The transporter associated with antigen processing (TAP) is a member of the family of ABC transporters that translocate a large variety of substrates across membranes. TAP transports peptides from the cytosol into the endoplasmic reticulum for binding to MHC class I molecules and for subsequent presentation to the immune system. Here we follow the lateral mobility of TAP in living cells. TAP's mobility increases when it is inactive and decreases when it translocates peptides. Because TAP activity is dependent on substrate, the mobility of TAP is used to monitor the intracellular peptide content in vivo. Comparison of the diffusion rates in peptide-free and peptide-saturated cells indicates that normally about one-third of all TAP molecules actively translocate peptides. However, during an acute influenza infection TAP becomes fully employed owing to the production and degradation of viral proteins. Furthermore, TAP activity depends on continuing protein translation. This implies that MHC class I molecules mainly sample peptides that originate from newly synthesized proteins, to ensure rapid presentation to the immune system.

MHC class I molecules usually present peptides derived from endogenous antigens that are bound in the endoplasmic reticulum. Loading of exogenous antigens on class I molecules, e.g., in cross-priming, sometimes occurs, but the intracellular location where interaction between the antigenic fragment and class I takes place is unclear. Here we show that measles virus F protein can be presented by class I in transporters associated with antigen processing-independent, NH 4 Clsensitive manner, suggesting that class I molecules are able to interact and bind antigen in acidic compartments, like class II molecules. Studies on intracellular transport of green fluorescent protein-tagged class I molecules in living cells confirmed that a small fraction of class I molecules indeed enters classical MHC class II compartments (MIICs) and is transported in MIICs back to the plasma membrane. Fractionation studies show that class I complexes in MIICs contain peptides. The pH in MIIC (around 5.0) is such that efficient peptide exchange can occur. We thus present evidence for a pathway for class I loading that is shared with class II molecules.MHC molecules display antigenic peptides on the cell surface for surveillance by T lymphocytes. MHC class I molecules present peptides to CD8 ϩ cytotoxic T cells, whereas MHC class II molecules present peptides to CD4 ϩ Th cells. The current dogma is that antigens from the extracellular fluid enter the exogenous processing pathway by endocytosis and are partially degraded in acidic endosomal or lysosomal structures to yield peptides that bind MHC class II molecules. This type of processing is inhibited by reagents that prevent endosomal acidification (chloroquine, NH 4 Cl) (1). In the endogenous processing pathway intracellular proteins are degraded in the cytosol by the proteasome complex, generating peptides that are transported from the cytoplasm into the lumen of the endoplasmic reticulum (ER) by the transporters associated with antigen processing (TAP), where they bind to nascent MHC class I heavy chain-␤ 2 -microglobulin (␤ 2 m) heterodimers. Fully assembled class I͞peptide complexes exit the ER and are transported through the Golgi to the cell surface by the constitutive secretory route. This processing pathway can be blocked by proteasome inhibitors or Brefeldin A (BFA), an inhibitor of anterograde ER-Golgi transport, but not by lysosomotropic agents. Thus, in general endogenous antigens are presented by MHC class I molecules, and exogenous antigens are displayed at the cell surface by MHC class II molecules. However accumulating evidence has shown that this dichotomy in presentation of antigen from endogenous and exogenous origin is not absolute. It was demonstrated that cytotoxic T lymphocyte (CTL) responses can be primed in vitro and in vivo with exogenous antigen (reviewed in refs. 2 and 3). At least two fundamentally different pathways for presentation of exogenous antigens by MHC class I molecules in vitro have been described: one involving access of exogenous antigen to...

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Prior to their association with major histocompatibility complex (MHC) class I molecules, peptides generated from cytosolic antigens need to be translocated by the MHC-encoded peptide transporter (TAP) into the lumen of the endoplasmic reticulum (ER). While class I molecules possess well-known binding characteristics for peptides, the fine specificity of TAP for its peptide substrates has not been analyzed in detail. Previously, we have studied the effect of amino acid variations at the N-terminal, the C-terminal, and the penultimate residue on the efficiency of peptide translocation. Using permeabilized cells, we have shown that TAP pre-selects peptides in an allele- and species-specific manner, for which only the C-terminal residue is crucial. This finding is confirmed in the present study by using microsomes containing different TAP. The influence of amino acid substitutions at positions 2 to 7 of 9-residue model peptides on TAP-dependent peptide translocation is systematically examined. Only a few amino acid substitutions at these positions affect the efficiency of peptide translocation significantly, e.g. Pro at position 2 or 3 negatively influences transport whereas Glu at positions 6 and 7 enhances transport. The differences in translocation by the rat TAP alleles a or u, mouse TAP and human TAP are, however, minor for the peptide with internal substitutions used in this study. These results show that the C-terminal residue essentially governs the species-specific substrate specificity of TAP.