Prior to binding to a high affinity peptide and transporting it to the cell surface, major histocompatibility complex class I molecules are retained inside the cell by retention in the endoplasmic reticulum (ER), recycling through the ER-Golgi intermediate compartment and possibly the cis-Golgi, or both. Using fluorescence microscopy and a novel in vitro COPII (ER-to-ERGolgi intermediate compartment) vesicle formation assay, we find that in both lymphocytes and fibroblasts that lack the functional transporter associated with antigen presentation, class I molecules exit the ER and reach the cis-Golgi. Intriguingly, in wild-type T1 lymphoma cells, peptide-occupied and peptidereceptive class I molecules are simultaneously exported from ER membranes with similar efficiencies. Our results suggest that binding of high affinity peptide and exit from the ER are not coupled, that the major histocompatibility complex class I quality control compartment extends into the Golgi apparatus under standard conditions, and that peptide loading onto class I molecules may occur in post-ER compartments.

The endoplasmic reticulum (ER) protein tapasin is essential for the loading of high-affinity peptides onto MHC class I molecules. It mediates peptide editing, i.e. the binding of peptides of successively higher affinity until class I molecules pass ER quality control and exit to the cell surface. The molecular mechanism of action of tapasin is unknown. We describe here the reconstitution of tapasin-mediated peptide editing on class I molecules in the lumen of microsomal membranes. We find that in a competitive situation between high-and low-affinity peptides, tapasin mediates the binding of the high-affinity peptide to class I by accelerating the dissociation of the peptide from an unstable intermediate of the binding reaction. IntroductionMHC class I molecules present antigenic peptides on the cell surface for surveillance by cytotoxic T lymphocytes. The peptides (usually 8-10 aa in length) are generated in the cytosol and transported into the endoplasmic reticulum (ER), where they bind to the newly synthesized class I heterodimer, which consists of the heavy chain (HC) and beta-2 microglobulin (b 2 m). For most class I allotypes, optimal peptide loading (prior to exit toward the cell surface) requires an interaction with the peptide loading complex (PLC), which consists of the peptide transporter TAP, the lectin chaperone calreticulin, the protein disulfide isomerases ERp57 and possibly PDI, and the class I-specific peptide loading factor, tapasin [1,2]. In the PLC, class I binds directly to tapasin [3]. This interaction is crucial for the loading of high-affinity peptides onto most (''tapasin-dependent'') class I allotypes, and thus for their expression at the cell surface. Tapasin-mediated peptide binding proceeds in an iterative manner such that low-affinity peptides that are bound initially are successively displaced by higher affinity ones. This optimization process is known as peptide editing [4].The structure of tapasin has recently been solved [5], but it is still unclear how it induces class I molecules to bind high-affinity peptides in the presence of an excess (estimated to be a 1000-fold, [6]) of low-affinity peptides. Its mechanism of action has been addressed in different experimental systems, which have yielded different and sometimes conflicting results. In cell-based assays, tapasin leads to the increased surface expression, thermostability, and persistence of class I-peptide complexes [4,[7][8][9], but some reports state that it does not influence the composition of the peptide pool bound to class I [10,11]. In assays that use whole cells, the peptide editing function of tapasin may be obscured by ER/Golgi quality control [12], a role of tapasin in the trafficking of class I to or from the Golgi apparatus (Springer et al., unpublished data) [13,14], class I loading by alternative pathways [2], and the metabolic stabilization of TAP by tapasin [3]. In search of the molecular mechanism of peptide editing, several groups have à These authors contributed equally to this work. 214therefore es...

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The interaction of T4 phage-encoded anti-sigma factor, asiA, andEscherichia coli ς70 was studied by using the yeast two-hybrid system. Truncation of ς70 to identify the minimum region involved in the interaction showed that the fragment containing amino acid residues proximal to the C terminus (residues 547 to 603) was sufficient for complexing to asiA. Studies also indicated that some of the truncated C-terminal fragments (residues 493 to 613) had higher affinity for asiA as judged by the increased β-galactosidase activity. It is proposed that the observed higher affinity may be due to the unmasking of the binding region of asiA on the sigma protein. Advantage was taken of the increased affinity of truncated ς70 fragments to asiA in designing a coexpression system wherein the toxicity of asiA expression in E. coli could be neutralized and the complex of truncated ς70 and asiA could be expressed in large quantities and purified.