SummaryTo determine the mechanism and structural consequences of peptide binding to class I molecules, we have studied the L d molecule of the mouse. Previous studies have shown that a significant proportion of surface and intracellular L a molecules can be detected in an alternative conformation designated Laalt, Ldalt molecules are non-ligand associated and show weak if any 32-microglobulin (32m) association. We report here that L a molecules have a relatively rapid surface turnover compared with other class I molecules and that exogenous peptide dramatically prolongs L d surface half-life. By contrast, Laalt molecules are stably expressed on the surface and their half-life is unaffected by exogenous peptide. To study the surface interaction of peptide with L a, live cells were incubated with iodinated peptides and L d molecules were precipitated from cells precoated with monoclonal antibody before lysis. Using this assay, peptide binding to surface L a molecules was found not to depend upon exchange with exogenous 32m, but did correlate with the level of 32m association. To study the intracellular interaction of peptide with L d, cell lysates were used. In cell lysates, peptide was found to convert Ldalt molecules to properly folded L d. This peptide-induced folding was almost complete at earlier but not later time points in pulse-chase analyses. Furthermore, conversion of Ldalt to L d was found to affect almost exclusively immature (Endo H s) class I molecules. Thus intrinsic properties of immature Ldalt molecules or their associated chaperonins are maintained in cell lysates that allow them to undergo de novo folding in vitro. These combined results demonstrate that immature Laalt molecules are precursors awaiting constituents such as peptide and 32m that influence folding, whereas surface Laalt molecules appear refractory to association with peptide, 32m, and consequent folding.

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In homozygous mice bearing I regions derived from haplotype k, only a single type of Ia molecule bearing the alloantigenic specificities Ia.7 and Ia.22 was found using techniques of sequential immune precipitation and tryptic peptide analysis. As suggested at the fourth Ir Gene Workshop (Sachs 1978), Ia.7 is considered here to be an antigenic determinant associated with I-E-subregion-encoded molecules, i.e., it is excluded from the I-C subregion. The I-C subregion is currently defined mainly by functional traits. It is now known that the I-E molecules are composed of an alpha chain encoded in the I-E subregion, and a beta chain encoded in the I-A subregion. Since the I-C subregion is not involved with the determination of these Ia molecules, and since in homozygotes there is apparently only a single type of molecule bearing both specificities Ia.7 and Ia.22, the term "I-E/C" molecule should probably be dropped in favor of the simpler designation I-E.

Newly synthesized class I heavy (H) chain/beta 2m heterodimers awaiting peptides in the endoplasmic reticulum are associated with the transporter associated with Ag processing (TAP). We present evidence here that calreticulin, but not calnexin, displays steady state association with class I/TAP complexes. To separate the ability of beta 2m to bind with TAP and calreticulin from that of H chain, we studied human cell lines that lack expression of beta 2m or H chain. Little if any H chain was detected in association with TAP and calreticulin in the beta 2m- cell line Daudi. By contrast, high levels of beta 2m are found with TAP and calreticulin in the H chain-deficient cell line LCL 721.221, even after preclearance of the trace amount of class IB protein expressed by this cell line. Thus, beta 2m appears to bind TAP in the absence of H chain, providing an elegant mechanism to retain beta 2m in the endoplasmic reticulum at the site of peptide loading. To investigate whether other molecules participate in the binding of beta 2m and H chain to TAP and calreticulin, we analyzed the deletion mutant cell line LCL 721.220, which lacks tapasin. In 721.220, TAP and calreticulin are not associated with each other. Also, in these cells, H chain/beta 2m are not associated with TAP, but H chain and a low level of beta 2m are associated with calreticulin. These results suggest that tapasin is an obligatory mediator of the assemblage of calreticulin/H chain/beta 2m with TAP.

Before peptide binding, a variety of endoplasmic reticulum (ER) proteins are associated with class I including calnexin, TAP, calreticulin, and tapasin. Although the selective functions of any one of these ER proteins have been difficult to define, individually or in combination they perform two general chaperone functions for class I. They promote assembly of the class I heterotrimeric molecule (heavy (H) chain, β2m, and peptide) and they retain incompletely assembled complexes in the ER. In this study, we present evidence that calreticulin clearly differs from calnexin in how it associates with class I. Regarding the structural basis of the association, the oligosaccharide moiety in the α1 domain and the amino acid residue at position 227 in the α3 domain were both found to be critical for the interaction of class I with calreticulin. Interestingly, calreticulin displayed sensitivity to class I peptide binding even in TAP-deficient human or mouse cells. Thus, calreticulin is clearly more specific than calnexin in the structures and conformation of the class I molecule with which it can interact.