Walker A Lysine Mutations of TAP1 and TAP2 Interfere with Peptide Translocation but Not Peptide Binding

Abstract

We generated mutants of the transporter associated with antigen-processing subunits TAP1 and TAP2 that were altered at the conserved lysine residue in the Walker A motifs of the nucleotide binding domains (NBD). In other ATP binding cassette transporters, mutations of the lysine have been shown to reduce or abrogate the ATP hydrolysis activity and in some cases impair nucleotide binding. Mutants TAP1(K544M) and TAP2(K509M) were expressed in insect cells, and the effects of the mutations on nucleotide binding, peptide binding, and peptide translocation were assessed. The mutant TAP1 subunit is significantly impaired for nucleotide binding relative to wild type TAP1. The identical mutation in TAP2 does not significantly impair nucleotide binding relative to wild type TAP2. Using fluorescence quenching assays to measure the binding of fluorescent peptides, we show that both mutants, in combination with their wild type partners, can bind peptides. Since the mutant TAP1 is significantly impaired for nucleotide binding, these results indicate that nucleotide binding to TAP1 is not a requirement for peptide binding to TAP complexes. Peptide translocation is undetectable for TAP1⅐TAP2(K509M) complexes, but low levels of translocation are detectable with TAP1(K544M)⅐TAP2 complexes. These results suggest an impairment in nucleotide hydrolysis by TAP complexes containing either mutant TAP subunit and indicate that the presence of one intact TAP NBD is insufficient for efficient catalysis of peptide translocation. Taken together, these results also suggest the possibility of distinct functions for TAP1 and TAP2 NBD during a single translocation cycle.The transporter associated with antigen processing (TAP) 1 is a critical component of the major histocompatibility complex (MHC) class I antigen presentation (1-3). TAP functions to translocate peptides from the cytosol to the ER. Binding of peptides to newly synthesized MHC class I molecules in the ER stabilizes the MHC class I heterodimer and allows transit of MHC class I-peptide complexes to the cell surface for immune surveillance by T cells (4). Two structurally related subunits of the TAP transporter, TAP1 and TAP2, form a complex on the ER membrane that is necessary and sufficient for peptide translocation from the cytosol into the ER. The cytosolic face of TAP1⅐TAP2 complexes contains a binding site for peptides (5), which can function in the sequestration of peptides derived from proteasomal proteolysis. A recently discovered protein called tapasin is associated with the TAP1⅐TAP2 complex (6, 7). Tapasin has been shown to enhance the expression level of TAP1 and increase peptide transport by TAP complexes (8). However, tapasin is not required for peptide binding by TAP1⅐TAP2 complexes or for translocation per se, since TAP1⅐TAP2 complexes expressed heterologously in insect cells can bind and transport peptides (9).TAP is a member of the ATP-binding cassette (ABC) family of transmembrane transport proteins (10). Members of this family transport various substrates acro...

Functional Non-equivalence of ATP-binding Cassette Signature Motifs in the Transporter Associated with Antigen Processing (TAP)

“…Different transport activities of Walker A mutants indicate, however, a functional asymmetry of the two TAP subunits (14,15,18). TAP1 and TAP2 also show an asymmetry in their C-loops, LSGGQ versus LAAGQ.…”

“…Indeed, beryllium fluoridetrapping studies provided direct evidence that both NBDs hydrolyze ATP during peptide translocation (17). Evidence for a functional asymmetry of the NBDs of TAP came from the analysis of mutations in the Walker A motif that interrupt peptide transport when introduced in TAP2 but still show low transport activity if placed in TAP1 (14,15,18). Furthermore, TAP complexes with two identical NBDs translocate peptides with a drastically decreased activity, whereas TAP complexes containing switched NBDs showed wild type activity (19,20).…”

Calreticulin recognizes misfolded HLA-A2 heavy chains

“…Peptides were obtained synthetically and labeled with FITC as described (11). Calreticulin was purified as described (12).…”

Use of chimeric proteins to investigate the role of transporter associated with antigen processing (TAP) structural domains in peptide binding and translocation

“…Other reports have described similar observations (11). These studies, taken together with reports that suggest reduced interactions of nucleotides with TAP2 NBD compared with TAP1 NBD (8,(11)(12)(13)(14)(15), raised the question of whether functional distinctions between TAP1 and TAP2 NBDs are important for coordinating the TAP transport cycle. Alternatively, the described differences might be a trivial consequence of structural differences between TAP1 and TAP2 NBDs, given that the structures are nonidentical (60% sequence identity), and therefore chemically distinct.…”

“…More recent reports described impaired peptide binding to mutant TAP complexes in which nucleotide binding was impaired (7). We examined the effects of nucleotides on peptide binding to wild-type TAP complexes or a mutant TAP1(K544M)͞TAP2 complex in which nucleotide binding to TAP1 was impaired (8). We showed that, at room temperature, peptide binding affinities and peptide dissociation kinetics were very similar for the TAP1(K544M)͞TAP2 mutant complex as for the wild-type complex, both in the presence and absence of nucleotides.…”