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(Received for publication, April 13, 1995; and in revised form, June 16, 1995) From the
The transporter associated with antigen processing (TAP)
transports short peptides from the cytosol to the endoplasmic
reticulum, where peptides assemble with class I molecules of the major
histocompatibility complex. TAP is comprised of two subunits, termed
TAP1 and TAP2. We produced recombinant vaccinia viruses that direct
synthesis of the TAP subunits, either individually or together.
Virus-encoded TAP is rapidly and efficiently assembled (t of 5
min or less) by cells and does not spontaneously assemble in detergent
extracts. By confocal immunofluorescence microscopy, TAP1 when
expressed alone or with TAP2 is largely, if not exclusively, localized
to the endoplasmic reticulum. Metabolic labeling with
[2-
CD8 The TAP genes are members of a large family of integral membrane
transporters referred to as ATP binding cassette (ABC) proteins since
each has a characteristic sequence associated with ATP binding. ATP
hydrolysis is believed to drive the transport of the wide variety of
substrates handled by the various family members(9) .
Typically, ABC transporters are comprised of a single subunit
containing two cytosolic ATP binding domains of approximately 300
residues and 12 hydrophobic domains believed to traverse the membrane,
with short peptides connecting the hydrophobic domains. The structure
of TAP is similar to other ABC proteins, with the most notable
difference being its division into two polypeptides, TAP1 and TAP2,
each containing a single ATP binding domain and 6 potential
transmembrane domains. The past 2 years have witnessed rapid
progress in understanding TAP-mediated translocation of peptides. Using
semi-intact and cell-free systems, the basic requirements for peptide
length and sequence have been
defined(10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23) .
There are still sizable gaps, however, in our knowledge of numerous
aspects of TAP, including its assembly, intracellular trafficking, and
precise mechanism of function. In the present study we explore some of
these issues using recombinant vaccinia viruses (rVVs) expressing each
TAP subunit or the two subunits simultaneously.
Figure 7:
TX114 phase partitioning of
TAP[1+2]. TX114 extracts from VV-TAP1 or
VV-TAP[1+2]-infected cells were incubated on ice (Tot.) or at 37 °C for 5 min and then partitioned into
detergent (Det.) or aqueous phases (Aq.), and
immunoreactive species were analyzed by
SDS-PAGE.
Figure 1:
Function of VV-encoded TAP subunits. A, T2 cells were infected with the indicated rVVs for 3 h
before testing in a 6-h cytotoxicity assay using secondary in vitro restimulated VV-specific T
The number of class I-peptide complexes necessary for lysis
by T These findings demonstrate that
VV-expressed TAP1 and TAP2 are functional, but only when expressed
together. This is concordant with findings in microsomal or
permeabilized cell systems that both subunits are required for peptide
transport into the ER above background levels (10, 18, 19) or formation of a functional
peptide binding site(21) .
Figure 2:
Assembly of VV-encoded TAP subunits. A, L929 cells infected for 4 h with the indicated rVVs were
labeled with [
Figure 5:
[2-
Following double
infection of cells with VV-TAP1 and VV-TAP2, TAP1 was always recovered
in higher amounts using the anti-TAP1 antiserum. Even if assembly
occurred with 100% efficiency this would be expected, since it is
statistically inevitable that some cells will be infected with a
greater number of VV-TAP1 virions (and vice versa). Following
infection with VV-TAP[1+2], enhanced recovery of TAP1
relative to TAP2 was observed in approximately half of the experiments.
Since we expect that TAP1 and TAP2 are translated at the same rate in
VV-TAP[1+2]-infected cells, this implies that a pool of
unassembled TAP1 (and possibly TAP2) exists. In the other experiments,
however, using the identical stock of VV-TAP[1+2], the
ratio of TAP1 to TAP2 was close to 1, indicating that TAP assembly can
be quite efficient, even when unnaturally overexpressed in the absence
of up-regulation of other gene products normally regulated in parallel
with TAP. The variability in the ratio of TAP1:TAP2 recovered might
reflect true variability, the efficiency of TAP assembly in
vivo, or artefactual variability in the preservation of the
heterodimers in biochemical procedures following detergent extraction.
The former possibility would be consistent with TAP assembly being a
regulated process. We next examined the rate of assembly of TAP
heterodimers by labeling VV-TAP[1+2]-infected L929 cells
for 1 min with [ TAP assembly is remarkable in that it occurs as swiftly as any
oligomeric membrane proteins we are aware of(46) . This
indicates that the complicated topology of multispanning membrane
proteins such as TAP need not limit their rate of assembly. Due to the
high levels of rVV expression, the present findings may represent an
upper limit for the rate of TAP assembly, which could be slower under
normal conditions when the concentration of newly synthesized subunits
in the ER is lower. Note that the experimental design precludes
determining the extent to which newly synthesized subunits pair with
new versus old subunits.
We first attempted to detect binding of
[
Figure 3:
Photolabeling of TAP1 and TAP2. A, detergent extracts from L929 cells co-infected for 3.5 h
with VV-TAP1 and VV-TAP2 and ATP depleted for 30 min by incubation in
carbonyl cyanide m-chlorophenylhydrazone-containing medium
were incubated with 8-azido[
Photolabeling of TAP was completely inhibited by EDTA. This is
probably due to depletion of Mg The nucleotide binding properties of TAP were
further examined by inhibition studies. As shown in Fig. 3B (quantitated in Fig. 3C), photolabeling was
prevented by preincubation with nucleotides in the order ATP =
CTP = UTP GTP > ADP for both the TAP1 and TAP2 proteins. AMP
did not significantly inhibit photolabeling. It is notable that
labeling of TAP1 and TAP2 was inhibited in parallel by the various
nucleotides, indicating that they either independently bind nucleotides
in a highly similar way or bind nucleotides cooperatively when
complexed. Based on its rank relative to other nucleotides and its
relatively high concentration in cells (1 mM), it is likely
that ATP is the most common substrate for TAP. Since GTP is present at
similar concentrations in the cytosol and has an apparent affinity of
roughly that of ATP, it is likely that GTP also serves as a TAP ligand in vivo. ATP was also the most potent inhibitor of the
COOH-terminal domains examined in previous
studies(47, 48) , but the rank order of inhibition by
other nucleotides differs from our findings and, indeed, between the
previous studies. These differences presumably reflect differences
between full-length and COOH-terminal fragments of TAP or between TAP
produced by mammalian cells versus insect cells or bacteria.
Figure 4:
Immunofluorescence localization of TAP.
VV-TAP[1+2]-infected L929 cells were
paraformaldehyde-fixed and detergent-permeabilized prior to reactivity
with antibodies specific for TAP or BiP, and suitable secondary anti-Ig
reagents were conjugated to fluorescein or Texas Red. Fluorescence was
simultaneously detected by confocal scanning laser
microscopy.
To further characterize TAP1
glycosylation, we examined the sensitivity of
[
Figure 6:
Absence of TAP1 binding to lectins. L929
cells were labeled for 15 min with [
Inclusion of
protease inhibitors (aprotinin, pepstatin, leupeptin,
1-chloro-3-tosylamido-7-amino-2-heptanone, phenylmethylsulfonyl
fluoride, and
Volume 270,
Number 36,
Issue of September 08, pp. 21312-21318, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
H]mannose demonstrates that TAP1 (but not
TAP2) possesses Asn-linked oligosaccharides, but the lack of binding of
[S]methionine-labeled TAP to concanavalin
A-agarose suggests that the glycosylated form represents a minor
population of TAP1. The two subunits of the assembled complex present
in detergent extracts photolabeled equally with
8-azido[
-P]ATP. Photolabeling of the two
subunits was inhibited in parallel by various di- and trinucleotides,
suggesting that their nucleotide binding sites function in a highly
similar manner. Incubation of detergent extracts at 37 °C results
in the rapid loss of TAP1 immunoreactivity, indicating either an
unusual sensitivity to proteases or an irreversible conformation
alteration.
T cells (T
) recognize
peptides, usually 8-10 residues in length, bound to major
histocompatibility complex (MHC) (
)class I
molecules(1) . Peptides are predominantly generated from a
cytosolic pool of proteins (2, 3) . Class I molecules
consist of a polymorphic integral membrane glycoprotein ( chain)
complexed to 
-microglobulin, a soluble nonglycosylated
protein. Both chains possess NH-terminal hydrophobic
sequences that target them co-translationally to the endoplasmic
reticulum (ER). Most antigenic peptides, having no such ER insertion
sequence, remain sequestered on the cytosolic side of the ER membrane
and require a specific transporter, termed TAP (acronymic for
transporter associated with antigen processing) to access class I
molecules. TAP is produced by the association of two MHC-encoded
subunits, termed TAP1 and
TAP2(4, 5, 6, 7) . The central
importance of TAP in T responses is most stunningly
shown by the severe depletion of T cells in mice with a targeted
disruption of the TAP1 gene(8) .
Cells and Viruses
The antigen
processing-deficient human cell lines T2 (24) and B6 (25) were maintained in Iscove's modified DMEM
supplemented with 7.5% (v/v) fetal bovine serum. L929 mouse fibroblasts
were maintained in DMEM supplemented with 7.5% (v/v) fetal bovine
serum. rVVs were propagated in thymidine kinase-deficient human 143B
osteosarcoma cells. rVVs expressing TAP1 (VV-TAP1) or TAP2 (VV-TAP2)
were produced by cloning cDNAs encoding the respective genes behind the
early/late VV p7.5 promoter into a modified pSC11 plasmid as
described(26) . The human TAP1 (a allotype) and TAP2 (b allotype) genes have been described(27) . A
double rVV expressing TAP1 and TAP2 genes under the
control of the p7.5 promoter (VV-TAP[1+2]) was produced
by the method described by Coupar et al.(1988)(28) .
Briefly, the TAP2 gene fragment ending with XhoI
sites was inserted into the SalI site of pTK-7.5B plasmid,
which contains the herpes simplex virus type I thymidine kinase gene
under the control of the VV promoter P-F. 143B cells infected with
TAP1-rVV were transfected by the pTK-7.5B plasmid containing the TAP2 insert. Double rVVs with the TAP1 gene inserted
in the VV tk gene and the TAP2 gene in the VV HindIII-F region were selected for thymidine kinase expression
by inclusion of aminopterin in the growth medium(29) . rVVs
encoding A/Puerto Rico/8/34 influenza virus HA and NP under the
control of the VV p7.5 promoter have been described(30) . rVVs
encoding a secreted form of NP and mouse ICAM-1 will be described
elsewhere.Cytofluorography
T2 cells were infected for 15 h
with rVVs and incubated for 15 min at 0 °C with
fluorescein-conjugated antibodies specific for HLA class I molecules
(W6/32; Accurate, Westbury, NY), -microglobulin (The
Binding Site, San Diego, CA), or mouse ICAM-1 (Pharmingen, San Diego,
CA). After washing once, cells were resuspended in PBS supplemented
with 10 µg/ml ethidium homodimer (Molecular Probes, Eugene, OR) and
analyzed using a FACSCAN (Becton Dickinson, San Jose, CA). Nonviable
cells fluorescently labeled with ethidium homodimer were excluded from
analysis.Cytotoxicity Assay
Target cells were infected with
VV as described(31, 32) . T were
generated from splenocytes derived from animals immunized with viruses
2-6 weeks previously by 7-day in vitro stimulation with
virus-infected autologous splenocytes as
described(31, 32) . Microcytotoxicity assays were
performed as described(31, 32) . Data are expressed as
percentage of specific release defined as ((experimental cpm -
spontaneous cpm)/(total cpm - spontaneous cpm))
100.
Immunofluorescence
VV-infected L929 cells were
prepared for immunofluorescence as described(33) . Double
antibody immunofluorescence was performed by incubating coverslips for
2 h at 20 °C with PBS containing 1 µg/ml of rabbit anti-TAP1
antibody. After washing with PBS, coverslips were incubated for 2 h at
20 °C with PBS containing 1% (v/v) Texas Red-conjugated goat
anti-rabbit IgG, washed and incubated for 2 h at 20 °C with PBS
containing 33% (v/v) rat hybridoma tissue culture supernatant
containing a mAb specific for immunoglobulin binding protein
(BiP)(34) . Coverslips were then washed, incubated for a
further 2 h at 20 °C with PBS containing 1% (v/v) fluorescein
isothiocyanate-conjugated rabbit anti-rat IgG, washed, and mounted in
Fluorsave (Calbiochem, San Diego, CA). Images of fluorescent staining
were acquired using a Bio-Rad MRC 600 confocal microscope, and hard
copies were produced as described (35) .Lectin Depletion
Detergent extracts were prepared
as described below, with the exception that the extraction buffer was
supplemented with 5 mM Ca and 5 mM Mn
. Extracts were incubated for 2 h at 4 °C
with agarose coupled to concanavalin A (ConA, Vector Laboratories,
Burlingame, CA), and immunoreactive species remaining in the
supernatant were collected as described below.
Metabolic Labeling
For metabolic labeling with
[S]methionine (Amersham Corp.), L929 cells were
infected as described(36) . Four hours post-infection, cells
were incubated for 30 min at 37 °C in serum-free, methionine-free
DMEM and then, after adjusting cells to a concentration of 1-2
10
/ml, for 5-15 min at 37 °C in 1 ml of
the same medium supplemented with 400 µCi of
[S]methionine. Cells were then pelleted and
chased at 37 °C in DMEM containing 1 mg/ml methionine. For labeling
with [2-
H]mannose (American Radiolabeled
Chemicals, St. Louis, MO), 1-2 10
/ml cells
were incubated in serum-free, glucose-free medium for 15 min at 37
°C in 1 ml of the same medium supplemented with 1 mCi of
[2-H]mannose. For all radiolabeling experiments,
cells were shifted to 0 °C after appropriate incubation periods
until detergent-extracted by 15 min of incubation at 0 °C in
150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40,
0.5% MEGA9, 1 mM phenylmethylsulfonyl fluoride, 10 mM Tris, pH 7.4. Following centrifugation at 15,000
g, supernatants were harvested and precleared by overnight
incubation with protein A-agarose preloaded with rabbit serum specific
for an irrelevant antigen. Supernatants were then incubated for 1 h
with protein A-agarose preloaded with the affinity-purified rabbit
anti-peptide antiserum RING4 produced by immunizing rabbits with a
synthetic peptide corresponding to the COOH-terminal 15 residues of
TAP1(5) . After extensive washing, proteins were eluted from
beads by boiling in SDS-PAGE sample buffer and analyzed by SDS-PAGE
using the conditions of Laemmli(37) . Digestion of samples with
endo-
-N-acetylglucosaminidase H (endo H) was performed as
described(36) . After acid fixing and staining with Coomassie
Blue to ensure that equivalent amounts of antibody were recovered from
samples, gels were dried at 80 °C under vacuum. For samples labeled
with [2-H]mannose, gels were incubated with
Amplify® (Amersham) prior to drying. Fluorographs of tritiated
samples were produced by exposing dried gels to preflashed film at
-70 °C. Dried gels containing
[S]methionine-labeled proteins were exposed to
PhosphorImager screens (Molecular Dynamics, Sunnyvale, CA), which were
imaged and quantitated using the associated hardware and software.
Fluorographs were digitized using a flatbed scanner and quantitated
using the PhosphorImager software. Hard copies of digital images were
produced using Adobe Photoshop software and a Kodak XL7700 printer.
Mice
Six- to eight-week-old BALB/cByJ
(H-2
) mice were obtained from Jackson Laboratories (Bar
Harbor, ME). Mice were immunized with 10
plaque forming
units of VV by intravenous injection.Phase Partitioning
VV-infected cells were labeled
with [S]methionine and detergent extracted as
described above with 1% Triton X-114 (Pierce) replacing Nonidet P40 and
MEGA9. Following centrifugation at 15,000
g,
supernatants were harvested, incubated on ice (total in Fig. 7) or underlaid with cushion solution (6% sucrose in 10
mM Tris, pH 7.4, 150 mM NaCl, 0.06% Triton X-114),
and incubated at 37 °C for 5 min, at which time clouding was
obvious (38) . Following centrifugation at 15,000
g at room temperature the aqueous phase and detergent phase (yellow
droplet at the bottom of the tube) were removed separately and diluted
5 times with normal extraction buffer, and species reactive with
anti-TAP1 antiserum were collected as described above.
Photolabeling
Photolabeling was performed
according to Hobson et al.(39) . 3.5 h post-infection,
cells were incubated for 30 min in serum-free, glucose-free medium
containing 1 mM carbonyl cyanide m-chlorophenylhydrazone to deplete ATP. Extracts were prepared
by incubating cells for 15 min at 0 °C in 250 mM sucrose,
50 mM KCl, 2 mM MgCl, 2 mM EGTA,
1% Triton X-100 (v/v), 1 mM phenylmethylsulfonyl fluoride,
aprotinin, leupeptin, 10 mM Tris, pH 6.8 and centrifuging at
15,000 g for 30 min. 100-µl aliquots of the
supernatant were placed in the wells of a 96-well flat bottom
microtiter plate, and 2 µl of
8-azido-[
-P]-ATP were added (ICN
Biomedicals, Costa Mesa, CA). Samples were irradiated for two 2.5-min
intervals, first at 245 nm (1250 microwatts/cm
) and then at
365 nm (1050 microwatts/cm), with a 1-min interval to
prevent excessive heating. Approximately 3 10
cells
were used for each labeling reaction. For competition experiments, the
same conditions were used, but samples were preincubated on ice with
various nucleotides. Samples were diluted to 1 ml with the extraction
buffer used for radioimmuno-collection, and the reactive species were
analyzed by SDS-PAGE as described above.
Function of rVV-encoded TAP1 and TAP2
The
function of rVV-produced TAP1 and TAP2 was examined by testing the
ability of rVVs to rescue the ability of T2 cells (which express
neither TAP subunit) to present VV antigens to mouse
H-2K-restricted, VV-specific T. To
enable T
recognition, cells were co-infected with a
rVV expressing K
chains. As previously
reported(31, 40) , presentation of
K-restricted VV antigens by T2 cells is minimal (Fig. 1A). Co-infection of cells with
VV-TAP[1+2] greatly increased lysis, despite the fact
that co-infection with an additional rVV would be expected to decrease
the rate of K biosynthesis. Target cell lysis was
K-restricted since cells infected by
VV-TAP[1+2] alone were not lysed. Co-infection of
VV-K-infected cells with either VV-TAP1 or VV-TAP2 alone
failed to restore antigen presentation. Triple infection of cells with
all three recombinants, however, enabled T recognition. The decreased restoration relative to double
infection with K
and VV-TAP[1+2] can be
attributed to both competition for gene expression between rVVs and the
decreased likelihood of triple infection versus double
infection. Notably, co-infection of VV-K infected cells
with either VV-TAP1 or VV-TAP2 alone failed to rescue antigen
presentation. This indicates that neither subunit alone is able to
rescue antigen presentation. In additional experiments using antigen
processing-deficient cells lacking one of the two subunits, infection
with the appropriate rVV expressing a single TAP subunit was able to
restore presentation of either VV antigens or a number of foreign
antigens to the appropriate T population (data not
shown).
. B, T2
cells infected overnight with the rVVs indicated were tested for
binding to saturating quantities of fluorescein-conjugated W6/32 mAb
specific for native class I molecules, fluorescein-conjugated sheep
anti-human
-microglobulin, or fluorescein-conjugated
rat anti-mouse ICAM-1 mAb. Cells were analyzed in a cytofluorograph,
and the log mean channel fluorescence of viable cells
(gated by exclusion of ethidium homodimer) is
shown.
in the cytotoxicity assay employed in this
study is not known but is possibly under 1000
complexes/cell(41) . To more quantitatively test the ability of
rVVs expressing TAP subunits to restore class I assembly, T2 cells were
infected overnight with rVVs at 37 °C, and the cell surface
expression of native endogenous class I molecules was determined by
flow cytometry. T2 cells cultured at 37 °C express considerable
amounts of HLA-A2 due to its association with peptides derived from ER
insertion sequences(42, 43) . Class I expression is
increased, however, by TAP expression(25) . As shown in Fig. 1B, the level of endogenous class I expression
detected by fluorescein-conjugated W6/32 (a mAb specific for native
class I molecules), or fluorescein-conjugated sheep
anti-
-microglobulin antibodies was roughly doubled by
infection with VV-TAP[1+2]. In contrast, infection with
rVVs expressing the individual subunits did not significantly increase
class I expression. The specificity of enhanced class I expression is
shown by the failure of TAP[1+2] expression to enhance
the surface expression of VV-encoded mouse ICAM-1, a non-MHC-associated
integral membrane glycoprotein.Assembly of rVV-encoded TAP
To biochemically
characterize TAP, affinity-purified rabbit antibodies raised to a
peptide corresponding to the 15 COOH-terminal residues of TAP1 were
used to recover TAP1 from detergent extracts. Following 15 min of
labeling with [S]methionine of cells infected
for 4 h with VV-TAP1, we detected a protein with a M
of 69,000 in SDS-PAGE (Fig. 2A). This is less
than the predicted M of 75,000 but in accord with
previously reported values(44, 45) . The mobility of
TAP1 was unchanged over a 2-h chase period (see Fig. 5B). The absence of this protein from cells
infected with a control VV confirms its identity as TAP1 (not shown).
Following infection with VV-TAP[1+2], or co-infection
with VV-TAP1 and VV-TAP2 a new band with the expected mobility of TAP2 (M 74,000) appeared (Fig. 2A). To
determine whether assembly of TAP occurred in detergent extracts, cells
infected with VV-TAP1 or VV-TAP2 were mixed and detergent-extracted,
and the species reactive with anti-TAP1 antibody were collected (Fig. 2B). This resulted in the recovery of TAP1 only.
Similarly, TAP(1+2) heterodimers were not detected when detergent
extracts from cells infected with either VV-TAP1 or VV-TAP2 were mixed.
Adding radiolabeled TAP2 in a 5-fold excess over TAP1 still did not
result in detectable complex formation. Based on these findings, we
conclude that under the conditions employed for detergent extraction,
all TAP heterodimers detected are formed in cells.
S]methionine for 15 min, and
material in detergent extracts reactive with anti-TAP1 antibodies was
collected and analyzed by SDS-PAGE. Only the region of the gel
containing TAP1 and TAP2 is shown. B, as in A, except extracts
were mixed prior to exposure to anti-TAP1 antibodies (middle),
or cells were mixed prior to exposure to extraction buffer (EB) (rightpanel). On the left are
reactive species present in cells infected with VV-TAP1, VV-TAP2, or
both rVVs. C, as in A, except cells were labeled for 1 min and
then placed on ice or incubated for 5 or 15 min at 37 °C prior to
detergent extraction and collection of antibody reactive
species.
H]mannose
labeling TAP1. A, L929 cells were labeled for 30 min with
[2-H]mannose 3.5 h post-infection with VV-TAP1 or
VV-HA and incubated at 0 °C (P) or 37 °C (C)
for 3 h. Species reactive with anti-TAP1 antibodies or the anti-HA mAb
H28-E23 (56) were collected, digested with endo H (+) or
mock-digested, and analyzed by SDS-PAGE. B, L929 cells were
labeled for 10 min with [S]methionine 3.5 h
post-infection with VV-TAP1 or VV-K
and incubated on ice or
at 37 °C for 120 min. TAP1 or K was collected from
detergent extracts, digested with endo H (+) or mock-digested, and
analyzed by SDS-PAGE.
S]methionine and chasing cells
for 5 or 15 min (Fig. 2C). The amount of TAP1 recovered
increased 3.4-fold within 5 min of initiating the chase. Since protein
synthesis in mammalian cells occurs at a rate of approximately 10
residues/s, synthesis of TAP is likely to require between 1 and 2 min.
This probably accounts for most of the increase in TAP1 recovery over
the chase period, although we cannot rule out the occurrence of
intrinsic alterations in TAP structure or the association of TAP1 with
molecular chaperones that limits antibody access to the COOH terminus.
Most notably, there was little increase in the ratio of TAP1:TAP2
recovered in either of the chase periods relative to the pulse. This
indicates that complex formation occurs extremely rapidly. These
findings were confirmed using the antigen-deficient human cell line B6,
which expresses only the TAP1 subunit. Following infection with
VV-TAP2, complex formation was again detected within 5 min (not shown).
Nucleotide Binding Properties of TAP
As members of
the ABC transporter family, TAP1 and TAP2 possess putative ATP binding
domains. ATP was previously demonstrated to bind to the nucleotide
binding domain of human TAP1 expressed in Escherichia coli and
to the COOH-terminal domains of mouse TAP1 and TAP2 expressed in Drosophilamelanogaster cells(47, 48) . Binding of ATP to intact TAP has
not, however, been reported.S]methionine-labeled, detergent-solubilized
TAP1 (expressed alone or with TAP2) to Affi-Gel Blue Sepharose (which
binds many ATP-binding proteins) or ATP-agarose by eluting bound
material with ATP and collecting TAP1 using anti-TAP1 antibodies. Both
matrices bound cellular or viral proteins that were released by ATP,
but we failed to detect TAP1 in either eluate (not shown). Since
similar failures have been reported for other ABC transporter family
members, we used 8-azido-[
-P]ATP to
photoaffinity-label TAP. Cell extracts prepared from L929 cells
co-infected with VV-TAP1 and VV-TAP2 were UV-irradiated in the presence
of 8-azido-[
-P]ATP, and species reactive
with TAP1 antiserum were analyzed by SDS-PAGE. This revealed labeling
of both TAP subunits (Fig. 3). The specificity of labeling is
demonstrated by the failure to recover labeled TAP in the absence of UV
irradiation (not shown) or when 1 mM unlabeled ATP was added
to samples prior to irradiation (Fig. 3B). Notably, the
ratio of
P-labeled TAP1 and TAP2 was similar to that
observed following [
S]methionine labeling, which
suggests that the subunits have a similar affinity for ATP.
-P]ATP and,
from left to right, EDTA, EDTA and
Mg
, EDTA and Mg
with
Ca
, or EGTA and Mg
, and the TAP
proteins were immunopurified and analyzed by SDS-PAGE. B, as
in A except samples were preincubated with the indicated
nucleotide prior to the addition of
8-azido-[
-P]ATP. C, quantitation
of data in B.
, since addition of
excess Mg
to EDTA-containing samples restored
labeling. A slight additional increase was observed upon addition of
Ca
to the Mg
repleted samples,
while Ca
alone had only a marginal effect on
photolabeling (Fig. 3A). These findings are in good
agreement with those reported for other ABC
transporters(49, 50, 51, 52) , which
have been interpreted to mean that each cytosolic ATP binding domain
possesses a Mg
binding site. The enhanced ATP binding
observed when Mg
is supplemented with Ca
is consistent with two possibilities: 1) the cytosolic domains
possess a Ca
binding site in addition to the
Mg
binding site, and 2) additional
Ca
-dependent factors in the extract contribute to ATP
binding to TAP.
Intracellular Localization of TAP
We examined the
intracellular localization of TAP by confocal immunofluorescence
microscopy of paraformaldehyde-fixed, detergent-solubilized VV-infected
L929 cells. Using the rabbit affinity-purified anti-TAP1 peptide
antiserum and a goat anti-rabbit Ig Texas Red-conjugated second
antibody, staining of cells infected with a control VV was at the low
levels observed when the rabbit antibody was omitted (not shown). By
contrast, staining of VV-TAP1 or VV-TAP[1+2] was quite
intense, yielding a classical ER pattern (Fig. 4). The ER
localization of TAP1 in VV-TAP[1+2]-infected cells was
confirmed by its nearly perfect co-localization with a rat antibody
specific for mouse BiP detected using rabbit anti-rat IgG
fluorescein-conjugated antibodies (Fig. 4). The ER localization
of TAP is consistent with previous data indicating that peptide
association occurs in an early secretory compartment (35) and
with findings that peptides transported via TAP into microsomes are
glycosylated(10) . It differs somewhat from the report that
considerable amounts of TAP are present in the cis-region of the Golgi
complex(53) . Additional experiments are required to determine
whether a minor portion of the intense TAP staining co-localizes with
the Golgi complex. It is possible, however, that transport of TAP to
the Golgi complex occurs too slowly for detection under the transient
conditions employed (5 h of infection). Also, it is worth noting that
interpretation of the immuno-EM images required a correction for
background staining to TAP-deficient cells and that the specificity of
staining of the Golgi complex was less certain than staining of the
ER(53) .
N-Linked Glycosylation of TAP
The ER is the site
of addition of Asn-linked oligosaccharides. TAP1 has three potential
sites for N-linked glycosylation (TAP2 has none). Using
hydropathy plots to identify the transmembrane regions of TAP1 and the
resulting ER versus cytosolic orientation of the
intertransmembrane regions, one Asn (Asn) is predicted to
be lumenally oriented and a potential target for Asn-linked
glycosylation. To investigate Asn-linked glycosylation of TAP1,
VV-TAP1- or VV-TAP[1+2]-infected L929 cells were
pulse-radiolabeled with [2-
H]mannose, and the
radiolabeled species reactive with anti-TAP1 antiserum were analyzed by
SDS-PAGE. [2-H]Mannose is the most specific
metabolic radiolabel for N-linked
oligosaccharides(54) , and the pulse labeling conditions should
further minimize its conversion to other metabolites. As seen in Fig. 5A, [2-H]mannose was
incorporated into influenza virus hemagglutinin (HA) collected from
cells infected with a rVV containing the HA gene. HA is an integral
membrane glycoprotein containing five N-linked
oligosaccharides. All of the label was removed from pulse-labeled HA by
endo H, whose specificity of digestion for immature high mannose
oligosaccharides is shown by its failure to digest a slower migrating
form of HA in the chase known to contain oligosaccharides modified by
Golgi complex-associated enzymes into endo H-resistant
forms(33, 36) . Labeled TAP1 was recovered from
detergent extracts derived from
[2-H]mannose-labeled VV-TAP1-infected cells (Fig. 5A) or from VV-TAP[1+2]-infected
cells (not shown). In the latter case, no label was associated with
TAP2, which is consistent with its absence of potential N-linked sites. Labeling of TAP1 decreased with the chase,
and, unlike HA, no shift in M was apparent. Label
was completely removed from pulse and chased samples by endo H, which
confirms that the label is incorporated into N-linked
oligosaccharides and indicates that the oligosaccharides do not undergo
modification by enzymes in the distal portions of the Golgi complex.
Based on densitometry of the fluororadiograph, TAP1 was labeled at the
level of HA. Assuming that the rates of HA and TAP1 synthesis are the
same and that one oligosaccharide is labeled in TAP1 versus five in HA, this would mean that
[2-H]mannose is incorporated into mannose at
approximately the level of HA, which suggests that only a
subpopulation is labeled.S]methionine-labeled TAP1 derived from
VV-TAP1-infected cells to digestion with endo H (Fig. 5B). While endo H induced a large shift in the
mobility of H-2K
(an integral membrane glycoprotein with
three N-linked oligosaccharides) treated in parallel, it had
no effect on the mobility of TAP1 in this experiment or on the mobility
of TAP1 or TAP2 from VV-TAP[1+2]-infected cells (not
shown). This finding is consistent with the prior observation that the
electrophoretic mobility of TAP produced by insect cells was unaffected
by tunicamycin inhibition of Asn-linked glycosylation(19) . The
lack of effect of endo H on TAP1 mobility has two plausible
explanations: the shift in M resulting from
detachment of a single oligosaccharide is too small to resolve by the
SDS-PAGE conditions utilized, or only a minor, undetected population of
TAP is glycosylated. In an attempt to maximize mobility difference
associated with the presence of N-linked oligosaccharides, we
labeled cells treated with inhibitors (bromoconduritol or
deoxynojirimycin) that prevent the removal of the glucose residue
present on the oligosaccharide initially transferred(55) . This
failed to alter the mobility of TAP1 in SDS-PAGE (not shown). Finally,
we incubated cell extracts prior to radioimmuno-collection with
ConA-agarose. ConA binds proteins containing Asn-linked
oligosaccharides, particularly those with high mannose
oligosaccharides. As seen in Fig. 6, incubation with
ConA-agarose did not remove
[S]methionine-labeled TAP1 from detergent
extracts. Under the same conditions a secreted form of influenza virus
NP containing a single Asn-linked oligosaccharide was nearly completely
depleted from extracts. Based on these findings, we provisionally
conclude that Asn-linked glycosylation of TAP is limited to a
subpopulation of molecules.
S]methionine
4 h post-infection with VV-TAP1 or VV-IS-NP (influenza virus NP with an
ER insertion sequence). Detergent extracts were incubated with agarose
coupled to ConA, and immunoreactive TAP or IS-NP present in
supernatants was analyzed by SDS-PAGE.
Detergent Phase Partitioning of TAP
Each TAP
subunit is thought to span the ER membrane six times. The membrane
association of TAP was examined by exposing TX114 extracts to a
temperature greater than the TX114 cloud point(38) . At
temperatures exceeding the cloud point, solutions partition into
detergent and water-rich phases. Integral membrane proteins partition
at least partially into the detergent-rich phase, while other proteins
are generally exclusively recovered from the aqueous phase. To our
surprise, incubation of Triton X-114 extract at 37 °C for as
briefly as 5 min decreased by more than 90% the amount recovered of
TAP1 or TAP[1+2] complex reactive with anti-TAP1
antiserum (Fig. 7). The remaining fraction of TAP was recovered
from both the detergent and soluble phases, which parallels the
distribution of influenza virus HA in additional experiments, while a
negative control protein, influenza virus NP, partitioned exclusively
in the aqueous phase (not shown). If the remaining TAP is
representative of the bulk of unrecovered TAP, these findings support
the idea that TAP is an integral membrane protein.2-macroglobulin) alone or as a mixture in the
extraction buffer did not block the loss of TAP under these conditions.
Nor was loss blocked by the addition of antigenic peptides, ATP, or
inhibitors of ATPase activity (not shown). A similar loss of TAP1 was
observed when TX100 or Nonidet P-40 extracts were incubated at 37
°C (not shown). Since these detergents phase partition only at 50
°C, the loss of immunoreactive TAP is related to temperature and
not phase partitioning per se. There are two explanations for
these findings. First, the COOH terminus of TAP1 (against which
anti-peptide TAP1 antibody is directed) may be cleaved by a protease
insensitive to the inhibitors used. It is notable that under the same
conditions, influenza virus NP, which is very sensitive to proteolysis,
was not digested (not shown). Thus, if loss of immunoreactive TAP
reflects proteolysis, a highly specific protease may be involved.
Second, elevated temperatures may induce an irreversible conformational
alteration or association with other factors in the extract, resulting
in diminished accessibility of the antibody to its determinant.
Regardless of the precise mechanism, the temperature-dependent decrease
in immunoreactive material may reflect a process that modulates TAP
function in vivo.
)
We are grateful to Linda Hendershot (St. Jude
Children's Research Hospital, Memphis, TN) for providing anti-BiP
mAb. Judy Stephens and Bethany Buschling provided outstanding technical
assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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