Reconstitution of peptide‐binding activity by TAP in proteoliposomes

Stephens, David B.; Androlewicz, Matthew J.

doi:10.1016/s0014-5793(97)01221-0

Cited by 7 publications

(6 citation statements)

References 25 publications

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“…Although substrate-binding activity is well conserved after reconstitution, ATP-dependent transport activity seems to be a very fragile property of TAP (28,30). Here we were able to demonstrate that reconstituted TAP not only binds peptides but also displays ATP-dependent transport activity (Fig.…”

Section: Methodsmentioning

confidence: 59%

Allosteric crosstalk between peptide-binding, transport, and ATP hydrolysis of the ABC transporter TAP

Gorbulev

Abele

Tampé

2001

Proc. Natl. Acad. Sci. U.S.A.

116

114

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The transporter associated with antigen processing (TAP) is essential for intracellular transport of protein fragments into the endoplasmic reticulum for loading of major histocompatibility complex (MHC) class I molecules. On the cell surface, these peptide-MHC complexes are monitored by cytotoxic T lymphocytes. To study the ATP hydrolysis of TAP, we developed an enrichment and reconstitution procedure, by which we fully restored TAP function in proteoliposomes. A TAPspecific ATPase activity was identified that could be stimulated by peptides and blocked by the herpes simplex virus protein ICP47. Strikingly, the peptide-binding motif of TAP directly correlates with the stimulation of the ATPase activity, demonstrating that the initial peptide-binding step is responsible for TAP selectivity. ATP hydrolysis follows Michaelis-Menten kinetics with a maximal velocity V max of 2 mol͞min per mg TAP, corresponding to a turnover number of approximately 5 ATP per second. This turnover rate is sufficient to account for the role of TAP in peptide loading of MHC molecules and the overall process of antigen presentation. Interestingly, sterically restricted peptides that bind but are not transported by TAP do not stimulate ATPase activity. These results point to coordinated dialogue between the peptide-binding site, the nucleotide-binding domain, and the translocation site via conformational changes within the TAP complex.membrane proteins ͉ transport mechanism ͉ ATPase ͉ antigen processing A n essential step in the major histocompatibility complex (MHC) class I-mediated cellular immune defense is the transport of antigenic peptides from the cytoplasm into the endoplasmic reticulum (ER) (reviewed by ref. 1). The transporter associated with antigen processing (TAP) translocates peptides that are mainly derived from the proteasomal degradation into the ER (reviewed by ref. 2). Subsequently, peptides bind to MHC class I molecules and are presented at the cell surface, where they are recognized by cytotoxic T lymphocytes.TAP belongs to the family of ATP-binding cassette (ABC) transporters that, under consumption of ATP, translocate a vast spectrum of substrates across membranes (3). On the basis of sequence comparison, TAP fits in the B subfamily of human ABC transporters (www.gene.ucl.ac.uk͞users͞hester͞abc.html), including the well-characterized P-glycoprotein (ABCB1). The TAP complex forms a heterodimer of TAP1 (ABCB2) and TAP2 (ABCB3), where each subunit comprises a hydrophobic transmembrane domain and a cytoplasmic nucleotide-binding domain (NBD) containing the Walker A͞B and C-loop consensus sequences. The transmembrane domains are thought to form a channel by which peptides are transported across the ER membrane driven by ATP hydrolysis. The coupling between NBDs, the substrate-binding site, and the translocation pathway is not understood and is a central topic to be studied for all ABC transporters.TAP is part of a macromolecular peptide translocation and loading complex that comprises TAP1, TAP2, tapasin, MHC class I hea...

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Section: Methodsmentioning

confidence: 59%

Allosteric crosstalk between peptide-binding, transport, and ATP hydrolysis of the ABC transporter TAP

Gorbulev

Abele

Tampé

2001

Proc. Natl. Acad. Sci. U.S.A.

116

114

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“…M) is slightly above the reported Kd (0.1-0.2 ? M) for peptide binding to TAP including the same B27#3 peptide used in our transport assays [31,32]. Therefore, tapasin alone is sufficient to increase the number of functional TAP heterodimers.…”

Section: Discussionmentioning

confidence: 98%

The N-terminal region of tapasin is required to stabilize the MHC class I loading complex

et al. 1999

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Tapasin mediates the binding of MHC class I molecules to the transporter associated with antigen processing (TAP). Deletion mutants of tapasin were used to examine the effect of tapasin on interactions within the MHC class I complex. Binding to TAP is mediated by the C-terminal region of tapasin. Michaelis-Menten analysis of peptide transport shows that this interaction is sufficient to increase TAP levels without significantly affecting the intrinsic translocation rate. Weak interactions exist between MHC class I molecules and TAP in the absence of tapasin, and between free heavy chains and TAP-tapasin complexes in the absence of g 2-microglobulin. The N-terminal 50 residues of tapasin constitute the key element which converts the sum of these weak interactions into a stable complex.

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“…TAP function is essential for antigen presentation by major histocompatibility complex class I molecules to cytotoxic T lymphocytes, since deficiency in one or both TAP subunits results in severely reduced major histocompatibility complex class I expression and function. As peptide transport by TAP in permeabilized cells requires ATP and is inhibited by non-hydrolyzable ATP analogues (16), TAP is thought to be an ATPase, although in experiments with isolated recombinant TAP NBD fragments, or with TAP dimers reconstituted in membranes, it has so far not been possible to provide direct evidence for this hypothesis (17)(18)(19)(20). However, nucleotide binding to both subunits assembled in TAP complexes (21), and to the recombinant TAP1 NBD (17,18) has been demonstrated directly; contradictory results have been reported with respect to the ATP binding capacity of the TAP2 NBD (17,18).…”

mentioning

confidence: 99%

Role of Nucleotides and Peptide Substrate for Stability and Functional State of the Human ABC Family Transporters Associated with Antigen Processing

Endert

1999

Journal of Biological Chemistry

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The transporters associated with antigen processing (TAP) belong to the family of ATP-binding cassette (ABC) transporters which share structural organization and use energy provided by ATP to translocate a large variety of solutes across cellular membranes. TAP is thought to hydrolyze ATP in order to deliver peptides to the endoplasmic reticulum where they can assemble with major histocompatibility complex class I molecules. However, initial binding of peptide substrates to TAP has been suggested to be ATP-independent. In this study, the effect of temperature, energetic nucleotides, and peptide on conformation and functional capacity of TAP proteins was examined. Incubation of insect cell microsomes overexpressing human TAP complexes or of human B cell microsomes at 37°C induced a rapid and irreversible structural change that reduced dramatically TAP reactivity with antibodies to transmembrane and nucleotide-binding domains and abolished peptide binding and transport by TAP. These alterations were inhibited almost completely by di-or trinucleotides, and partially by high affinity peptides, suggesting that complete nucleotide dissociation inactivates TAP complexes. Experiments with isolated TAP subunits and fragments suggested that TAP complex stabilization by nucleotides may depend on their binding to the TAP1 subunit. Thus, the cellular level of functional TAP complexes may be regulated by nucleotide concentrations. It is speculated that this regulation may serve to prevent induction of autoimmunity by stressed cells with low energy levels.

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Reconstitution of peptide‐binding activity by TAP in proteoliposomes

Cited by 7 publications

References 25 publications

Allosteric crosstalk between peptide-binding, transport, and ATP hydrolysis of the ABC transporter TAP

Allosteric crosstalk between peptide-binding, transport, and ATP hydrolysis of the ABC transporter TAP

The N-terminal region of tapasin is required to stabilize the MHC class I loading complex

Role of Nucleotides and Peptide Substrate for Stability and Functional State of the Human ABC Family Transporters Associated with Antigen Processing

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