To attribute the different nucleotide binding behaviour of NBD1 and NBD2 to specific sequences,we generated chimeric TAP1 and TAP2 polypeptides in which either the nonhomologous C-terminal
Trang 1The distinct nucleotide binding states of the transporter associated with antigen processing (TAP) are regulated by the nonhomologous C-terminal tails of TAP1 and TAP2
Hicham Bouabe* and Michael R Knittler
Institute for Genetics, University of Cologne, Germany
The transporter associated with antigen processing (TAP)
delivers peptides into the lumen of the endoplasmic
reticu-lum for binding onto major histocompatibility complex
class I molecules TAP comprises two polypeptides,TAP1
and TAP2,each with an N-terminal transmembrane domain
and a C-terminal cytosolic nucleotide binding domain
(NBD) The two NBDs have distinct intrinsic nucleotide
binding properties In the resting state of TAP,the NBD1
has a much higher binding activity for ATP than the NBD2,
while the binding of ADP to the two NBDs is equivalent To
attribute the different nucleotide binding behaviour of
NBD1 and NBD2 to specific sequences,we generated
chimeric TAP1 and TAP2 polypeptides in which either the
nonhomologous C-terminal tails downstream of the Walker
B motif,or the core NBDs which are enclosed by the
con-served Walker A and B motifs,were reciprocally exchanged
Our biochemical and functional studies on the different TAP chimeras show that the distinct nucleotide binding beha-viour of TAP1 and TAP2 is controlled by the nonhomolo-gous C-terminal tails of the two TAP chains In addition,our data suggest that the C-terminal tail of TAP2 is required for
a functional transporter by regulating ATP binding Further experiments indicate that ATP binding to NBD2 is important because it prevents simultaneous uptake of ATP
by TAP1 We propose that the C-terminal tails of TAP1 and TAP2 play a crucial regulatory role in the coordination of nucleotide binding and ATP hydrolysis by TAP
Keywords: antigen presentation; transporter associated with antigen processing; endoplasmic reticulum; peptide trans-port; nucleotide binding domains
The transporter associated with antigen processing (TAP)
translocates antigenic peptides from the cytosol into the
lumen of the endoplasmic reticulum where the peptides are
loaded onto the major histocompatibility complex (MHC)
class I molecules [1] Cytotoxic T lymphocytes identify
and eliminate cells harbouring pathogens by monitoring
the peptide–MHC class I complex at the cell surface
TAP-deficient cell lines show low MHC class I cell surface
expression demonstrating the essential role of TAP for
MHC class I-restricted antigen presentation [1] TAP
belongs to the ATP binding-cassette (ABC) family of
transporters that use ATP hydrolysis to move a remarkable
variety of substrates across cellular membranes [2] TAP is
an endoplasmic reticulum membrane protein consisting of two subunits,TAP1 and TAP2,each of which has an N-terminal transmembrane domain (TMD) and a C-terminal cytosolic nucleotide binding domain (NBD) The four-domain (two TMDs,two NBDs) structure appears
to be general in the ABC-transporters although the chain composition making up the four domains is variable within the superfamily The TMDs are involved in substrate interaction and translocation whereas the NBDs energize the transport by ATP hydrolysis Several conserved sequence motifs common to the NBDs of all ABC-transporters have been identified,including the Walker A and B motifs,which are involved in ATP binding and hydrolysis,the Q- andD -loop,the signature motif and the switch region (Fig 1A) Studies on several different ABC transporters [3–9] describe distinct functional and biochemical properties for the two NBDs of a single transporter In the case of TAP we showed, under experimental conditions not allowing nucleotide hydrolysis,that TAP1 has a much higher ATP binding activity than TAP2 [10] Similar results were reported by others observations [11–14] Models of the transport cycle of TAP were proposed in which the NBDs bind and hydrolyze nucleotides in an alternating and strongly interdependent manner [10,12,15] Reconstitution of purified human TAP into proteoliposomes has recently allowed the measurement
of the ATPase activity of the transporter [16] The authors calculated that a single TAP complex hydrolyses about five ATP molecules per second to transport two to three peptides,
a rate that is compatible with a requirement for ATP hydrolysis by both TAP chains for a single transport cycle
Correspondence toM R Knittler,Institute for Genetics,University of
Cologne,Zu¨lpicher Strasse 47,50674 Cologne,Germany.
Fax: + 49 221 4705015,Tel.: + 49 221 470 5292,
E-mail: Knittler@uni-koeln.de
Abbreviations: ABC,ATP binding-cassette; CFTR,cystic fibrosis
transmembrane conductance regulator; FACS,fluorescence-activated
cell sorting; MHC,major histocompatibility complex; NBD,
nucleotide binding domain; TAP,transporter associated with
antigen processing; tapasin,TAP-associated glycoprotein;
TMD,transmembrane domain.
*Present address: Max von Pettenkofer-Institut fu¨r Hygiene und
Medizinische Mikrobiologie,Mu¨nchen,Pettenkofer Str 9a,
80336 Mu¨nchen,Germany.
(Received 18 July 2003,revised 17 September 2003,
accepted 23 September 2003)
Trang 2The different nucleotide binding behaviours of TAP1
and TAP2 are intrinsic properties of their NBDs [9] Thus,
the critical sequences responsible must be sought within the
NBDs themselves The core NBDs of TAP containing the
ATP binding-cassette between the Walker A and B motifs have an overall sequence homology of about 75% The most variable part of the core NBDs,in other ABC trans-porters as well as TAP,lies within the helical subdomains
Fig 1 Chimeric TAP variants: sequence exchange and expression in T2 cells (A) Amino acid alignment of the NBDs of rat TAP1aand rat TAP2a Sequences were retrieved from the GenBank database (GenBank X57523 and X63854) and aligned using the software VECTOR NTI (Informax) Identical residues are marked by black boxes while grey boxes indicate similar residues The conserved sequence motifs termed Walker A (WA), Q-loop,signature motif,Walker B (WB), D -loop and switch region (switch) as well as the a 6 - and b 11 -region are indicated on top of the aligned sequences The sequences of the core NBDs containing Walker A motif,Q-loop,signature motif,Walker B motif and D -loop (residues 506–652 in TAP1 and residues 494–639 in TAP2) and the C-terminal tails downstream the D -loop (residues 653–725 in TAP1and residues 640–703 in TAP2) that were mutually exchanged between TAP1 and TAP2 are underlined in black In addition,the amino acid sequence encoded by exon 11 (GenBank AL732652) is underlined by a dashed black line (residues 658–725 in TAP1 and residues 645–703 in TAP2) A vertical line behind the
D -loop indicates the breakpoint of the truncated TAP2 chain 2DV (after residue 639 in TAP2) The region of the truncated alternative C-terminal tail in the human splice variant TAP2iso (KTLWKFMIF,in the single amino acid letter code),which is encoded by exon 12,is indicated and underlined by a grey line (B) Expression and schematic overview of wild-type and chimeric TAP subunits T2 transfectants were lysed,separated by SDS/PAGE and blotted onto nitrocellulose as described (see Materials and methods) Western blots were probed for the different TAP chains by using antisera D90 (C-term NBD1),116/5 (C-term NBD2) and antibody MAC 394 (core NBD2) A pictorial overview of the wild-type TAP and the different chimeric TAP subunits termed 1V2,2V1,1C2 and 2C1 is shown at the bottom of the analysis TMDs and NBDs of TAP1 are indicated
in black while the corresponding domains of TAP2 are indicated in grey.
Trang 3between the Walker A and Walker B motifs It has been
suggested that this approximately 100 amino acid long
region containing the Q-loop and the signature motif is
mainly involved in interactions with the TMDs rather than
in the catalytic process of the NBDs [17] Low sequence
homology is also a characteristic feature of the C-terminal
tails directly downstream of the conservedD-loop
compri-sing 64 residues in NBD2 and 73 in NBD1 of rat TAP
(Fig 1A) The overall sequence similarity of these
NBD-segments is below 30% Structural analysis of the NBD1 of
human TAP showed that the C-terminus is close to the
nucleotide binding site and might play an important role in
modulating the catalytic function [18]
To identify the sequence region that imposes the distinct
nucleotide binding and accordingly the different
function-ality of NBD1 and NBD2,we generated TAP1 and TAP2
chimeras in which either the nonhomologous C-terminal
tails (residues 640–703 in TAP2 and residues 653–725 in
TAP1) or the core NBDs (residues 494–639 in TAP2 and
residues 506–652 in TAP1) were mutually exchanged For
biochemical and functional characterization,we established
T2 cell lines that stably express either single TAP chains or
different combinations of wild-type and chimeric
transpor-ter subunits Our findings demonstrate that the distinct
nucleotide binding behaviour of the TAP-NBDs is
deter-mined by the nonhomologous C-terminal tails A chimeric
NBD2 with the C-terminal tail of NBD1 exhibits the ATP
binding capacity and the function of wild-type NBD1 This
indicates that TAP2 has a catalytically active ATP
binding-cassette,which is functionally regulated by the C-terminal
tail In accordance with this,we found that truncated TAP2
chains deprived of their C-terminal tails retain the ability to
bind to ADP but cannot mediate the transport function
of TAP Furthermore,our findings indicate that the
C-terminal control of nucleotide interaction in NBD1 is
more complex than in NBD2 A chimeric NBD1 with the
C-terminal tail of NBD2 shows a nucleotide binding
behaviour similar to NBD2 but is defective in exchanging
ADP to ATP We also provide evidence that ATP binding
in TAP2 prevents simultaneous uptake of ATP by TAP1
Based on our data,we propose that structural influence
from the C-terminal tails and the conformational cross-talk
between the core NBDs build the mechanistic scaffold for
the alternating catalytic cycle of ATP binding and
hydro-lysis of TAP
Materials and methods
Cell lines and cell culture
T2 is a human lymphoblastoid cell line that lacks both TAP
genes,and expresses only the HLA-A2 and -B5 class I
molecules [19] Transfectants of T2 containing rat TAP1a
and rat TAP2a wild-type chains [20] were cultured in
IMDM (Gibco BRL) supplemented with 10% FCS (BIO
Whittaker) and 1 mgÆmL)1G418 (PAA,Co¨lbe) T2 cells
expressing single TAP chimeras 1N2 or 2N1 (formerly
named 1/2 and 2/1) [9] were cultured in the same medium,
whereas transfectants containing chimeric TAP variants
1–2N1 and 2–1N2 (formerly named 1–2/1 and 2–1/2) [9]
were grown in IMDM supplemented with puromycin
(750 ngÆmL)1)
Cloning and expression of chimeric TAP1 and TAP2 chains
The 2.6 and 2.4 kb EcoRI fragments containing full-length cDNA from rat TAP1aand TAP2a,respectively [1,21] were cloned into the multiple cloning site of pBluescript KS+ (Stratagene) The QuickChangeTMSite directed mutagenesis procedure (Stratagene) was used to create a ScaI site in TAP1
at position 1904 and in TAP2 at position 1943 (position 1 is the A of the first AUG) For TAP1 we used the comple-mentary primers 5¢-GGACGATGCCACCAGTACTCTG GATGCTGGCAACC-3¢ and 5¢-GGTTGCCAGCATCC AGAGTACTGGTGGCATCGTCC-3¢ and for TAP2 the complementary primers 5¢-GGATGAGGCTACCAGTAC TCTGGACGCCGAGTGCG-3¢ and 5¢-CGCACTCGGC GTCCAGAGTACTGGTAGCCTCATCC-3¢ All primers were purchased from ARK/Sigma The chimeric TAP construct 1V2 was created by ligation of the 1.6 kb ScaI-fragment containing the C-terminus of TAP2 to the 3.8 kb ScaI-fragment containing the TMD and core NBD of TAP1
In the case of 2V1, the 1.8 kb ScaI-fragment containing the C-terminus of TAP1 was ligated to the 3.8 kb ScaI-fragment containing the TMD and core NBD of TAP2 To restore the original amino acid sequence,a further site-directed muta-genesis was performed using the complementary primers for variant 1V2 5¢-GGACGATGCCACCAGTGCCCTG GACGCCGAGTGCG-3¢ and 5¢-CGCACTCGGCGTC
comple-mentary primers 5¢-GGATGAGGCTACCAGTGCCCT GGATGCTGGCAACC-3¢ and 5¢-GGTTGCCAGCATC CAGGGCACTGGTAGCCTCATCC-3¢ for 2V1 The resulting TAP constructs were cloned into the EcoRI site of pHbApr1neo [22] and sequenced fully in both directions Chimera 1V2 encoded residues 1–652 of TAP1 and residues 640–703 of TAP2 and chimera 2V1 encoded residues 1–639
of TAP2 and residues 653–725 of TAP1 TAP variants 1C2 and 2C1 were created by the same site-directed mutagenesis procedure using the cDNA templates of the chimeric variants TAP 1/2 (TAP1N2) and TAP 2/1 (TAP2N1) [9] Chimera 1C2 encoded residues 1–505 and 653–725 of TAP1 and residues 494–639 of TAP2 and chimera 2C1 encoded residues 1–493 and 640–703 of TAP2 and residues 506–652 of TAP1 The exchange was performed at the amino acid sequence positions 647 in TAP1/2 (TAP1N2) and 636 in TAP2/1 (TAP2N1) The C-terminal deletion construct TAP2DV was created by introducing a stop codon at position 1919 of the wild-type TAP2 sequence with site-directed muta-genesis Therefore,we used the complementary primers 5¢-GGATGAGGCTACCAGTGC TC TGGACGCCTAG TGCGAGCAGGC-3¢ and 5¢-GCCTGCTCGCACTAGG CGTCCAGAGCACTGGTAGCCTCATCC-3¢ All TAP constructs were transfected into T2 cells by electroporation using a Bio-Rad gene pulser at 270 V and 500 lF After selection with G418 (1 mgÆmL)1) for 4–6 weeks,stable transfectants were subcloned and screened for TAP chain expression by Western blotting
Antibodies 116/5 is a polyclonal rabbit antiserum recognizing the C-terminus of rat TAP2 chains [20] D90 is a polyclonal rabbit antiserum recognizing the C-terminus of rat TAP1
Trang 4chains [21] MAC 394 is a monoclonal mouse antibody
(mAb) against rat TAP2a[23] derived from immunization
with recombinant His-tagged cytoplasmic domain of rat
TAP2a MAC 394 fails to detect TAP2u due to the
polymorphic residues at position 538 and 539 in the core
NBD (M R Knittler,unpublished results) 4E is a
conformation-dependent mouse mAb,which
recogni-zes an epitope common to all HLA-B and -C antigens
[24]
Immunoprecipitation and Western blotting
Cells (5· 106) were washed twice in ice-cold NaCl/Pi
(1.7 mM KH2PO4,10 mM Na2HPO4,140 mM NaCl,
2.7 mM KCl),pH 7.5,prior to solubilization in lysis
buffer [NaCl/Pi,pH 7.5,containing 1% Triton X-100
(Sigma)] Immunoprecipitations with anti-rat TAP2 (116/
5) were performed as described previously [23]
Immuno-precipitates were washed with NaCl/Pi,1% Triton
X-100 and eluted with 10 mM Tris/HCl pH 8.8
Western blotting treated with specific primary antibody
Bands were visualized with horseradish
peroxidase-conjugated secondary antibodies (goat anti-rabbit
IgG–HRP) and enhanced chemiluminescence substrate
(Amersham)
Transport assay and peptide cross-linking
Cells (2· 106) were permeabilized with streptolysin O
(SLO) (2 UÆmL)1; Murex) After washing with NaCl/Pi,
0.5 lM radioiodinated peptide S8 (TVDNKTRYR),
10 mM ATP,and incubation buffer [50 mM Hepes
pH 7.5,250 mM sucrose,150 mM CH3COOK,5 mM
(CH3COO)2MgÆ4H2O,1 mMdithiothreitol,1 mMPefabloc
(Boehringer Mannheim),1.8 lgÆmL)1 aprotinin (Sigma)]
were added and incubated for 10 min at 37C Following
lysis with 20 mM Tris/HCl pH 7.5,500 mM NaCl,0.1%
Nonidet P-40 (Sigma),transported glycosylated peptides
were isolated with Con A-sepharose (Pharmacia) and
quantitated by gamma counting [25] For peptide
cross-linking permeabilized cells were incubated with 1 lM
radioiodinated and HSAB-conjugated peptide S8
Cross-linking was induced by irradiation with a UV lamp at
254 nm for 5 min on ice Cells were lysed by adding 1%
Triton X-100 in NaCl/Pi
Nucleotide binding assays
The nucleotide binding assay was performed as described
previously [26] Nucleotide binding experiments were
performed with N6-coupled ATP-, ADP- and
AMP-agarose (Sigma) using Triton X-100 solubilized cell
membranes For photolabelling of TAP with
radiola-belled 8-azido-ATP,membranes of cells were prepared
and resuspended in 250 mM sucrose,50 mM KCl,2 mM
MgCl2, 2 mMEGTA and 10 mMTris pH 6.8 [26]
Mem-branes corresponding to 3· 106cells in a final volume of
100 lL were incubated with 2 lM 8-azido-ATP[a-32P] or
8-azido-ATP[c-32P] (ICN Biomedicals) for 5 min at 4C
Cross-linking was induced by irradiation with a UV lamp
at 254 nm for 5 min at 4C
Preparation of microsomal membranes Microsomes from 108T2 cells expressing chimeric and wild-type TAP proteins were generated by a sucrose gradient fractionation [27] Cells were washed twice with ice cold NaCl/Pi,resuspended in 10 mL of 10 mMTris,pH 7.4 with protease inhibitor cocktail (CompleteTMProtease Inhibitor, Roche) and incubated on ice for 10 min The lysed cells were then homogenized and centrifuged at 800 g for 5 min
at 4C The resulting supernatants were resuspended in
5 mL 1.3M sucrose buffer [20 mMHepes pH 7.5,25 mM
CH3COOK,5 mM (CH3COO)2MgÆ4H2O,1 mM dithio-threitol,protease inhibitor (mix)] and centrifuged again at
800 g at 4C for 10 min The supernatants were then centrifuged at 68 000 g at 4C for 2 h and the membrane pellets resuspended in 800 lL of 0.25M sucrose buffer Afterwards,5.6 mL of 2.5M sucrose gradient buffer was added and the suspension overlaid carefully with 2.9 mL of
2Mand 2.9 mL of 1.3Msucrose buffer About 800 lL of 0.25Msucrose buffer was carefully loaded on the top of the gradient The sucrose gradient was centrifuged at 100 000 g for 16 h at 4C The microsomes were collected at the interface between the 2Mand 1.3Msucrose buffer,diluted
in 20 mM Hepes buffer [20 mM Hepes (pH 7.5),25 mM
CH3COOK,5 mM (CH3COO)2MgÆ4H2O,1 mM dithio-threitol and protease inhibitor cocktail],homogenized and centrifuged at 68 000 g at 4C for 1 h The microsomal pellets were resuspended in 200 lL 20 mM Hepes buffer Finally,aliquots of 30–50 lL were snap frozen in liquid nitrogen and stored at –80C
Flow cytometry Experiments were performed as described previously [26]
Results
The nonhomologous C-terminal tails of the NBDs control the distinctive nucleotide binding properties
of TAP1 and TAP2
To identify the sequence region within the NBDs that imposes the different nucleotide binding properties of TAP1 and TAP2,we constructed chimeric TAP chains by exchanging either the highly homologous core NBDs (termed C1 and C2) or the less homologous C-terminal tails downstream of the Walker B motif (termed V1 and V2) corresponding essentially to exon 11 (Fig 1A) The cDNAs for the chimeric TAP chains were stably transfected into TAP-negative human T2 cells The expression of chimeric TAP polypeptides was analyzed in the transfectants by Western blot (Fig 1B) with antisera D90 and 116/5,which recognize the C-terminal 14 amino acids of rat TAP1 [21] and C-terminal 15 amino acids of rat TAP2 [20],respect-ively,and the monoclonal antibody MAC 394 [23] which binds specifically to the core NBD of TAP2 (see Materials and methods) The chimeric TAP chains termed 2V1 and 1C2 which both have the C-terminal tail of TAP1 and the core NBD2 were detected both by the antiserum D90 (Fig 1B,upper) and antibody MAC 394 (Fig 1B,bottom), whereas the chimeric TAP chains termed 1V2 and 2C1, which both have the C-terminal tail of TAP2 were
Trang 5recognized by the antiserum 116/5 (Fig 1B,middle) With
the exception of the chimera 2V1,where the expression is
low,all the chimeras were expressed to roughly the same
amount as wild-type TAP chains (Fig 1B)
Membrane lysates from T2 cell lines expressing the
chimeric TAP chains were incubated with ATP- and
ADP-agarose beads [26] Bound proteins were eluted and
analyzed by Western blotting The binding of the chimeric
TAP polypeptides to the nucleotide agaroses was compared
with that of wild-type TAP subunits and of the TAP
chimeras 1N2 and 2N1 with switched NBDs (formerly
named TAP 1/2 and 2/1 [9]) (Fig 2) The latter chimeras
confirmed that distinct nucleotide binding behaviour is an
inherent property of the NBDs [9] Thus,TAP1 and 2N1,
both with NBD1,bound efficiently to ATP- as well as to
ADP-agarose whereas TAP2 and 1N2,both with NBD2,
bound only to ADP-agarose (Fig 2,top and second panels,
left and right) The new TAP chimeric polypeptides 2V1 and
1C2 both bound to ATP- as well as ADP-agarose (Fig 2,
left column,third and fourth panels from top),whereas the
chimeras 1V2 and 2C1 bound only to ADP-agarose (Fig 2,
right column,third and fourth panels from top) Thus,the
chimeric NBD consisting of the core NBD2 with the
C-terminal segment of TAP1 confers the nucleotide binding
behaviour of wild-type TAP1 whereas the chimeric NBD1
bearing the core NBD1 and the C-terminal segment of
TAP2 confers the characteristic ADP binding of wild-type
TAP2 Taken together,our data suggest that in the resting
state of the transporter the nonhomologous C-terminal
segments of TAP1 and TAP2,and not the core NBDs,
determine the distinct nucleotide binding properties of the
two polypeptides
Functional correlation between the C-terminal regulated nucleotide binding and the transport activity of TAP From our previous experiments [9] we observed that chimeric transporter variants with two identical NBDs are not functional for peptide transport In contrast to functional TAP molecules such chimeras have the same nucleotide binding properties on both polypeptides,either TAP1-like (ATP and ADP) or TAP2-like (ADP only) depending on the construct We asked whether simply exchanging the C-terminal segment on one chain of such disabled transporters with two identical NBDs,and thus modulating the nucleotide binding activity of this chain, could lead to rescue of the transport function We therefore created TAP variants with two identical core NBDs but two different C-terminal tails by coexpressing either wild-type TAP1 with 2C1 (TAP variant 1–2C1) or wild-type TAP2 with 1C2 (TAP variant 2–1C2) The expression levels of these TAP variants were similar to that of wild-type TAP and of the original nonfunctional TAP variants 1–2N1 and 2–1N2 (Fig 3A,top) and showed normal subunit assembly (Fig 3A,bottom)
We measured the peptide transport function of the new chimeric transporters in the Neefjes assay [25] using the iodinated model peptide S8 (TVDNKTRYR,in the single amino acid letter code) In Fig 3B,TAP variant 2–1C2 showed a significant recovery in transport activity when compared to the original variant 2–1N2 with identical C-terminal segments The transport efficiency of variant 2–1C2 was 40–55% of that of wild-type TAP Thus,the chimeric polypeptide 1C2 seems to acquire not only the intrinsic nucleotide binding behaviour but also nearly the full function of wild-type TAP1 In contrast,however,
no peptide transport above background was seen for TAP variant 1–2C1 (Fig 3B) The contrasting peptide transport activities of these reciprocally chimeric TAP molecules were also reflected in different surface expression levels of mature MHC class I molecules determined by FACS analysis (Fig 3C) Thus,although polypeptide 2C1 adopts the nucleotide binding behaviour of TAP2 (Fig 2),the chimeric chain is not able to express the functional properties of the wild-type TAP2 polypeptide
The ability to exchange ADP for ATP is a prerequisite for the function of the TAP-NBDs
In photo-cross-linking experiments with radioactive 8-azido-ATP,the wild-type TAP1–TAP2 complex shows a characteristic ratio of ATP binding to the two chains of about 5 : 1 in favour of TAP1 ([10],Fig 4A,top panel and Fig 4B) reflecting the nucleotide binding capacities of the two polypeptides in the TAP complex [10,14,28] A similar pattern of ATP-labelling was found for the functional TAP variant 2–1C2 where the labelling efficiency for the 1C2 polypeptide is fourfold higher than for the associated wild-type TAP2 (Fig 4A,top panel,and Fig 4B) In contrast,in the case of the inactive TAP variant 1–2C1,ATP-cross-linking was detectable exclusively for the wild-type TAP1 polypeptide (Fig 4A,top panel) In confirmation of previous suggestions that the function of the peptide binding site of TAP is conformationally linked to the NBDs of both TAP chains,the defective transporter 1–2C1
Fig 2 Nucleotide binding properties of wild-type and chimeric TAP
subunits Membrane fractions of T2 transfectants were resuspended in
lysis buffer containing 1% Triton X-100 and incubated with different
nucleotide agaroses Bound proteins were eluted with SDS-sample
buffer and analyzed in Western blots probed for the C-terminal tail
of TAP1 with antiserum D90 (TAP1,2N1,1C2 and 2V1) or the
C-terminal tail of TAP2 with antiserum 116/5 (TAP2,1N2,2C1 and
1V2) TAP variants are indicated by pictograms.
Trang 6was also found to be unable to bind free peptide in a
photo-cross-linking assay,while normal peptide binding was seen
for the functional chimeric transporter 2–1C2 (Fig 4A,
middle panel) These results together were consistent with
the idea that the chimeric NBD1 in 2C1 is locked in a
conformation that does not allow the binding of ATP during the transport cycle
To investigate this,we performed affinity chromato-graphy with ADP-agarose for the chimeric 1C2 and 2C1 polypeptides as well as the wild-type TAP subunits The
Fig 3 Functional properties of different chimeric transporter variants (A) Expression levels and schematic overview of wild-type and chimeric TAP transporters (top panel) T2 transfectants were lysed in buffer containing 1% Triton X-100 Lysates were separated by SDS/PAGE and blotted onto nitrocellulose Western blot analysis was performed as described in Fig 1B TAP variants are indicated by pictograms Subunit assembly of the TAP variants 2–1C2 and 1–2C1 (bottom panel) Transfected T2 cells were lysed in 1% Triton X-100 and TAP complexes were immunoprecipitated with anti-TAP2 serum 116/5 Immunoisolated proteins were separated on an SDS gel and analyzed in Western blots probed for TAP1- (D90) or TAP2-NBD (116/5) (B) TAP-mediated peptide transport Transfected and nontransfected T2 cells were permeabilized with streptolysin O and incubated in transport buffer containing ATP and radioiodinated peptide S8 for 10 min at 37 C Bar graphs show the recovered amount of transported labelled peptides as counts per minute (cpm) and represent the average values of experiments carried out in duplicate (C) Surface expression of MHC class I molecules Cells were incubated with mAb 4E that recognizes HLA-B5 followed by fluorescein isothiocyanate-labelled secondary antibody Surface expression of HLA-B5 was detected by flow cytometry (shaded peaks) Mean values of the fluorescence intensity are indicated Background staining was determined by incubating only with secondary antibody (nonshaded peaks).
Trang 7ADP-bound polypeptides were eluted with increasing
concentrations of free MgATP (0–1.0 mM) (Fig 4C) As
can be seen from Fig 4C,the wild-type TAP1 and TAP2
chains could both be released from the ADP-agarose by
MgATP,and as expected [9,10],much more efficiently in the
case of TAP1 than of TAP2 (Fig 4C,top left and right)
The functional chimeric chain,1C2,was as efficiently eluted
by MgATP as was the wild type TAP1 (Fig 4C,bottom
left),but the nonfunctional chimera,2C1,could not be
detectably eluted even at the highest MgATP concentration
(Fig 4C,bottom right) Thus,in contrast to the chimeric
NBD of 1C2 and the wild-type domains,the chimeric NBD
of 2C1 appears to have lost the ability to exchange ADP for
ATP The specificity of ADP-binding was tested by the
addition of free MgADP All TAP chains showed a 40–50%
release at 1 mMMgADP (data not shown)
TAP2 exerts allosteric control over the nucleotide
binding of TAP1
Current working models propose that ATP binding and
hydrolysis in the TAP-NBDs alternate in a cooperative
fashion [10,12,15] From vanadate trapping experiments it
has been speculated that during the transport cycle,ATP
binding and hydrolysis in NBD2 are involved in the
regulation of ATP binding by NBD1 [12] The experimental
procedure used,however,does not directly demonstrate
ATP binding by TAP2 and does not distinguish between
vanadate-trapped TAP molecules that were generated in the
presence and absence of preceding ATP metabolism [29,30]
Our construction of an ATP binding chimeric variant of
NBD2 (Figs 2 and 3) could provide a direct experimental
strategy to investigate whether TAP2 exerts allosteric
control over the nucleotide binding of TAP1 We therefore
established an appropriate TAP variant in T2 cells [T2(1–
2V1)] with wild-type TAP1 and the ATP binding chimera,
2V1 (Fig 1B) For comparison we also set up the reciprocal
T2 transfectant [T2(2–1V2)],with wild-type TAP2 and the
chimera 1V2,which contains the chimeric NBD1 (Fig 1B)
T2(1–2V1) cells appear to express lower levels of TAP than
T2(2–1V2) and T2(TAPwt) but show the same balanced
expression (Fig 5A,left) and assembly (Fig 5A,right
panel) of both TAP subunits Photo-cross-linking of
8-azido-ATP was performed on membrane preparations
from both these cell lines and assessed for labelling of TAP
polypeptides as before (Fig 5B) For variant 1–2V1 we
found a clear ATP cross-link corresponding to the chimera
2V1 but,in contrast to wild-type transporter,essentially no
ATP cross-link to TAP1 (Fig 5B) ATP binding thus
appears to be interchanged between the two subunits when
compared to the wild-type transporter Thus,binding of
ATP to the chimeric TAP2 chain seems to interfere with
ATP binding to TAP1,presumably via a conformational
interaction between the two NBDs No detectable ATP
cross-linking was observed for variant 2–1V2,suggesting
that binding of ADP by variant 1V2 does not shift the
nucleotide binding behaviour of wild-type TAP2 from ADP
to ATP
We compared the transport activity of TAP variant
1–2V1 and 2–1V2 with that of wild-type TAP (Fig 5C,left)
As expected,T2(2–1V2) was transport-inactive,however,
peptide translocation was clearly detectable in the T2(1–2V1)
cell line,consistent with the elevated HLA-B5 surface expression data The reduced level of peptide translocation
by T2(1–2V1) cells (15–20% of wild-type TAP) may be at least partially attributed to the reduced TAP expression noted above (Fig 5A,left) though there is probably a residual functional deficit as well Nevertheless,the finding that 1–2V1 forms a functional transporter strongly suggests that indeed the chimeric NBD2 in chimera 2V1 adopts a conformation reflecting a functional ATP binding state
The C-terminal tail of the NBD2 is essential for ATP binding and the catalytic function of TAP
Our results have shown that the C-terminal segment is directly involved in the functional regulation of nucleotide binding in rat TAP2 However,Yan et al have described a human TAP2 splice variant,named TAP2iso,which lacks essentially the entire C-terminal tail encoded by the exon 11 but nevertheless forms an active transporter in conjunction with TAP1 [31] We therefore asked whether the core NBD
of rat TAP2 might be able to bind nucleotide and retain catalytic function without the C-terminal tail,by creating a truncated TAP2 variant (2DV) lacking the C-terminal 64 amino acids The variant 2DV can be expressed in T2 cells, showing an apparent molecular weight of about 55 kDa on SDS gels (Fig 6A,left),and has similar,ADP-restricted nucleotide binding activity to that of wild-type TAP2 in a nucleotide-agarose binding assay (Fig 6A,right) In con-trast to wild-type TAP2,however,ADP-agarose bound 2DV could not be detectably released with free MgATP (Fig 6B,compare left and right panels) and thus lacks the normal ability of wild-type TAP2 to allow nucleotide exchange Wild-type TAP2 and 2 DV showed a half-maximal elution from ADP-agarose with 1 mM free MgADP (data not shown) We expressed the truncated TAP2 chain together with wild-type TAP1 (TAP variant 1–2DV) (Fig 7A) and tested the subunit assembly (Fig 7B) and the activity of this transporter variant in peptide transport (Fig 7C) Removal of the C-terminal tail in TAP2 had apparently no influence on the assembly of the two TAP chains (Fig 7B) but abolished the translocation of radiolabelled peptides completely (Fig 7C) Thus,in the absence of the C-terminal segment,the core NBD2 alone, while retaining the competence to bind ADP,cannot exchange ADP for ATP or support the transport function
of TAP
Discussion
Previous studies have shown that the two NBDs of ABC-transporter TAP are not equivalent either in terms of nucleotide binding or function [10,12,13,32] The core NBDs of TAP1 and TAP2,containing the ATP binding-cassettes with the essential Walker A and B motifs,while not identical are highly conserved whereas the C-terminal segments of both TAP subunits,essentially encoded by exon 11 (Fig 1A),have low sequence homology to each other To investigate whether the distinctive nucleotide binding behaviour of TAP1 and TAP2 can be attributed to the sequence differences between the C-terminal tails,or between the core NBDs,we created chimeric TAP chains by exchanging one or other of these segments between TAP1
Trang 8and TAP2 (Fig 1) We were able to show that in the resting
state the distinctive nucleotide binding behaviours of TAP1
and TAP2 depend directly on the divergent C-terminal tails
(Fig 2) A chimeric NBD2 with the C-terminal segment of
TAP1 adopts the ATP binding behaviour of wild-type
NBD1 whereas a corresponding chimeric NBD1 shows the
characteristic ADP binding properties of wild-type NBD2
(Fig 2) Further,the chimeric NBD2 acquires not only the nucleotide binding behaviour but also the functional properties of NBD1 (Figs 3 and 5) and can participate in
a functional transporter This result shows for the first time that the core NBD of TAP2,normally seen only as an ADP-binding structure,has indeed a potentially catalytically active ATP binding-cassette,which must normally be tightly
Trang 9controlled by the C-terminal tail Moreover,the
function-ality of variant 2–1C2 (Fig 3),shows that the two core
NBD2s can form functional interfaces similar to those in
wild-type TAP
The sequence differences within the ATP
binding-cas-settes of TAP1 and TAP2 make apparently no contribution
to the functional asymmetry of the NBDs,contrary to
previous proposals [12,18] In the RAD50 homodimer, the
signature motifs are adjacent to the opposing Walker A sites
and the serines of each signature motif form hydrogen
bonds with the c-phosphate of the ATP bound by the
opposing subunit [33] This kind of molecular bridging is
thought to be generally important to promote subunit
assembly and ATP hydrolysis of NBDs in
ABC-transport-ers Based on these and other studies Karttunen et al [12]
proposed that the serine in the canonical signature motif of
TAP1 (LSGGQ) (Fig 1A) forms a hydrogen bond with the
c-phosphate of ATP bound to TAP2 and is involved in the
stimulation of ATP hydrolysis during the transport cycle
This residue is an alanine (LAVGQ in rat and LAAGQ in
human) in TAP2 which would disallow such a hydrogen
bonding interaction,thus contributing to the functional
asymmetry of the two chains Our demonstration that the
2–1C2 transporter is functional,despite having alanine in
the signature motif on both chains,excludes this model In
line with this,it was shown for the sulfonylurea receptor
(SUR) that the serines in the signature motif are not
required for ATPase activity but seem to be involved in
transducing structural information between the
ABC-transporter domains [34] The somewhat lowered transport
activity of TAP variant 2–1C2 (Fig 3) could be due to
alterations in the signature motif-dependent cooperation
between the TAP domains Recent experiments on mutated TAP chains in which the serine and the second glycine of the TAP1 signature motif and the glycine of the TAP2 signature motif were exchanged by alanine showed that the signature motifs are required for peptide translocation but not peptide binding [35] From our results in Fig 3 it is suggestive that the substitution of the glycines rather than substitution of the serine caused the observed defect in peptide transport Results reported for SUR [6,36,37], appear to be highly relevant to the TAP case The two splice variants,2A and 2B,of this tandem ABC-protein differ in their sequence only for the 42 amino acid-long C-terminal tails of NBD2,which are encoded by different exons SUR2B has a much higher nucleotide binding activity than SUR2A and it is suggested that this functional difference arises from an interaction between the C-terminal tails and their respective NBDs [38]
A sequence of about seven amino acids in the b11-strand of the C-terminal tail of NBD2 is necessary and sufficient to confer the different nucleotide binding and functional properties of SUR2A and 2B [39] As the sequence of the TAP-NBD2 is homologous to the C-terminal NBD of SUR,regulation of nucleotide binding in NBD2 of TAP may be based on a similar mechanism Following the proposed working model for SUR [39] polar and charged residues in the middle portion of the b11 region (Fig 1A) may be also critical to control the distinct nucleotide binding
of the NBDs in TAP1 and TAP2 As the residues of the b11 region with charge differences in the two TAP-NBDs are located within a distance that allows interaction with the Walker A motifs over a short distance [18],one possibility might be that the conformation of the phosphate binding loop in NBD1 and NBD2 is differently affected by electrostatic interactions and thereby regulates the distinct nucleotide binding in the TAP molecule Thus,it will be of interest to find out whether sequence differences in the b11 -strands (Fig 1A) of TAP1 and TAP2 are directly involved
in the distinct nucleotide binding and function of the NBDs
of TAP
It might be also possible that the C-terminal tails control the ATP binding to the nucleotide binding pocket by other sequence elements than the b11 region Crystal structure analysis of the human TAP-NBD1 shows that the end part
of the a6 region points structurally into the nucleotide binding pocket and is close to the sequences of the Walker A and B motif [18] Most interestingly,the a6 regions in the NBDs of TAP1 and TAP2 are characterized by differences
in sequence and also in length (Fig 1A) Thus,the a6region could function as conformational regulator of the C-terminal tail in controlling the access of ATP to the nucleotide binding pocket during the peptide transport cycle Indeed,our own studies suggest that the a6region is important for the distinct nucleotide binding and function
of the two TAP-NBDs (S Ehses and M R Knittler, unpublished results) and might have a steric effect for arranging the critical sequence elements in the nucleotide binding pocket of the NBDs in TAP1 and TAP2
The switch region (Fig 1A) is another defined sequence element in the C-terminal segment,postulated to sense c-phosphate binding [40],which could contribute to func-tional asymmetry [18] TAP2 contains the consensus sequence of the switch region whereas TAP1 has a glutamine in place of the conserved histidine found in most
Fig 4 Nucleotide- and peptide-binding properties of chimeric TAP
variants (A) Biochemical characteristics of different TAP variants To
assess ATP binding capacity of wild-type and chimeric transporters
(top panel) membrane fractions of T2 transfectants were incubated
with 2 l M radiolabelled 8-azido-ATP for 5 min at 4 C After UV
cross-linking and lysis in 1% Triton X-100,TAP variants were
immunoprecipitated with either anti-TAP1 (D90) or anti-rat TAP2
(116/5) serum and separated on an SDS gel The peptide binding
activity of the TAP variants (middle panel) was analyzed by substrate
cross-linking Microsomal fractions were resuspended in binding
buf-fer and incubated with 1 l M iodinated and HSAB-conjugated peptide
S8 After cross-linking,cells were lysed and TAP was immunoisolated
with anti-TAP2 or anti-TAP1 serum Migration behaviour and
amount of TAP chains was controlled by Western blots of the
cor-responding lysates (bottom panel) probed with a mixture of anti-TAP1
and anti-TAP2 serum TAP variants are indicated by pictograms.
(B) The results of the ATP cross-link experiment were quantified by
phosphoimager Peak integrals of TAP1- and TAP2-ATP complexes
were plotted in arbitrary units (C) ADP to ATP exchange in wild-type
and chimeric TAP chains Membrane fractions of T2 transfectants
expressing single wild-type (TAP1 or TAP2,top) or chimeric TAP
chains (1C2 or 2C1,bottom) were resuspended in lysis buffer and
incubated with ADP-agarose Bound TAP chains were eluted with
increasing concentrations (0–1.0 m M ) of MgATP The nucleotide
matrix and the eluted fractions were analyzed in Western blots probed
for TAP1 (D90) and TAP2 (116/5) Enhanced chemiluminescence
fluorographs were quantified by densitometric scanning and the
obtained peak integrals were plotted in arbitrary units.
Trang 10vertebrates However,our own experiments suggest that
sequence differences in this region are not responsible for
the distinctive nucleotide binding of the TAP subunits
(H Bouabe and M R Knittler,unpublished finding)
Functional importance of the C-terminal tail of NBDs
has been discussed for several ABC-transporters [41–44]
Our experiments on the truncated TAP2 variant 2DV
demonstrate that,although the core NBD2 retains the
ability to bind to ADP,the C-terminal tail of NBD2 is
indispensable for the active transport function of TAP (Figs 6 and 7) In P-glycoprotein,only small C-terminal deletions of up to 23 amino acids leave a functional transporter [41],while truncation of the C-terminal 50–60 amino acids in the cystic fibrosis transmembrane conduct-ance regulator (CFTR) severely impairs the ability of the NBD2 to bind and/or hydrolyse ATP [45]; similarly,that the impairment of variant 2DV may primarily involve regulation of ATP binding We also found a decreased
Fig 5 Allosteric cross-talk between core NBDs in the TAP complex (A) Expression levels of transporter variants 1–2V1 and 2–1V2 (left) T2 transfectants were lysed in buffer containing 1% Triton X-100 Lysates were analyzed in Western blots probed with antiserum D90 (C-term NBD1),antiserum 116/5 (C-term NBD2) and antibody MAC 394 (core NBD2) TAP variants are indicated by pictograms Subunit assembly of the TAP variants 1–2V1 and 2–1V2 (right panel) Transfected T2 cells were lysed in 1% Triton X-100 TAP complexes were immunoprecipitated with antibody MAC 394 and analyzed by Western blots probed for the terminal tail of TAP1 with antiserum D90 (TAPwt and 1–2V1) or the C-terminal tail of TAP2 with antiserum 116/5 (2–1V2) (B) Nucleotide binding properties of wild-type TAP1 and TAP2 when expressed in a combination with chimeric TAP chains Membrane fractions of T2 cells expressing wild-type or chimeric transporters were lysed in 50 m M Tris/HCl
pH 7.5,150 m M NaCl,3 m M MgCl 2 ,1% Triton X-100 containing 2 l M of radiolabelled 8-azido-ATP After UV cross-linking,TAP variants were immunoprecipitated with an anti-TAP1 serum and separated by SDS/PAGE (C) Peptide transport activity by TAP variants 1–2V1 and 2–1V2 Streptolysin O -permeabilized T2 cells expressing wild-type TAP or chimeric transporter variants were incubated with iodinated reporter peptide S8
at 37 C for the times indicated,the reactions were stopped by adding cold lysis buffer containing 1% Triton X-100 and peptides were quantitated
by gamma counting (left panel) Peptide supply by wild-type TAP and chimeric transporter variants to HLA-B5 molecules was analyzed by FACS analysis (right panel) as described in Fig 3C.