FEBS 29795
FEBS Letters 579 (2005) 4413–4416
Exploring the minimal functional unit of the transporter
associated with antigen processing
Joachim Koch, Renate Guntrum, Robert Tampé*
Institute of Biochemistry, Biocenter, Johann Wolfgang Goethe-University Frankfurt, Marie-Curie-Strasse 9, D-60439 Frankfurt a.M., Germany
Received 18 May 2005; revised 25 June 2005; accepted 11 July 2005
Available online 19 July 2005
Edited by Felix Wieland
Abstract TAP, an ABC transporter in the ER membrane, provides antigenic peptides derived from proteasomal degradation to
MHC class I molecules for inspection by cytotoxic T lymphocytes at the cell surface so as to trace malignant or infected cells.
To investigate the minimal number of transmembrane segments
(TMs) required for assembly of the TAP complex based on
hydrophobicity algorithms and alignments with other ABC
transporters we generated N-terminal truncation variants of human TAP1 and TAP2. As a result, a 6 + 6 TM core-TAP complex represents the minimal functional unit of the transporter,
which is essential and sufficient for heterodimer assembly, peptide binding, and peptide translocation into the ER. The TM1
of both, core-TAP1 and core-TAP2 are critical for heterodimerization of the complex.
2005 Federation of European Biochemical Societies. Published
by Elsevier B.V. All rights reserved.
Keywords: ABC-transporter; Antigen processing; MHC class I;
Peptide-loading complex; Peptide transport
1. Introduction
The survival of vertebrates eminently depends on the adaptive immune system to protect the organism against invaders
or cancer. For this purpose, the MHC class I-dependent
pathway of antigen processing can trigger elimination of affected cells by cytotoxic T lymphocytes (CTL) upon presentation of antigenic peptides at the cell surface. The majority of
peptides are derived from proteasomal degradation [1] and,
after further trimming by TPPII [2], translocated into the
ER lumen via the transporter associated with antigen processing (TAP) [3]. After loading MHC class I molecules with
high-affinity peptides, peptide-MHC complexes are shuttled
to the plasma membrane for inspection by CTL [3–5]. In
the absence of a functional TAP transporter, most class I
molecules are not loaded with peptides and as a consequence
retained in the ER and ultimately directed to proteasomal
degradation [6–8]. An efficient loading of high-affinity peptides onto class I molecules hence depends on the formation
*
Corresponding author. Fax: +49 69 798 29495.
E-mail address: [email protected] (R. Tampé).
Abbreviations: ABC, ATP-binding cassette; CTL, cytotoxic T-lymphocyte; MHC, major histocompatibility complex; NBD, nucleotidebinding domain; TAP, transporter associated with antigen processing;
TM, transmembrane segment; TMD, transmembrane domain
of a macromolecular peptide-loading complex consisting of
TAP, MHC class I, tapasin, ERp57, and calreticulin [9,10].
TAP is a member of the family of ATP-binding cassette
(ABC) transporters and comprises a heterodimer of TAP1
(81 kDa) and TAP2 (76 kDa) (see Fig. 1A). Both subunits
are divided into a transmembrane domain (TMD) and a nucleotide-binding domain (NBD), which energizes peptide transport by ATP hydrolysis [11]. We could show that the TMD
can be functionally separated into a core-complex of two times
six transmembrane segments (TM), which is sufficient for peptide recognition and transport, and N-terminal domains essential for the assembly of the peptide-loading complex by
recruiting the adapter protein tapasin [12].
We determined in this study the minimal number of TMs
needed for the formation of a functional heterodimeric TAP
complex on the basis of N-terminal truncation studies. These
data are of particular interest in the light of other ABC transporters (see Transporter Classification Database, TCDB, at
http://tcdb.ucsd.edu/tcdb), which have less than two times six
TMs such as the Escherichia coli macrolide antibiotic exporter
MacB [13] and the lipoprotein exporter LolC/LolE [14], respectively.
2. Materials and methods
2.1. Cloning and expression of TAP constructs
To generate deletion mutants of human TAP1 (mini5TAP1, D2
–211) and TAP2 (mini5TAP2, D2–174), the primer pairs
5 0 -CAGCTCGAGATGACTGACTGGATTCTACAAGATG-3 0 , and
5 0 -CTGCTCGAGTCATTCTGGAGCATCTGCAGGAGCC-3 0 , and
5 0 -CAGGCGGCCGCATGCGTGTGATTGACATCCTG-3 0 , and 5 0 were
CTGGCGGCCGCTCAGAGCTGGGCAAGCTTCTGC-3 0
used, respectively. Different combinations of TAP1 and TAP2 (mini5TAP1/mini5TAP2, mini5TAP1/mini6TAP2, and mini6TAP1/mini5TAP2; the generation of mini6TAP was described previously [12])
were cloned into the expression vector pFASTBac Dual (Invitrogen).
Recombinant baculoviruses were generated using the Bac-to-Bac
Baculovirus Expression system according to the manufacturerÕs instructions (Invitrogen).
2.2. Cell culture and microsome preparation
Insect cells (Spodoptera frugiperda (Sf9)) were grown in Sf900II medium (Invitrogen) following standard procedures. Infection with recombinant baculovirus and preparation of microsomes were performed as
described [15].
2.3. Peptide-transport assays
2.5 · 106 Sf9 cells were incubated with 0.01% of saponin in a total
volume of 50 ll AP-buffer (5 mM MgCl2 in PBS) for 1 min at 27 C.
Cells were washed twice with AP-buffer to remove the excess of detergent. Transport was performed with 500 nM of fluorescein-labeled
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.febslet.2005.07.006
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J. Koch et al. / FEBS Letters 579 (2005) 4413–4416
3. Results and discussion
Fig. 1. Design and expression of truncated TAP variants. (A) Topological model of human TAP based on hydrophobicity analysis and
sequence alignment with human MDR1. TMs of the N-terminal
domains are shown as open cylinders, those being part of the coredomain of the complex as shaded cylinders [12]. The sites used to
generate different deletion mutants of TAP1 and TAP2 are indicated. (B)
Microsomes (50 lg protein/lane) prepared from baculovirus infected Sf9
cells were analyzed by SDS–PAGE and immunoblotting with the human
TAP1- and TAP2-specific mAbs 148.3 and 435.3, respectively. Bound
antibodies were visualized by chemiluminiscence imaging.
peptide (RRYQNSTUL; N-core glycosylation site is underlined and U
indicates fluorescein coupled via a cysteine residue) in the presence of
10 mM ATP or 1 U of apyrase for 3 min at 32 C. Peptide transport
was stopped with 1 ml of AP-buffer supplemented with 10 mM EDTA.
After centrifugation at 1500 · g, membranes were solubilized in 1 ml of
lysis buffer (50 mM Tris/HCl, 150 mM NaCl, 5 mM KCl, 1 mM CaCl2,
1 mM MnCl2, 1% NP-40; pH 7.5) for 10 min at 27 C. Insoluble proteins were removed by centrifugation at 20 000 · g and glycosylated
peptides were bound to concanavalin A Sepharose overnight at 4 C.
After double washing of the beads with lysis buffer, bound peptides
were eluted with 200 mM of methyl-a-D -mannopyranoside in lysis buffer for 30 min at 27 C and quantified in a 96-well plate reader (Fluorostar Galaxy, BMG Labtechnologies, Jena, Germany).
The TMD of most ABC transporters comprises six transmembrane segments (TM); however, extra N-terminal extensions have evolved, which are related to secondary functions.
As for TAP, the 6 + 6 TM core complex (annotation of helices
based on hydrophobicity algorithms and alignments with other
ABC transporters of subfamily B) is sufficient to allow for peptide binding and transport, whereas the N-terminal domains
are important for assembly of the peptide-loading complex
via recruitment of the adapter protein tapasin [12]. A comparison of different structures of membrane transporters made it
evident that 7–10 TMs are required to form a transport pathway [17]. Proteins with less TMs need oligomerization to pattern a tunnel through the membrane. On the basis of these
observations, we investigated a further possible subdivision
of the core-domains of TAP1 and TAP2.
As truncation mutants of human TAP1 and TAP2 proved
useful to examine the structural organization of the peptideloading complex and the structure–function relationship of
the heterodimeric complex [12], several deletion mutants of
TAP1 and TAP2 with five or six remaining TMs were generated (mini5/5TAP (D2–211/D2–174), mini5/6TAP (D2–211/
D2–122), and mini6/5TAP (D2–166/D2–174)) (Fig. 1A). These
TAP variants were heterologously expressed in insect cells
(Sf9) from a single baculovirus encoding both TAP1 (p10 promoter) and TAP2 (polyhedrin promoter) as shown by immunoblotting using human TAP1- or TAP2-specific mouse
monoclonal antibodies (Fig. 1B).
To elucidate whether peptide transport was preserved in
these truncated TAP complexes we analyzed TAP-specific peptide transport in semi-permeabilized cells. Therefore, Sf9 cells
expressing various TAP constructs were permeabilized with
saponin and used in peptide translocation assays with a fluorescein-labeled peptide (RRYQNSTUL), containing an N-core
glycosylation site to monitor its translocation into the ER-lumen. Surprisingly, although nearly equal amounts of the
TAP constructs were expressed as shown by immunoblotting
with TAP1- and TAP2-specific monoclonal antibodies (inlet
2.4. Peptide-binding assays
Microsomes (100 lg protein) were incubated with 2 lM of the fluorescein-labeled RRYUKSTEL peptide in the absence or presence of a
400-fold excess of non-labeled competitor peptide (RRYQKSTEL) in
100 ll of AP-buffer for 15 min on ice. After solubilization with 1% SDS
in PBS, membrane associated peptides were quantified in a 96-well
plate reader. Peptides specifically bound to TAP were determined as
the amount of labeled peptide, which was displaced from the peptide-binding pocket.
2.5. Co-immunoprecipitation
Microsomes (750 lg protein) were solubilized in 1 ml of lysis buffer
(50 mM Tris/HCl, 150 mM NaCl, 5 mM MgCl2, 1% digitonin; pH 7.5)
for 60 min on ice. Insoluble proteins were removed by centrifugation
at 100 000 · g for 45 min at 4 C. Solubilized TAP1 and TAP2 were
co-immunoprecipitated on magneto beads (Dynabeads M-280 sheep
anti-mouse IgG, Dynal Biotech), which were loaded with the TAP2-specific mouse monoclonal antibody 435.3 [15] prior to incubation with solubilized protein. After incubation for 2 h at 4 C, the beads were washed
three times with 1 ml of washing buffer (50 mM Tris/HCl, 150 mM NaCl,
2 mM EDTA, 0.01% digitonin; pH 7.5). Proteins associated with the
beads were eluted with 40 ll of SDS sample buffer containing 300 mM
DTT and subsequently analyzed by SDS–PAGE and immunoblotting
with the TAP1-specific rabbit antiserum 1p2. The polyclonal antibody
1p2 was raised against the peptide epitope ETEFFQQNQTGNIMSR
according to Nijenhuis and Hämmerling [16].
Fig. 2. Peptide transport of truncated TAP variants in semi-permeabilized cells. Peptide translocation assays were performed with
fluorescein-labeled peptide (500 nM; RRYQNSTCUL) in the presence
of MgATP (10 mM, black bars) or apyrase (1 U, open bars) for 3 min
at 32 C. Aliquots of the cells expressing TAP truncation variants
(5 · 105 cells/lane) were separated by SDS–PAGE and analyzed by
immunoblotting using mAbs 148.3 and 435.3 (figure inlet).
J. Koch et al. / FEBS Letters 579 (2005) 4413–4416
Fig. 2), all of the TAP variants were deficient in peptide transport when compared to wt TAP (Fig. 2). Similar results were
obtained using isolated microsomes and radiolabeled peptide
(RRYQNSTEL) in an experimental setup as described previously (data not shown; [12]). Note, mini6/6TAP shows the
same transport activity as wt TAP [12].
Since TAP-dependent peptide translocation into the ER is a
dynamic process, which requires multiple structural rearrangements within the TMDs and the NBDs [3], we next analyzed
peptide binding of the TAP constructs, representing the initial
step in peptide transport. Isolated microsomes were incubated
with 2 lM of fluorescein-labeled peptide (RRYUKSTEL;
KD = 93 ± 9 nM [18]) in saturation binding experiments. The
extent of TAP-specific peptide binding was determined as the
amount of fluorescent peptide, which could be competed from
the binding pocket of the complex with an excess of non-labeled peptide (RRYQKSTEL; KD = 213 ± 21 nM [12]). Both,
mini5/6TAP and mini6/5TAP showed less than 20% of specific
peptide binding activity when compared to wt TAP (Fig. 3).
Mini6/6TAP has the same peptide binding affinity as wt TAP
[12]. However, no binding above background was detected
for mini5/5TAP (Fig. 3). The peptide binding pocket is localized in the cytosolic loops between TM4 and TM5 and a
stretch of 15 amino acids C-terminal of TM6 of the core domains of TAP1 and TAP2 [16]. All of these regions are present
in the TAP constructs displayed in our study.
Both, TAP1 and TAP2 contribute to peptide binding [19],
indicating that heterodimerization is a prerequisite for peptide
binding. Therefore, impaired activity of the truncation variants
could be due to a deficiency in heterodimer formation. We
therefore performed co-immunoprecipitation experiments with
a TAP2-specific antibody. Surprisingly, mini5/6TAP and
mini6/5TAP formed heterodimers as shown by immunoblotting with a TAP1-specific rabbit polyclonal antibody, whereas
mini5/5TAP did not (Fig. 4). Worth mentioning, an additional
non-identified band of about 72 kDa was detected in the detergent-solubilized fraction of mini5/5TAP. Our data clearly demonstrate that impaired peptide transport and peptide binding
of mini5/6TAP and mini6/5TAP are not caused by a loss of
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Fig. 4. Heterodimerization of truncated TAP variants. Microsomes
(100 lg protein) were solubilized with 1% digitonin; TAP1 and TAP2
were co-immunoprecipitated on magneto beads with the TAP2-specific
mouse mAb 435.3. Proteins associated with the beads were eluted
and analyzed by SDS–PAGE and immunoblotting with the TAP1specific rabbit antiserum 1p2. S, solubilized fraction; IP, immune
precipitate; N, negative control using a myc-specific mAb for immune
precipitation.
the assembly of the heterodimeric complex but due to a disturbance of dynamic processes within the heterodimer.
On sequence level the TMs in the core-TAP complex align
well with MsbA and P-glycoprotein, of which the X-ray and
low resolution structure were solved, respectively [20,21].
Although it is a matter of debate whether the structure of
the MsbA homodimer does indeed reflect the native domain
interface, also because the dimer interface would be too small
to confer thermodynamic stability [20,22], the packing of the
TMs of the MsbA monomer is similar to that observed for
P-glycoprotein [20]. Here, we provide biochemical evidence
that for the core-TAP complex also the TM1 are needed to
form a stable heterodimer interface.
Taken together, our data point out that the core-TAP complex is essential and sufficient for heterodimerization of the
TAP complex, peptide binding and transport with the TM1
of core-TAP1 and core-TAP2 being crucial for heterodimerization. However, it remains unclear how these TMs contribute to
the formation of an active transport complex. In spite of the
existence of ABC transporters with less than 6 TMs, such as
the E. coli macrolide antibiotic exporter MacB [13] and the
lipoprotein exporter LolC/LolE [14], respectively, our data
moreover our data show clearly that a 6 + 6 TM core-domain
represents the minimal active transporter for human TAP.
Further structural characterization of the TMDs of the TAP
heterodimer and related ABC transporters, especially with respect to the relative orientation of the TMs to one another at
the dimer interface, will be essential for the understanding of
how the substrate translocation pathway is formed.
Acknowledgements: We thank Eckhard Linker for excellent technical
assistance as well as Rupert Abele and Hans Bäumert for critical comments on the manuscript. The Deutsche Forschungsgemeinschaft
supported this work.
References
Fig. 3. Peptide binding of truncated TAP variants. Microsomes
(100 lg protein) were incubated with fluorescein-labeled peptide
(2 lM; RRYCUKSTEL) in the absence (black bars) or presence (open
bars) of a 400-fold excess of non-labeled competitor peptide
(RRYQKSTEL) for 15 min at 4 C.
[1] Rock, K.L., Gramm, C., Rothstein, L., Clark, K., Stein, R., Dick,
L., Hwang, D. and Goldberg, A.L. (1994) Inhibitors of the
proteasome block the degradation of most cell proteins and the
generation of peptides presented on MHC class I molecules. Cell
78, 761–771.
[2] Reits, E., Neijssen, J., Herberts, C., Benckhuijsen, W., Janssen,
L., Drijfhout, J.W. and Neefjes, J. (2004) A major role for TPPII
4416
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
J. Koch et al. / FEBS Letters 579 (2005) 4413–4416
in trimming proteasomal degradation products for MHC class I
antigen presentation. Immunity 20, 495–506.
Abele, R. and Tampé, R. (2004) The ABCs of immunology:
structure and function of TAP, the transporter associated with
antigen processing. Physiology (Bethesda) 19, 216–224.
Pamer, E. and Cresswell, P. (1998) Mechanisms of MHC class Irestricted antigen processing. Annu. Rev. Immunol. 16, 323–358.
Yewdell, J.W., Reits, E. and Neefjes, J. (2003) Making sense of
mass destruction: quantitating MHC class I antigen presentation.
Nat. Rev. Immunol. 3, 952–961.
Hughes, E.A., Hammond, C. and Cresswell, P. (1997) Misfolded
major histocompatibility complex class I heavy chains are
translocated into the cytoplasm and degraded by the proteasome.
Proc. Natl. Acad. Sci. USA 94, 1896–1901.
Salter, R.D. and Cresswell, P. (1986) Impaired assembly and
transport of HLA-A and -B antigens in a mutant TxB cell hybrid.
EMBO J. 5, 943–949.
Townsend, A., Ohlen, C., Bastin, J., Ljunggren, H.G., Foster, L.
and Karre, K. (1989) Association of class I major histocompatibility heavy and light chains induced by viral peptides. Nature
340, 443–448.
Antoniou, A.N., Ford, S., Pilley, E.S., Blake, N. and Powis, S.J.
(2002) Interactions formed by individually expressed TAP1 and
TAP2 polypeptide subunits. Immunology 106, 182–189.
Ortmann, B. et al. (1997) A critical role for tapasin in the
assembly and function of multimeric MHC class I-TAP complexes. Science 277, 1306–1309.
Gorbulev, S., Abele, R. and Tampé, R. (2001) Allosteric crosstalk
between peptide-binding, transport, and ATP hydrolysis of the
ABC transporter TAP. Proc. Natl. Acad. Sci. USA 98, 3732–
3737.
Koch, J., Guntrum, R., Heintke, S., Kyritsis, C. and Tampé, R.
(2004) Functional dissection of the transmembrane domains of
the transporter associated with antigen processing (TAP). J. Biol.
Chem. 279, 10142–10147.
Kobayashi, N., Nishino, K., Hirata, T. and Yamaguchi, A. (2003)
Membrane topology of ABC-type macrolide antibiotic exporter
MacB in Escherichia coli. FEBS Lett. 546, 241–246.
[14] Narita, S., Kanamaru, K., Matsuyama, S. and Tokuda, H. (2003)
A mutation in the membrane subunit of an ABC transporter
LolCDE complex causing outer membrane localization of lipoproteins against their inner membrane-specific signals. Mol.
Microbiol. 49, 167–177.
[15] Meyer, T.H., van Endert, P.M., Uebel, S., Ehring, B. and Tampé,
R. (1994) Functional expression and purification of the ABC
transporter complex-associated with antigen-processing (TAP) in
insect cells. FEBS Lett. 351, 443–447.
[16] Nijenhuis, M. and Hämmerling, G.J. (1996) Multiple regions of
the transporter associated with antigen processing (TAP)
contribute to its peptide binding site. J. Immunol. 157, 5467–
5477.
[17] Liu, Y., Engelman, D.M. and Gerstein, M. (2002) Genomic
analysis of membrane protein families: abundance and conserved
motifs. Genome Biol. 3, 1–12.
[18] Neumann, L. and Tampé, R. (1999) Kinetic analysis of peptide
binding to the TAP transport complex: evidence for structural
rearrangements induced by substrate binding. J. Mol. Biol. 294,
1203–1213.
[19] Androlewicz, M.J., Ortmann, B., van Endert, P.M., Spies, T. and
Cresswell, P. (1994) Characteristics of peptide and major histocompatibility complex class-I/beta(2)-microglobulin binding to
the transporters associated with antigen processing (TAP1 and
TAP2). Proc. Natl. Acad. Sci. USA 91, 12716–12720.
[20] Rosenberg, M.F., Callaghan, R., Modok, S., Higgins, C.F. and
Ford, R.C. (2005) Three-dimensional structure of P-glycoprotein: the transmembrane regions adopt an asymmetric
configuration in the nucleotide-bound state. J. Biol. Chem.
280, 2857–2862.
[21] Chang, G. (2003) Structure of MsbA from Vibrio cholera: a
multidrug resistance ABC transporter homolog in a closed
conformation. J. Mol. Biol. 330, 419–430.
[22] Stenham, D.R., Campbell, J.D., Sansom, M.S., Higgins, C.F.,
Kerr, I.D. and Linton, K.J. (2003) An atomic detail model for the
human ATP binding cassette transporter P-glycoprotein derived
from disulfide cross-linking and homology modeling. FASEB J.
17, 2287–2289.