Export of antigenic peptides from the endoplasmic reticulum intersects with retrograde protein translocation through the Sec61p channel.

Antigenic peptides are translocated by the TAP peptide transporter from the cytosol into the endoplasmic reticulum (ER) for loading onto MHC class I molecules. Peptides that fail to bind need to be removed from the ER. Here we provide evidence that peptide export utilizes the Sec61p translocon as demonstrated by blocking this channel with bacterial exotoxin. Peptide export interferes with the retrotranslocation of beta2-microglobulin from the ER to the cytosol, suggesting similar pathways for the disposal of proteins and oligopeptides. Peptide export requires ATP supply to the ER lumen but is independent of ATP hydrolysis.

A viral ER-resident glycoprotein inactivates the MHC-encoded peptide transporter.

Human cytomegalovirus inhibits peptide import into the endoplasmic reticulum (ER) by the MHC-encoded TAP peptide transporter. We identified the open reading frame US6 to mediate this effect. Expression of the 21 kDa US6 glycoprotein in human cytomegalovirus-infected cells correlates with the inhibition of peptide transport during infection. The subcellular localization of US6 is ER restricted and is identical with TAP. US6 protein is found in complexes with TAP1/2, MHC class I heavy chain, beta2-microglobulin, calnexin, calreticulin, and tapasin. TAP inhibition, however, is independent of the presence of class I heavy chain and tapasin. The results establish a new mechanism for viral immune escape and a novel role for ER-resident proteins to regulate TAP via its luminal face.

The rational design of TAP inhibitors using peptide substrate modifications and peptidomimetics.

The major histocompatibility complex (MHC)-encoded transporter associated with antigen processing (TAP) translocates peptides from the cytosol into the lumen of the endoplasmic reticulum. This step precedes the binding of peptides to MHC class I molecules and is essential for cell surface expression of the MHC class I/peptide complex. TAP has a broad sequence specificity and a preference for peptides of around 9 amino acids. To synthesize inhibitors for TAP, we studied various alterations of the peptide substrate. The results indicate that TAP is stereospecific and that peptide bonds engineered into isosteric structures can improve translocation of the peptide. Furthermore, TAP is able to translocate peptides with large side chains that correspond to a peptide of approximately 21 amino acids in extended conformation. Peptides with longer side chains compete for the peptide binding site of TAP but fail to be translocated. Therefore, they represent the first rationally designed inhibitors of TAP.

Generation, intracellular transport and loading of peptides associated with MHC class I molecules.

MHC class I molecules present antigenic peptides that are mostly derived from endogenous cytosolic proteins. Recent studies addressing the function of the proteasome and its activator complexes have advanced our understanding of the cytosolic processing of peptides. Transporters associated with antigen processing (TAPs) translocate these peptides to the endoplasmic reticulum where MHC class I molecules, which are retained in transient complexes with chaperones and TAPs, await them for binding. The sequence specificity and the peptide length preference of TAPs roughly meet the requirements of class I molecules in a range of different species, suggesting evolutionary shaping of the specificity of TAPs.

Translocation of long peptides by transporters associated with antigen processing (TAP).

The major histocompatibility complex (MHC)-encoded transporters associated with antigen processing (TAP) translocate peptides from the cytosol into the lumen of the endoplasmic reticulum (ER) where they associate with MHC class I molecules. The length of class I-binding peptides is usually 8-11 amino acids, but examples of significantly longer peptides have been described. The preferred lengths and upper and lower size limits for peptides translocated by TAP have not been determined in detail because in the currently used test systems, peptides are subject to proteolytic degradation. In the present study, three sets of individual peptides or partially randomized peptide libraries ranging between 6 and 40 residues were used that contained a radiolabeled tyrosine and a consensus sequence for ER-specific N-glycosylation at opposite ends, thus ensuring that only nondegraded peptides were monitored in the transport/glycosylation assay. For three different transporters, rat TAP1/2a, rat TAP1/2u and hTAP, the most efficient ATP-dependent transport was observed for peptides with 8-12 amino acids. Hexamers and longer peptides of up to 40 amino acids were also translocated, albeit less efficiently. For two of the three sets of peptides analyzed, rat TAP1/2a showed a less stringent length selection than rat TAP1/2u and human TAP. The superior transport of the decamer of the TNKT.. Y series was not due to faster degradation or less efficient glycosylation of shorter or longer length variants. A binding assay with TAP-containing microsomes revealed a high affinity for the radiolabeled decamer (KD = 580 nM), while other length variants were clearly inferior in their binding affinities. Thus, TAP binds and preferentially translocates peptides with a length suitable for binding to MHC class I molecules, but peptides that are considerably longer may also be substrates. About 10(5) peptide binding sites per cell equivalent of microsomes were determined, providing an estimate for the number of TAP complexes in the ER membrane.