(Received for publication, August 19, 1996, and in revised form, December 11, 1996)
From the Nuffield Department of Clinical Biochemistry and Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom
The ATP-binding cassette transporters associated
with antigen presentation (Tap1 and Tap2) mediate the transport of
peptide fragments across the endoplasmic reticulum membrane of
mammalian cells. Tap1 and Tap2 are closely related to one another and
are believed to function as a heterodimer. Each protein possesses a
hydrophobic domain predicted to span the membrane multiple times and a
highly conserved nucleotide-binding domain. We have assessed the
transmembrane topology of Tap1 by expressing a series of fusions to a
reporter protein, the mature form of -lactamase in Escherichia coli. From these data a topological model can be derived in which Tap1 spans the membrane eight times, with several large loops exposed
in the lumen of the endoplasmic reticulum and with both the N and C
termini (including the nucelotide-binding domain) residing in the
cytoplasm.
Cytotoxic T lymphocytes (CTLs)1
continually survey cells for changes in cytosolic content. Antigens
from cytoplasmic proteins are presented to the CTLs at the cell surface
in the form of peptide fragments complexed with major
histocompatibility class I and 2-microglobulin molecules
(1). These trimeric complexes are recognized by the T cell receptor on
the CTLs. To assemble this trimeric complex, the peptide fragments
which are normally generated by the proteasome in the cytoplasm, must
be translocated into the lumen of the endoplasmic reticulum (ER). Two
proteins, Tap1 and Tap2, are required for this transport process (2,
3). Tap1 and Tap2 each consist of a hydrophobic domain predicted to span the membrane multiple times, and an ATP-binding domain, which is
believed to couple the energy of ATP hydrolysis to peptide transport.
Tap1 and Tap2 function as a heteromer (4-6) and are members of the
ATP-binding cassette (ABC) superfamily of transporters (7).
The transmembrane domains of ABC transporters typically (although there
are exceptions; see below) consist of 12 clearly defined, putative
membrane-spanning segments, which could, potentially, span the lipid
bilayer. For a number of ABC transporters, both prokaryotic and
eukaryotic, this predicted topology has been confirmed experimentally
(8-10). The N-terminal hydrophobic domains of Tap1 and Tap2 appear to
differ from those of other ABC transporters in that the potential
membrane-spanning segments are not clearly defined by conventional
algorithms and appear to number more than 12 (for the Tap1-Tap2
complex). To clarify this situation we have analyzed the transmembrane
topology of the human Tap1 protein using a genetic approach in which a
reporter protein (the mature form of -lactamase) was fused to a
series of defined points along the length of the Tap1 protein. The
orientation of the
-lactamase with respect to the membrane was
assessed by its ability to confer ampicillin resistance when expressed
in Escherichia coli. This approach, and the related
phoA method, have been used, successfully, to study many
other membrane proteins (11-13). The data generate a model in which
Tap1 spans the membrane eight times with large extracellular (lumenal)
hydrophilic loops and the N and C termini, including the
nucleotide-binding domain, located in the cytoplasm. This predicted
organization differs from that of many other ABC transporters, and its
functional implications are considered.
E. coli strain
DH5F
: endE44
hsdR17(rK
mK+)
supE44 thi-1 recA1 gyrA(Nalr) relA1
(lacIAYZ-argF)U169 deoR
(
80dlac
(lacZ)M15) was used routinely. For certain studies the protease-deficient strain CH1790 (htpR155::Tn10
lon ilv his
supo strA
proC galOP::IS1
bio
[Bam-N+]) (10) was used.
Plasmid pYJ1 contains the tap1 cDNA (14) in the general
cloning vector pKG18. pYZ4 and pYZ5 are two plasmids designed to facilitate the cloning and generation of C-terminal -lactamase fusions to eukaryotic genes.
Restriction endonucleases and DNA modifying enzymes were purchased from New England Biolabs and used according to the manufacture's instructions. The oligonucleotides used in this study are listed in Table I.
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A total of 39 in-frame Tap1--lactamase fusions were generated. Two approaches to
generating these fusions were used, a random method and a directed
method.
Random fusions were generated as follows. The tap1 gene was
cloned into plasmid pYZ4 (15) under control of the inducible E. coli lac UV5 promoter and a bacterial ribosome binding site, generating pYZtap1 (Fig. 1). pYZ
tap1 was cleaved at its unique HindIII site
(located near the C-terminal end of the postulated hydrophobic domain)
and progressive deletions introduced into the tap1 gene with
exonuclease Bal-31 (Fig. 2A). These randomly truncated derivatives of tap1 were fused to the coding
sequence of the mature form of -lactamase (Fig. 2A) and
transformed into E. coli strain DH5
F
, selecting for
kanamycin resistance. The resultant colonies were screened for
ampicillin resistance at high cell density (see below) to determine
whether they expressed an in-frame Tap1-
-lactamase fusion. Plasmids
that conferred resistance were sequenced to determine the
Tap1-
-lactamase fusion junctions. This approach generated 29 independent, in-frame
-lactamase fusions between amino acids 11 and
342 of Tap1 (designated ptap11 to ptap342, respectively).
Directed Tap1--lactamase fusions were constructed as follows. Two
fusions were constructed taking advantage of unique PvuII and HindIII restriction endonuclease sites within the
tap1 gene. The coding sequence of the mature form of
-lactamase, excised from pYZ5 as a PvuII-EcoRI
fragment (Fig. 1B), was inserted directly into the
tap1 gene of plasmid pYZtap1 at the unique
PvuII and HindIII sites (trimmed to blunt ends
with mung bean nuclease). These fusions were named according to the
site of the fusion junctions, ptap378 and
ptap433, respectively. Eight additional directed
Tap1-
-lactamase fusions were generated by the PCR (Fig.
2B) and named according to the position of the fusion
junctions, ptap40 to ptap748. The DNA sequences
of all PCR-amplified DNA and the fusion junctions were determined.
Plasmids containing
in-frame Tap1--lactamase fusions were identified by their ability to
confer ampicillin resistance on E. coli at high cell
density. At high cell density all in-frame fusions, whether to
intracellular or extracellular domains of Tap1, will confer ampicillin
resistance due to release of
-lactamase from a proportion of the
cells by lysis. Stationary-phase cultures were washed once in Luria
broth (LB; Ref. 16) and resuspended in the original volume of LB. 10 µl of the cell suspension was spotted onto an LB agar plate
containing 50 µg/ml kanamycin and 100 µg/ml ampicillin and
incubated overnight at 37 °C. Resistance was defined as confluent
cell growth within the spotted area.
Because the target for ampicillin is extracellular, isolated cells will
be ampicillin-resistant only if they express -lactamase fused to an
extracellular domain. To determine whether a Tap1-
-lactamase fusion
junction was intracellular or extracellular, cells were screened for
ampicillin resistance at low cell density. An overnight culture of
cells was washed once in LB, diluted, and 10 µl of each dilution
spotted, in duplicate, onto eight LB agar plates containing 50 µg/ml
kanamycin and different concentrations of ampicillin: 0, 2.5, 5, 7.5, 10, 25, 50, 100, or 200 µg/ml. Several dilutions (10
5,
10
6, and 10
7) were used so that one would
generate separate colonies (5-15 per spot) representing isolated
bacteria. Plates were incubated overnight at 30 °C or until colonies
appeared on plates with no ampicillin. Incubation at 30 °C rather
than 37 °C was used to avoid overgrowth of bacterial colonies, which
might lead to appearance of satellite ampicillin-sensitive colonies.
For each Tap1-
-lactamase fusion a maximum ampicillin resistance was
assigned as the highest ampicillin concentration at which the fusion
permitted growth.
Cells (~108), grown to
midlog phase in LB plus 50 µg/ml kanamycin, were harvested by
centrifugation and lysed in SDS sample buffer at 90 °C (16).
Proteins were separated by SDS-polyacrylamide gel electrophoresis,
transferred onto HybondTM-C Super transfer membranes (Amersham Corp.)
and fusion proteins detected using rabbit anti--lactamase antibodies
(5 Prime
3 Prime, Inc., Boulder, CO) using enhanced
chemiluminescence (ECL; Amersham).
The Tap1 protein consists of an N-terminal hydrophobic domain and
a C-terminal hydrophilic domain (the nucleotide-binding domain). A
hydrophobicity plot of Tap1 predicts 10 clusters of hydrophobic amino
acids that are of sufficient length to span the membrane (labeled as
A to J in Fig. 3). To assess which
of these segments actually traverse the membrane, and hence the
transmembrane topology of Tap1, fusions between defined points in Tap1
and the mature form of -lactamase were generated. When expressed in
E. coli,
-lactamase acts as reporter of transmembrane
topology.
-Lactamase breaks down ampicillin, an antibiotic whose
target is extracellular (i.e. periplasmic in E. coli). If
-lactamase is fused to a point in Tap1 which is
periplasmic (equivalent to the lumen of the ER), ampicillin is
hydrolyzed and the cells are ampicillin resistant. If
-lactamase is
fused to a point in Tap1 which is intracellular (cytoplasmic), cells
are ampicillin-sensitive.
Twenty nine in-frame Tap1--lactamase fusions within the
transmembrane domain of Tap1 were generated by random approaches. A
directed approach was then taken to generate an additional 10 fusions
to defined points in the sequence to ensure complete coverage of the
entire transmembrane domain (see "Experimental Procedures"). At
least one fusion was generated to each of the hydrophilic loops separating the 10 predicted transmembrane segments of Tap1. The precise
fusion junctions, and their relation to the predicted transmembrane
segments, are shown in Fig. 3.
To assess the cellular location of the Tap1--lactamase fusion
junctions, the maximum ampicillin resistance conferred by each of the
fusions was assessed (Table II). Those fusions, which
conferred resistance to ampicillin when plated at low density, were
considered to be to fusions to an extracellular portion of Tap1
(equivalent to the ER lumen). The absolute level of ampicillin
resistance differed considerably between fusions due to differences in
levels of protein synthesis, stability, and/or folding. Nevertheless, any level of resistance implies an extracellular location. Those fusions, which conferred no ampicillin resistance when cells were plated at low density, were considered to place
-lactamase in a
cytoplasmic location. To exclude the possibility that such fusions failed to confer ampicillin resistance because no fusion protein was
made, rather than because the fusion junction was intracellular, two
tests were performed. First, their ability to confer ampicillin resistance at high cell density was assessed. If
-lactamase is synthesized but remains intracellular some cells lyse, releasing
-lactamase to hydrolyze ampicillin, which allows neighboring cells
to grow when cells are plated at high density. All the fusions conferred resistance at high cell density, indicating that they do
indeed express Tap1-
-lactamase fusion proteins. Second, the production of
-lactamase was examined by Western blotting (Fig. 4). All fusions were shown to produce
-lactamase
fusion proteins, although the predicted full-length fusions could not
always be detected due to proteolysis. Transferring selected fusions
into a protease-deficient strain, CH1790, showed no difference in
protein degradation (data not shown). Although degradation meant that full-length protein could not be detected, the full-length fusion protein must initially be synthesized in order for ampicillin resistance to be detected (as it was for all fusions). Furthermore, degradation cannot generate false positives (i.e. cannot
indicate a fusion is to an extracellular segment when it is not)
because by definition, the high level ampicillin resistance conferred by extracellular Tap1-
-lactamase fusions demands that the
-lactamase moiety is synthesized and translocated.
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Based on the above data the transmembrane topology of Tap1 expressed in
E. coli can be determined. The predicted hydrophobic amino
acids clusters (A to J in Fig. 3) were considered
to be actual membrane-spanning segments when fusions upstream and
downstream of the hydrophobic amino acid clusters located -lactamase
on opposite sides of the membrane. For example hydrophobic cluster B
was designated as a membrane-spanning segment because an upstream
-lactamase fusion (to amino acid 40) was periplasmic, while a downstream
-lactamase fusion (to amino acid 66) was cytoplasmic. On
this analysis, only 8 of the 10 predicted membrane-spanning segments
traverse the membrane. These are indicated in Fig. 3. Clusters
D and E do not appear to span the membrane. Some
-lactamase fusions were within potential membrane-spanning segments.
These fusions conferred levels of ampicillin resistance consistent with the location of the preceding hydrophilic loop, presumably because the
residual portion of the membrane-spanning segment present in the fusion
was not sufficiently long to span the entire membrane or because
topogenic information was present downstream of the actual
membrane-spanning segment. For example, the fusion to amino acid 210 is
located extracellularly, since it does not contain all the information
required to transfer membrane-spanning segment F across the membrane:
the downstream fusion, to amino acid 220, contains all the required
topogenic elements and is located at the cytoplasmic face of the
membrane. This additional topogenic information is presumably outside
the hydrophobic membrane-spanning segments.
Fig. 5 (B and D) shows the
topological model generated for Tap1 derived from these data. Tap1
expressed in E. coli spans the membrane eight times with
several large extracellular loops.
Tap1 and Tap2 are related proteins which together form the Tap peptide transporter of the endoplasmic reticulum required for Class I-mediated antigen presentation. The transmembrane topology of Tap1 predicted from its primary sequence is, on first inspection, different from that of many other ABC transporters. To clarify the transmembrane topology of Tap1 we used an experimental approach, the toplogy reporter-protein system, which has been used extensively to monitor the transmembrane topology of membrane proteins (12), including many from eukaryotic cells (15, 17-22). Although this approach requires heterologous expression in E. coli, this species has previously been used to express functional eukaryotic transport proteins (23, 24), and several eukaryotic polytopic membrane proteins have been shown to fold correctly in the E. coli membrane (19, 22). However, it is possible that the topology of Tap1 expressed in E. coli differs from that in mammalian membraanes.
A topological map of Tap1 was generated, with eight membrane-spanning segments and both the N and C termini located in the cytoplasm. This places the nucleotide-binding domain in the cytoplasm, in agreement with the location of this domain predicted by in situ antibody labeling experiments (5). The distribution of positive charges around the first membrane-spanning segment (three arginines preceding it and two succeeding it before the next membrane-spanning segment see Fig. 3) is consistent with the positive inside rule (25), while the distribution of positive charges around subsequent membrane-spanning segments is less adherent to this rule as is the case with other eukaryotic polytopic membrane proteins (26).
Although the -lactamase and the related phoA methods for
mapping transmembrane topology have been informative for membrane proteins, they involve fusing truncated versions of Tap1 to
-lactamase and any model generated must be considered within this
limitation. For example, if Tap1 has C-terminal topological
determinants these would be deleted in fusion proteins and may
influence the folding observed. Nevertheless, this has not proved a
problem in determining the folding of other polytopic membrane proteins
using this method where data have been confirmed by other, biochemical
approaches (9, 17, 27). The topological model presented here is
consistent with other available data. Perhaps significantly, the
topology of Tap1 is very similar to that determined for the MalF
protein, an E. coli ABC transporter for maltose. MalF has
eight membrane-spanning segments arranged along the polypeptide in a
similar 3:2:2:1 order (Fig. 5) with similar large extracellular
(lumenal) loops (8).
The transmembrane topology of Tap differs from the paradigm for ABC transporters, although several other exceptions have been reported (e.g. Refs. 8, 28, and 29). More importantly, the model places several large loops in the lumen of the endoplasmic reticulum. This is unusual for ABC transporters, where the large loops are generally cytoplasmic, but may reflect the fact the Tap interacts with the major histocompatibility class I molecule in the ER lumen (30-33). One of these loops contains two hydrophobic segments (clusters D and E), which were initially predicted to span the membrane. However since the experimental data suggest they do not traverse the membrane they may associate with the lumenal face of the membrane or be buried within the tertiary or quaternary structure of a protein complex in the ER.
Tap1 and Tap2 are closely related in primary sequence. However, Tap2 is
slightly shorter than Tap1 (by 40 amino acids). Both tap1
and tap2 genes are organized in 11 exons. Comparison of the length of coding sequence within each exon shows that difference in
length between the hydrophobic domains of the two proteins is mainly
due to differences within the first exon. An optimal alignment of the
Tap1 and Tap2 sequences (Fig. 6) indicates that hydrophobic cluster E is absent in Tap2 and that there is also considerable divergence in the region around and including hydrophobic cluster D. These are the two hydrophobic clusters of Tap1 which do not
appear to span the membrane. Thus, it seems likely that Tap2 has the
same transmembrane topology as Tap1 but that the large lumenal loop
containing hydrophobic clusters D and E is much reduced in size. As
Tap1, but not Tap2, interacts with the major histocompatibility class I
molecule in the ER (30, 32, 33), it is tempting to speculate that this
large hydrophobic loop of Tap1 plays a role in this interaction. The
topology for Tap1 determined here provides a working model to
facilitate further structure-function analysis.
We are grateful to Alain Townsend and John Trowsdale for the tap1 cDNA and Tim Eilliot, Sebastian Springer, Kenny Linton, and Jenny Broome-Smith for helpful discussions.
Since submitting this work, studies have appeared indicating the peptide binding site of Tap is in a region that our model would predict to be extracellular (i.e. in the ER lumen) (Momberg, F., Armandola, E. A., Post, M., and Hammerling, G. J. (1996) J. Immunol. 156, 1756-1763; Nijenhuis, M., and Hammerling, G. J. (1996) J. Immunol. 157, 5467-5477). As the peptide binding site is expected to be cytoplasmic, the topology of Tap1 expressed in E. coli is not fully consistent with these data. This remains to be resolved.