The endoplasmic reticulum (ER) is the site where proteins enter the secretory pathway. Proteins are translocated into the ER lumen in an unfolded state and require protein chaperones and catalysts of protein folding to attain their final appropriate conformation. A sensitive surveillance mechanism exists to prevent misfolded proteins from transiting the secretory pathway and ensures that persistently misfolded proteins are directed towards a degradative pathway. In addition, those processes that prevent accumulation of unfolded proteins in the ER lumen are highly regulated by an intracellular signaling pathway known as the unfolded protein response (UPR). The UPR provides a mechanism by which cells can rapidly adapt to alterations in client protein-folding load in the ER lumen by expanding the capacity for protein folding. In addition, a variety of insults that disrupt protein folding in the ER lumen also activate the UPR. These include changes in intralumenal calcium, altered glycosylation, nutrient deprivation, pathogen infection, expression of folding-defective proteins, and changes in redox status. Persistent protein misfolding initiates apoptotic cascades that are now known to play fundamental roles in the pathogenesis of multiple human diseases including diabetes, atherosclerosis and neurodegenerative diseases.

The endoplasmic reticulum (ER) is a multifunctional organelle which co-ordinates protein folding, lipid biosynthesis, calcium storage and release. Perturbations that disrupt ER homeostasis lead to the misfolding of proteins, ER stress and up-regulation of a signaling pathway called the ER stress response or the unfolded protein response (UPR). The UPR is characterized by the induction of chaperones, degradation of misfolded proteins and attenuation of protein translation. Age-related declines and activity in key molecular chaperones and folding enzymes compromise proper protein folding and the adaptive response of the UPR. This review will highlight age-related changes in the protein folding machinery and in the UPR.

The mechanism, in molecular terms of protein quality control, specifically of how the cell recognizes and discriminates misfolded proteins, remains a challenge. In the secretory pathway the folding status of glycoproteins passing through the endoplasmic reticulum is marked by the composition of the N-glycan. The different glycoforms are recognized by specialized lectins. The folding sensor UGGT acts as an unusual molecular chaperone and covalently modifies the Man9 N-glycan of a misfolded protein by adding a glucose moiety and converts it to Glc1Man9 that rebinds the lectin calnexin. However, further links between the folding status of a glycoprotein and the composition of the N-glycan are unclear. There is little unequivocal evidence for other proteins in the ER recognizing the N-glycan and also acting as molecular chaperones. Nevertheless, based upon a few examples, we suggest that this function is carried out by individual proteins in several different complexes. Thus, calnexin binds the protein disulfide isomerase ERp57, that acts upon Glc1Man9 glycoproteins. In another example the protein disulfide isomerase ERdj5 binds specifically to EDEM (which is probably a mannosidase) and a lectin OS9, and reduces the disulfide bonds of bound glycoproteins destined for ERAD. Thus the glycan recognition is performed by a lectin and the chaperone function performed by a specific partner protein that can recognize misfolded proteins. We predict that this will be a common arrangement of proteins in the ER and that members of protein foldase families such as PDI and PPI will bind specifically to lectins in the ER. Molecular chaperones BiP and GRp94 will assist in the folding of proteins bound in these complexes as well as in the folding of non-glycoproteins.

Recognition and elimination of misfolded proteins are essential cellular processes. More than thirty percent of the cellular proteins are proteins of the secretory pathway. They fold in the lumen or membrane of the endoplasmic reticulum from where they are sorted to their site of action. The folding process, as well as any refolding after cell stress, depends on chaperone activity. In case proteins are unable to acquire their native conformation, chaperones with different substrate specificity and activity guide them to elimination. For most misfolded proteins of the endoplasmic reticulum this requires retro-translocation to the cytosol and polyubiquitylation of the misfolded protein by an endoplasmic reticulum associated machinery. Thereafter ubiquitylated proteins are guided to the proteasome for degradation. This review summarizes our up to date knowledge of chaperone classes and chaperone function in endoplasmic reticulum associated degradation of protein waste.

Cotranslational translocation of polypeptides into the ER is controlled by the dynamic interaction of ribosome and translocon components. Analysis of the steps involved in this process by high resolution techniques such as gel electrophoresis is precluded by the high molecular masses of these complexes. We show, here, that modifications to standard native electrophoresis protocols can overcome these problems and lead to an increase in mass range and resolution. Using the modified technique, we show that ER ribosome anchored membrane protein (RAMP) complexes resolve into 3 stable and semistable complexes which range in size between 4 and 8 MDa and are sensitive to relevant concentrations of divalent metals. We demonstrate the molecular composition of the complexes and identify a number of modular components that differentiate them. The components that are common to all three RAMP complexes include the OST translocon subcomplex, Glucosidase I and microtubule tethering protein CLIMP63. The two larger complexes further include the kinesin motor binding protein p180 and Sec61, and the largest complex includes the TRAP translocon component and apoptotic regulator BAP31. On the lumenal side, the BiP cochaperone ERdj3 resides with the three RAMP complexes. Our observations may hint at how subcompartmentalization is achieved in the ER membrane continuum.