(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-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.
CD8 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) .
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. 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.
The number of class I-peptide complexes necessary for lysis
by T 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.
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 [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.
Figure 5:
[2-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.
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 [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).
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
[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.
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[-
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.
Photolabeling of TAP was completely inhibited by EDTA. This is
probably due to depletion of Mg, 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.
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
[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.
Figure 6:
Absence of TAP1 binding to lectins. L929
cells were labeled for 15 min with [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.
Inclusion of
protease inhibitors (aprotinin, pepstatin, leupeptin,
1-chloro-3-tosylamido-7-amino-2-heptanone, phenylmethylsulfonyl
fluoride, and 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.