We demonstrate the existence of a large endoplasmic reticulum (ER)-localized multiprotein complex that is comprised of the molecular chaperones BiP; GRP94; CaBP1; protein disulfide isomerase (PDI); ERdj3, a recently identified ER Hsp40 cochaperone; cyclophilin B; ERp72; GRP170; UDP-glucosyltransferase; and SDF2-L1. This complex is associated with unassembled, incompletely folded immunoglobulin heavy chains. Except for ERdj3, and to a lesser extent PDI, this complex also forms in the absence of nascent protein synthesis and is found in a variety of cell types. Cross-linking studies reveal that the majority of these chaperones are included in the complex. Our data suggest that this subset of ER chaperones forms an ER network that can bind to unfolded protein substrates instead of existing as free pools that assembled onto substrate proteins. It is noticeable that most of the components of the calnexin/calreticulin system, which include some of the most abundant chaperones inside the ER, are either not detected in this complex or only very poorly represented. This study demonstrates an organization of ER chaperones and folding enzymes that has not been previously appreciated and suggests a spatial separation of the two chaperone systems that may account for the temporal interactions observed in other studies. INTRODUCTIONTo travel along the secretory pathway and eventually reach their appropriate cellular destinations, newly synthesized secreted and membrane-bound proteins must fold and assemble correctly. Failure to do so results in their retention in the endoplasmic reticulum (ER) and eventual degradation. The proper conformational maturation of nascent secretory pathway proteins is both aided and monitored by a number of ER chaperones and folding enzymes in a complex process termed ER quality control . The components and mechanisms of action of two major chaperone systems have been best studied. The first system is dependent on the presence of both monoglucosylated N-linked glycans and unfolded regions on nascent glycoproteins. The resident ER protein UDP-glucosyltransferase (GT) binds to the unfolded regions and adds a single glucose to the deglucosylated glycan (Trombetta and Parodi, 1992), which in turns provides the binding site for the ER chaperones calnexin and calreticulin (Sousa et al., 1992;. Cleavage of this glucose by the resident ER protein glucosidase II (Kornfeld and Kornfeld, 1985) abrogates the calnexin/calreticulin binding site (Trombetta and Parodi, 1992;Hebert et al., 1995). If during the ensuing time the nascent chain folds, UDP-GT will not rebind and the protein will be released from the ER. However, if folding is not complete or correct folding is unable to occur, the cycle will repeat itself (Sousa et al., 1992;Hebert et al., 1995).The second major ER chaperone system is only dependent on the presence of unfolded regions on proteins containing hydrophobic residues, which are recognized by the ER chaperone BiP (Flynn et al., 1991;Blond-Elguindi et al., 1993). In fact, some calnexin/calretic...

We identified a mammalian BiP-associated protein, BAP, using a yeast two-hybrid screen that shared low homology with yeast Sls1p/Sil1p and mammalian HspBP1, both of which regulate the ATPase activity of their Hsp70 partner. BAP encoded an ϳ54-kDa protein with an N-terminal endoplasmic reticulum (ER) targeting sequence, two sites of N-linked glycosylation, and a C-terminal ER retention sequence. Immunofluorescence staining demonstrated that BAP co-localized with GRP94 in the endoplasmic reticulum. BAP was ubiquitously expressed but showed the highest levels of expression in secretory organ tissues, a pattern similar to that observed with BiP. BAP binding was affected by the conformation of the ATPase domain of BiP based on in vivo binding studies with BiP mutants. BAP stimulated the ATPase activity of BiP when added alone or together with the ER DnaJ protein, ERdj4, by promoting the release of ADP from BiP. Together, these data demonstrate that BAP serves as a nucleotide exchange factor for BiP and provide insights into the mechanisms that control protein folding in the mammalian ER.

Amphetamine is an indirect dopamine receptor agonist and increases glutamate release in the striatum. Activation of group I metabotropic glutamate receptors (mGluRs) upregulates cAMP response element-binding protein (CREB

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