Three-dimensional Structure of Transporter Associated with Antigen Processing (TAP) Obtained by Single Particle Image Analysis

Abstract

The transporter associated with antigen processing (TAP) is an ATP binding cassette transporter responsible for peptide translocation into the lumen of the endoplasmic reticulum for assembly with major histocompatibility complex class I molecules. Immunoaffinity-purified TAP particles comprising TAP1 and TAP2 polypeptides, and TAP2 particles alone were characterized after detergent solubilization and studied by electron microscopy. Projection structures of TAP1؉2 particles reveal a molecule ϳ10 nm across with a deeply staining central region, whereas TAP2 molecules are smaller in projection. A three-dimensional structure of TAP reveals it is isolated as a single heterodimeric complex, with the TAP1 and TAP2 subunits combining to create a central 3-nm-diameter pocket on the predicted endoplasmic reticulum-lumenal side. Its structural similarity to other ABC transporters demonstrates a common tertiary structure for this diverse family of membrane proteins. ATP binding cassette (ABC)1 transporters are ubiquitous and can form a significant component of an organism's genome, for example 2% in Escherichia coli (1). They are defined by the ABC domain, a nucleotide hydrolysis domain whose activity powers substrate transport (2), and are classically composed of at least four domains, two transmembrane domains (TMDs) and two cytoplasmic and soluble nucleotide binding domains (NBDs). These domains can be expressed together as a single modular polypeptide or separately.TAP is an ABC transporter expressed in the endoplasmic reticulum (ER), which supplies peptides from the cytosol for binding by MHC class I molecules (3-8). In the absence of TAP the assembly of MHC class I and, consequently, antigen presentation are both severely impaired (9 -12). TAP is expressed as two polypeptide subunits, TAP1 and TAP2, each composed of one TMD and one NBD, which together form the functional transporter. Models for the predicted structure of TAP have relied on the sequence-based prediction of multiple transmembrane-spanning regions in the TMDs (13), the location of point mutations or naturally occurring polymorphisms affecting peptide selection and transport (14 -17), or the cytosolic or ER lumenal orientation of TAPs engineered to contain reporter molecules (18,19). More recently, expression of truncated TAP polypeptides suggests interactions between both the TMDs and NBDs of . Nevertheless, no direct structural imaging of this crucial molecule to the development of cellular immune responses has yet been obtained.There is a need for structural studies of whole ABC transporters to permit insights into their overall structure and functions. Single particle image analysis has successfully been applied to molecules of comparable size to TAP (23-26). Furthermore, low resolution electron microscopic structures of the ABC transporters P-glycoprotein and MRP1 have been determined using a combination of single particle and crystallographic analysis (27,28). Recently a structure for MsbA, a prokaryotic half-ABC transporter, has been published, all...

Structural and Functional Fingerprint of the Mitochondrial ATP-binding Cassette Transporter Mdl1 from Saccharomyces cerevisiae

“…The full size transporters PDR5 (63) and CFTR (61) showed particle dimensions that fit (homo) dimers, whereas for particles of the full size transporter P-glycoprotein a monomeric state was consistent (64). Dimensions of the half-size transport complex TAP1/TAP2 and YvcC implicate heterodimeric or homodimeric structures, respectively (49,53). The Mdl1 structure is clearly different from the very recently solved x-ray structure of the ABC transporter Sav1866 from S. aureus, which is found in an outward facing conformation with the two NBDs in close contact (56).…”

Two highly divergent ancient allelic lineages of the transporter associated with antigen processing (TAP) gene in Xenopus: further evidence for co‐evolution among MHC class I region genes

“…the human and rodent sequences (data not shown). It was proposed that exon 1 containing the transmembrane domains [28], is responsible for forming TAP heterodimers [29], and a recent study using single-particle analysis supports TAP forming a pore in the ER membrane [30]. Despite the differences in amino acid sequence, human-rodent interspecies TAP1 and TAP2 heterodimers seem to function properly [25].…”

Purification and Crystallization of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

“…In contrast to progress with bacterial ABC transporters, eukaryotic ABC transporters have remained relatively resistant to high resolution structural analysis, although the CFTR structure herein represents the fourth eukaryotic ABC transporter for which low resolution three-dimensional structural data have been obtained (8,46,47). Projection data (two-dimensional) for a fifth eukaryotic ABC transporter have also been obtained (8), and a projection map for the P-glycoprotein in two-dimensional crystals in lipid membranes was also published (48).…”