24 and this study) provides a molecular basis for the requirement of glucosidase activity in the release of MHC class I proteins from TAP molecules. I assembly intermediates in the ER and show that glycan processing is functionally coupled to release of MHC class I proteins from peptide transport molecules. Most major histocompatibility complex (MHC) class I proteins are expressed around the cell surface in association with 2 microglobulin (2m) molecules and processed peptides (1, 2). Assembly of MHC class I protein complexes occurs in the endoplasmic reticulum (ER) and is proposed to be initiated by association IkB alpha antibody of newly translated CYC116 (CYC-116) MHC class I heavy chains with calnexin (3, 4, 5, 6, 7), a lectin-like chaperone molecule (8, 9, 10). In the murine system, 2m proteins associate with calnexinCHC to form calnexinCHCC2m complexes, followed by addition of peptides generated by proteosome processing of cytosolic proteins and transported into the ER lumen by TAP 1/2 (transporter associated with antigen presentation) heterodimers (11); addition of peptide to HCC2m complexes has been suggested to trigger their dissociation from calnexin and facilitate their egress from your ER (7, 12, 13, 14, 15). Immature glycan chains on nascent polypeptides have the structure Glc3Man9GlcNAc2 (Glc, glucose; Man, mannose; GlcNAc, indicated that 7% and 15% of total radiolabeled H-2Kb proteins coprecipitated with calnexin and calreticulin chaperones in splenic T-cell lysates (Fig. CYC116 (CYC-116) ?(Fig.11were determined by densitometric scanning and are expressed as the percentage of total labeled H-2Kb proteins in splenic T-cell lysates. Multiple exposures of autoradiographs were scanned to ensure linearity. Molecular Requirements for Assembly of Newly Synthesized Murine Class I Proteins with Calreticulin. Next, we examined the molecular requirements for assembly of newly synthesized class I proteins with calreticulin using splenic T cells from mice genetically deficient in expression of 2m (2m ?/?) or TAP (TAP ?/?) molecules. As shown in Fig. ?Fig.22except that lysates were precleared with the indicated antibodies, then sequentially precipitated with anti-calreticulin (Crt). ((23), who showed that calnexin association and assembly of MHC class I protein complexes in BW thymoma cells was greatly reduced by cas treatment (23); interestingly, however, normal class I assembly was observed in the glucosidase II-deficient BW variant, BW PHAR2.7, in which calnexin association (17) and calreticulin association (35) is severely impaired. Thus, it appears that option pathways exist for the assembly of class I proteins that do not require glucosidase activity and calnexin/calreticulin associations that are variably utilized, depending on the cell type. The molecular basis for normal MHC class I assembly in glucosidase-deficient cells is usually unknown but has been suggested to involve expression of other chaperones that are up-regulated under ER stress conditions (23). It is unknown if calnexin and calreticulin function redundantly in the ER quality control system or if they perform distinct molecular functions in the folding/assembly of newly synthesized glycoproteins. Peterson (19) recently demonstrated that the population of cellular proteins bound to calreticulin partially overlaps those bound to calnexin; and, at least for one protein, the influenza computer virus hemagglutinin protein, assembly with calnexin and calreticulin was indistinguishable, as measured by disulfide bond formation and conformation analysis. In the current study, we demonstrate by several criteria that calnexin and calreticulin associate with unique MHC class assembly intermediates in the ER, suggesting that calnexin and calreticulin may perform specific functions in the formation of class I heavy chainC2mCpeptide complexes. Whether or not newly synthesized class I proteins interact successively with calnexin and calreticulin chaperones remains to be decided. The data in the current study show that unlike calnexin, calreticulin interacts primarily with class IC2m heterodimers, and, importantly, that the vast majority of class I proteins associated with calreticulin in splenic T cells are simultaneously assembled with TAP. These results are in agreement with recently reported findings CYC116 (CYC-116) by CYC116 (CYC-116) Cresswell and coworkers (24) studying human class ICcalreticulinCTAP interactions (24). Importantly, the current study files that deglucosylation of N-linked glycans is an important step in the disassembly of MHC class I proteins from both calreticulin and TAP molecules..