A Subset of Chaperones and Folding Enzymes Form Multiprotein Complexes in Endoplasmic Reticulum to Bind Nascent Proteins

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

Probing for Membrane Domains in the Endoplasmic Reticulum: Retention and Degradation of Unassembled MHC Class I Molecules

Quality control of protein biosynthesis requires ER-retention and ER-associated degradation (ERAD) of unassembled/misfolded molecules. Although some evidence exists for the organization of the ER into functionally distinct membrane domains, it is unknown if such domains are involved in the retention and ERAD of unassembled proteins. Here, it is shown that unassembled MHC class I molecules are retained in the ER without accumulating at ER-exit sites or in the ERGIC of β2m(−/−) cells. Furthermore, these molecules did not cluster in the ER membrane and appeared to be highly mobile even when ERAD or their association with calnexin were inhibited. However, upon ATP depletion, they were reversibly segregated into an ER membrane domain, distinct from ER exit sites, which included calnexin and COPII, but not the ERGIC marker protein p58. This quality control domain was also observed upon prolonged inhibition of proteasomes. Microtubules were required for its appearance. Segregation of unfolded proteins, ER-resident chaperones, and COPII may be a temporal adaptation to cell stress