Abstract
The transporter associated with antigen processing (TAP) plays a critical role in the MHC class I antigen presentation pathway. TAP translocates cellular peptides across the endoplasmic reticulum membrane in an ATP hydrolysis-dependent manner. We used FRET spectroscopy in permeabilized cells to delineate different conformational states of TAP in a native subcellular membrane environment. For these studies, we tagged the TAP1 and TAP2 subunits with enhanced cyan fluorescent protein and enhanced yellow fluorescent protein, respectively, C-terminally to their nucleotide binding domains (NBDs), and measured FRET efficiencies under different conditions. Our data indicate that both ATP and ADP enhance the FRET efficiencies but that neither induces a maximally closed NBD conformation. Additionally, peptide binding induces a large and significant increase in NBD proximity with a concentration dependence that is reflective of individual peptide affinities for TAP, revealing the underlying mechanism of peptide-stimulated ATPase activity of TAP. Maximal NBD closure is induced by the combination of peptide and non-hydrolysable ATP analogs. Thus, TAP1-TAP2 NBD dimers are not fully stabilized by nucleotides alone, and substrate binding plays a key role in inducing the transition state conformations of the NBD. Taken together, these findings show that at least three steps are involved in the transport of peptides across the endoplasmic reticulum membrane for antigen presentation, corresponding to three dynamically and structurally distinct conformational states of TAP. Our studies elucidate structural changes in the TAP NBD in response to nucleotides and substrate, providing new insights into the mechanism of ATP-binding cassette transporter function.
Highlights
transporter associated with antigen processing (TAP) proteins tagged with CFP and YFP were used for FRET spectroscopy measurements
T2Y, or T1C/T2Y were detected using a fluorometer, which showed that the fusion of ECFP or EYFP to TAP1 or TAP2 did not alter the spectral properties of the fluorescent proteins (Fig. 2A)
The attachment of ECFP or EYFP at the C terminus does not affect the function of TAP, which allowed us to use the tagged TAP1TAP2 complexes to study nucleotide binding domains (NBD) interactions in permeabilized cells
Summary
TAP proteins tagged with CFP and YFP were used for FRET spectroscopy measurements. Results: Conformational changes of the nucleotide binding domains (NBD) were measurable in permeabilized cells. We used FRET spectroscopy in permeabilized cells to delineate different conformational states of TAP in a native subcellular membrane environment For these studies, we tagged the TAP1 and TAP2 subunits with enhanced cyan fluorescent protein and enhanced yellow fluorescent protein, respectively, C-terminally to their nucleotide binding domains (NBDs), and measured FRET efficiencies under different conditions. TAP1-TAP2 NBD dimers are not fully stabilized by nucleotides alone, and substrate binding plays a key role in inducing the transition state conformations of the NBD Taken together, these findings show that at least three steps are involved in the transport of peptides across the endoplasmic reticulum membrane for antigen presentation, corresponding to three dynamically and structurally distinct conformational states of TAP. We investigated the movement of TAP NBDs during the catalytic cycle using a FRET approach, which allows us to monitor the distance changes between the TAP1-TAP2 NBDs in a native cellular membrane environment under different conditions without the need of biochemical sample preparation
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