIVenv164RS, respectively; panels
E-G show syncytia in cells transfected with plasmids SIVenv6RS,
SIVenv13RS, and SIVenv103RS, respectively; and panel H shows
the co-culture of HUT 78 cells with untransfected HeLa T4 cells.
Arrows indicate typical syncytia.
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Interaction of
COP with SIV Env Proteins--
To investigate
the mechanism involved in the observed differences in retrieval of SIV
mutant proteins, we analyzed the interaction of the Env proteins with
COP proteins.
COP is a 110-kDa protein that is associated with
non-clathrin-coated vesicles and the Golgi complex (28) and has been
implicated in the transfer of proteins from the ER to the Golgi complex
(29). We developed an assay using saponin permeabilization which
enabled us to determine binding of the retrieved or retained proteins
to
COP. Quantitation of the amount of
COP-associated Env
proteins showed that proteins encoded by plasmids 164RS, 37RS, and 18RS
bound to
COP at least at 3- to 5-fold higher levels than the wt Env
protein (Figs. 8A, lanes
2-4, and 8B). In contrast, the KK motif-containing
proteins that were not retained showed lower binding to
COP (Figs.
8A, lanes 1 and 5-7, and
8B). The amount of radiolabeled
COP detected in
Env-expressing cells was less than that of the coprecipitated Env
proteins, presumably because most of the
COP is unlabeled due to
the 3-h labeling time used. To confirm the identity of
COP, we
transfected cells with a plasmid DNA expressing
COP, and the lysate
was immunoprecipitated using
COP antibodies (Fig. 8A,
lane 8). The intracellular expression levels of the Env
proteins were similar with all the constructs (Fig. 8C).
These results demonstrate that an increased binding to
COP is
observed in the proteins with a functional ER retention motif.

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Fig. 8.
Intracellular interaction of COP with SIV
Env proteins having an ER-retrieval signal. Transfected cells were
labeled with [35S]methionine and -cysteine for 3 h
and permeabilized with saponin for 3 min. The cells were incubated with
COP antibody for 30 min, lysed in CHAPS buffer, and processed as
described under "Experimental Procedures," and the proteins were
resolved using SDS-PAGE. A, coimmunoprecipitation of COP
with SIV Env proteins. Lanes 1-7 show immunoprecipitates
with COP antibody in cells transfected with plasmids SIV wt Env,
164RS, 37RS, 18RS, 103RS, 13RS and 6RS; lane 8, expression
of COP in cells transfected with COP DNA. B, the
amount of SIV Env proteins interacting with COP was quantified using a PhosphorImager and is graphically represented. C,
comparison of intracellular expression levels of SIV Env proteins
analyzed by immunoprecipitation with SIV antiserum. Lanes
1-7, cells transfected with plasmids SIV wt Env, 164RS, 37RS,
18RS, 103RS, 13RS and 6RS, respectively. Pre denotes the
precursor protein, COP denotes the position of the COP
protein.
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DISCUSSION |
Most previous studies of ER retrieval and retention have analyzed
proteins with cytoplasmic tails of more than 15 amino acids although
there are examples of retained proteins having only 10 amino acids in
the cytoplasmic tail (10, 30, 31). Chimeric proteins used to analyze
the di-lysine motif had at least 15 amino acids in the cytoplasmic
tail, and attempts to place the lysines closer to the membrane were not
reported. The present results provide evidence that potential retrieval
signals in the SIV Env proteins with a tail length of 6 or 13 amino
acids are not functional, whereas such a signal is functional in
proteins with longer cytoplasmic domains. Structural modeling (24) and
topogenic analyses of the HIV-1 envelope protein (25) indicated that
the cytoplasmic domain has the propensity to form two amphipathic
helical structures. Peptides which correspond to the amphipathic region
were found to interact with membranes (32, 33), indicating that such amphipathic regions may promote membrane association. The mutant protein encoded by plasmid SIVenv103RS contains a cysteine residue that
is the site for palmitoylation (34), and this modification was
postulated to enable the amphipathic regions to tightly associate with
cellular membranes. Our observation that a retrieval signal placed near
the predicted amphipathic helical region of the SIV Env protein is not
recognized by the cellular retention machinery may be explained by the
close association of this region with cellular membranes.
The finding that di-lysines introduced at different positions in the
SIV envelope protein result in proteins that have different cellular
localization phenotypes raises several possibilities. When lysines are
positioned very near to the membrane spanning domain, they may not be
recognized because of the lack of some structural features.
Alternatively, when the signal is located near the membrane, a cellular
protein may sterically mask the signal and thereby result in lack of
recognition. A masking of a retention signal has been reported to occur
during the assembly of the
and
chains of human high affinity
receptor for immunoglobulin E (35). The retention or retrieval of
proteins in the ER is a consequence of the interaction of the di-lysine
motif with the members of the 7-subunit coatomer complex (9-11). It
has been shown that di-lysine-containing proteins bind to
, 
and
(10),
COP (36), and
and
(37) subunits of the coatomer
complex. Our data indicate that the Env proteins which are retained
intracellularly exhibited higher binding to
COP than the proteins
which were transported to cell surfaces, suggesting that this
interaction is involved in the mechanism for the ER retrieval of SIV
Env proteins.
The cell surface transport of several viral proteins can be modulated
by the length of the cytoplasmic tail. Deletions or changes in the
cytoplasmic tails of VSV G (38), paramyxovirus HN (39), and
paramyxovirus F (40) proteins have been shown to inhibit or delay the
cell surface delivery of those proteins. VSV G mutants that were
retained in the ER had highly charged C termini and a lysine occupying
the
3 position (38). In contrast, for HIV and SIV, the truncation of
the cytoplasmic tail does not have a significant effect on cell surface
transport (13, 41-43). Furthermore, we have confirmed that the lack of
cleavage and cell surface transport of proteins expressed from plasmids
SIVenv18RS and SIVenv37RS were not due to the truncations in the
cytoplasmic tail. We found that all mutants in which the di-lysine
motif was changed to other amino acids (
3 and
4 FF or PF) were
processed and transported to the cell surface, confirming the strict
requirement for lysines in the retrieval signal. Several studies have
indicated that the amino acids surrounding the KK motif did not
contribute significantly to the retrieval phenomenon (2, 5, 44). Although the mutants we have described have different amino acids at
the
1 and
2 positions, these or similar amino acids are present in
other proteins that were found to be retrieved to the ER by the KK
motif at the
3 and
4 positions (2, 44). Hence, we conclude that the
lack of retention of the mutant proteins expressed by plasmids
SIVenv6RS, SIVenv13RS, and SIVenv103RS is due to the location of the KK
motif in a short cytoplasmic tail or near an amphipathic helical
region, which prevented efficient binding of these proteins to the
coatomer complex.
Recently it has been reported that specific tyrosine residues in the
cytoplasmic tail of the envelope protein of HIV-1 are responsible for
endocytosis of the protein (45). Mutations or deletion of these
tyrosine residues resulted in a dramatic increase in cell surface
expression, and this phenomenon was attributed to the reduced rate of
endocytosis (45). This phenomenon is not likely to account for the
differences that we observed in the expression of proteins at the cell
surface because SIVenv103RS, which was expressed at the cell surface,
and SIVenv164RS, which was retained intracellularly, contain both of
the tyrosine motifs thought to be responsible for endocytosis.
Furthermore, other mutants that are expressed at the cell surface
(SIVenv6RS and SIVenv13RS) as well as those that are not expressed at
the cell surface (SIVenv18RS and SIVenv37RS) all contain a tyrosine
residue that is conserved in many SIV and HIV isolates (21).
In summary, we have provided evidence that the ability of lysine
residues at
3 and
4 positions to function as an efficient retrieval
signal depends on the length and structure of the cytoplasmic domain.
The mutants in which the KK motif was functional failed to undergo
proteolytic processing, were not detected on the cell surface, and were
defective in inducing cell fusion. Thus, our studies provide evidence
that there is a requirement for lysines to be placed at a minimum
distance from the membrane spanning domain.
We thank Dr. Preston Marx for providing
antiserum to SIV and Dr. Thomas Kreis for providing the cDNA for
COP. The following reagents were obtained through the AIDS Research
and Reference Program, Division of AIDS, NIAID, National Institutes of
Health: HeLa T4 cells were from Dr. Richard Axel; HUT 78 cells were
from Drs. Adi Gazdar and Robert Gallo; and vaccinia virus (vVTF7-3) was from Dr. Bernard Moss. We also thank Lawrence Melsen for help in
the use of the modulation imaging system and preparation of figures and
Tanya Cassingham for help in preparing the manuscript.