Tài liệu Báo cáo khoa học: The distinct nucleotide binding states of the transporter associated with

The 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- and D￾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 to M. 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: [email protected]

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)

Eur. J. Biochem. 270,4531–4546 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03848.x